@article {pmid34544122,
year = {2021},
author = {Seher, TD and Nguyen, N and Ramos, D and Bapat, P and Nobile, CJ and Sindi, SS and Hernday, AD},
title = {AddTag, a two-step approach with supporting software package that facilitates CRISPR/Cas-mediated precision genome editing.},
journal = {G3 (Bethesda, Md.)},
volume = {11},
number = {9},
pages = {},
doi = {10.1093/g3journal/jkab216},
pmid = {34544122},
issn = {2160-1836},
support = {/NH/NIH HHS/United States ; //NIH/ ; //National Institute of Allergy and Infectious Diseases/ ; //NIAID/ ; /GM/NIGMS NIH HHS/United States ; R15AI137975/GM/NIGMS NIH HHS/United States ; //Pew Biomedical Scholar Award/ ; //Pew Charitable Trusts/ ; },
abstract = {CRISPR/Cas-induced genome editing is a powerful tool for genetic engineering, however, targeting constraints limit which loci are editable with this method. Since the length of a DNA sequence impacts the likelihood it overlaps a unique target site, precision editing of small genomic features with CRISPR/Cas remains an obstacle. We introduce a two-step genome editing strategy that virtually eliminates CRISPR/Cas targeting constraints and facilitates precision genome editing of elements as short as a single base-pair at virtually any locus in any organism that supports CRISPR/Cas-induced genome editing. Our two-step approach first replaces the locus of interest with an "AddTag" sequence, which is subsequently replaced with any engineered sequence, and thus circumvents the need for direct overlap with a unique CRISPR/Cas target site. In this study, we demonstrate the feasibility of our approach by editing transcription factor binding sites within Candida albicans that could not be targeted directly using the traditional gene-editing approach. We also demonstrate the utility of the AddTag approach for combinatorial genome editing and gene complementation analysis, and we present a software package that automates the design of AddTag editing.},
}

@article {pmid34542686,
year = {2021},
author = {Gupta, R and Kazi, TA and Dey, D and Ghosh, A and Ravichandiran, V and Swarnakar, S and Roy, S and Biswas, SR and Ghosh, D},
title = {CRISPR detectives against SARS-CoV-2: a major setback against COVID-19 blowout.},
journal = {Applied microbiology and biotechnology},
volume = {},
number = {},
pages = {},
pmid = {34542686},
issn = {1432-0614},
support = {BT/PR26301/GET/119/258/2017//department of biotechnology , ministry of science and technology/ ; BT/P/Budget/RD-74/2017//department of science and technology, government of west bengal/ ; },
abstract = {The emergence of SARS-CoV-2 has brought the world to a standstill, and till date, effective treatments and diagnostics against this idiosyncratic pathogen are lacking. As compared to the standard WHO/CDC qPCR detection method, which consumes several hours for detection, CRISPR-based SHERLOCK, DETECTR, and FELUDA have emerged as rapid diagnostic tools for the detection of the RNA genome of SARS-CoV-2 within an hour with 100% accuracy, specificity, and sensitivity. These attributes of CRISPR-based detection technologies have taken themselves one step ahead of available detection systems and are emerging as an inevitable tool for quick detection of the virus. Further, the discovery of Cas13s nucleases and their orthologs has opened a new corridor for exploitation of Cas13s as an antiviral therapy against SARS-CoV-2 and other viral diseases. One such approach is Prophylactic Antiviral CRISPR in huMAN cells (PACMAN), which needs a long haul to bring into therapy. The approval of SHERLOCK as the first CRISPR-based SARS-CoV-2 test kit by the FDA, for emergency diagnosis of COVID-19 patients, has given positive hope to scientists that sooner human trials of CRISPR-based therapy will be ratified. In this review, we have extensively reviewed the present CRISPR-based approaches, challenges, and future prospects in the light of diagnostics and therapeutics against SARS-CoV-2. KEY POINTS: • The discovery of Cas12 and Cas13 siblings allowed scientists to detect the viral genes. • Cas13d's identification aided scientists in precisely cleaving the SARS-CoV-2 ssRNA. • CRISPR-Cas system acts as "molecular detector and antiviral proctor."},
}

@article {pmid34541453,
year = {2021},
author = {Liu, W and Li, L and Jiang, J and Wu, M and Lin, P},
title = {Applications and challenges of CRISPR-Cas gene-editing to disease treatment in clinics.},
journal = {Precision clinical medicine},
volume = {4},
number = {3},
pages = {179-191},
doi = {10.1093/pcmedi/pbab014},
pmid = {34541453},
issn = {2516-1571},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-associated systems (Cas) are efficient tools for targeting specific genes for laboratory research, agricultural engineering, biotechnology, and human disease treatment. Cas9, by far the most extensively used gene-editing nuclease, has shown great promise for the treatment of hereditary diseases, viral infection, cancers, and so on. Recent reports have revealed that some other types of CRISPR-Cas systems may also have surprising potential to join the fray as gene-editing tools for various applications. Despite the rapid progress in basic research and clinical tests, some underlying problems present continuous, significant challenges, such as editing efficiency, relative difficulty in delivery, off-target effects, immunogenicity, etc. This article summarizes the applications of CRISPR-Cas from bench to bedside and highlights the current obstacles that may limit the usage of CRISPR-Cas systems as gene-editing toolkits in precision medicine and offer some viewpoints that may help to tackle these challenges and facilitate technical development. CRISPR-Cas systems, as a powerful gene-editing approach, will offer great hopes in clinical treatments for many individuals with currently incurable diseases.},
}

@article {pmid34541154,
year = {2017},
author = {Chen, F and Ding, X and Feng, Y and Seebeck, T and Jiang, Y and Davis, GD},
title = {Improving CRISPR Gene Editing Efficiency by Proximal dCas9 Targeting.},
journal = {Bio-protocol},
volume = {7},
number = {15},
pages = {e2432},
doi = {10.21769/BioProtoc.2432},
pmid = {34541154},
issn = {2331-8325},
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems function as an adaptive immune system in bacteria and archaea for defense against invading viruses and plasmids (Barrangou and Marraffini, 2014). The effector nucleases from some class 2 CRISPR-Cas systems have been repurposed for heterologous targeting in eukaryotic cells (Jinek et al., 2012 ; Cong et al., 2013 ; Mali et al., 2013 ; Zetsche et al., 2015). However, the genomic environments of eukaryotes are distinctively different from that of prokaryotes in which CRISPR-Cas systems have evolved. Mammalian heterochromatin was found to be a barrier to target DNA access by Streptococcus pyogenes Cas9 (SpCas9), and nucleosomes, the basic units of the chromatin, were also found to impede target DNA access and cleavage by SpCas9 in vitro (Knight et al., 2015 ; Hinz et al., 2015 ; Horlbeck et al., 2016 ; Isaac et al., 2016). Moreover, many CRISPR-Cas systems characterized to date often exhibit inactivity in mammalian cells and are thus precluded from gene editing applications even though they are active in bacteria or on purified DNA substrates. Thus, there is a need to devise a means to alleviate chromatin inhibition to increase gene editing efficiency, especially on difficult-to-access genomic sites, and to enable use of otherwise inactive CRISPR-Cas nucleases for gene editing need. Here we describe a proxy-CRISPR protocol for restoring nuclease activity of various class 2 CRISPR-Cas nucleases on otherwise inaccessible genomic sites in human cells via proximal targeting of a catalytically dead Cas9 (Chen et al., 2017). This protocol is exemplified here by using Campylobacter jejuni Cas9 (CjCas9) as nuclease and catalytically dead SpCas9 (SpdCas9) as proximal DNA binding protein to enable CjCas9 to cleave the target for gene editing using single stranded DNA oligo templates.},
}

@article {pmid34541105,
year = {2017},
author = {Nomura, T and Sakakibara, H},
title = {Targeted Mutagenesis Using RNA-guided Endonucleases in Mosses.},
journal = {Bio-protocol},
volume = {7},
number = {12},
pages = {e2359},
doi = {10.21769/BioProtoc.2359},
pmid = {34541105},
issn = {2331-8325},
abstract = {RNA-guided endonucleases (RGENs) have been used for genome editing in various organisms. Here, we demonstrate a simple method for performing targeted mutagenesis and genotyping in a model moss species, Physcomitrella patens, using RGENs. We also performed targeted mutagenesis in a non-model moss, Scopelophilla cataractae, using a similar method (Nomura et al., 2016), indicating that this experimental system could be applied to a wide range of mosses species.},
}

@article {pmid34537403,
year = {2021},
author = {Ricroch, AE and Martin-Laffon, J and Rault, B and Pallares, VC and Kuntz, M},
title = {Next biotechnological plants for addressing global challenges: The contribution of transgenesis and new breeding techniques.},
journal = {New biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.nbt.2021.09.001},
pmid = {34537403},
issn = {1876-4347},
abstract = {The aim of this survey is to identify and characterize new products in plant biotechnology since 2015, especially in relation to the advent of New Breeding Techniques (NBTs) such as gene editing based on the CRISPR-Cas system. Transgenic (gene transfer or gene silencing) and gene edited traits which are approved or marketed in at least one country, or which have a non-regulated status in the USA, are collected, as well as related patents worldwide. In addition, to shed light on potential innovation for Africa, field trials on the continent are examined. The compiled data are classified in application categories, including agronomic improvements, industrial use and medical use, namely production of recombinant therapeutic molecules or vaccines (including against Covid-19). The data indicate that gene editing appears to be an effective complement to 'classical' transgenesis, the use of which is not declining, rather than a replacement, a trend also observed in the patenting landscape. Nevertheless, increased use of gene editing is apparent. Compared to transgenesis, gene editing has increased the proportion of some crop species and decreased others amongst approved, non-regulated or marketed products. A similar differential trend is observed for breeding traits. Gene editing has also favored the emergence of new private companies. China, and prevalently its public sector, overwhelmingly dominates the patenting landscape, but not the approved/marketed one, which is dominated by the USA. The data point in the direction that regulatory environments will favor or discourage innovation.},
}

@article {pmid34529358,
year = {2021},
author = {Huang, RS and Lai, MC and Lin, S},
title = {Ex Vivo Expansion and CRISPR-Cas9 Genome Editing of Primary Human Natural Killer Cells.},
journal = {Current protocols},
volume = {1},
number = {9},
pages = {e246},
doi = {10.1002/cpz1.246},
pmid = {34529358},
issn = {2691-1299},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Gene Knockout Techniques ; Humans ; Immunotherapy, Adoptive ; Killer Cells, Natural ; },
abstract = {Natural killer (NK) cells are potent innate immune cells that provide the surveillance and elimination of infected, stressed, and malignant cells. The unique immune recognition mechanisms and functions of NK cells make them an attractive cell type for immunology research and adoptive immunotherapy. However, primary NK cells are challenging to culture ex vivo and lack efficient genetic tools, hindering the research of NK cells and the development of NK cell therapeutics. Here we describe methods for the freeze-thaw process, feeder-free ex vivo expansion, CRISPR-Cas9 genome editing, and functional characterizations of primary human NK cells. Our protocol enables ∼30-fold and ∼2000-fold average expansion rates from 1 × 107 cryopreserved NK cells in 14 and 28 days, respectively. We also detail methods for CRISPR gene knockout and knockin by nucleofection of Cas9 ribonucleoproteins (RNP) and DNA repair templates. Gene knockout by Cas9 RNP nucleofection can be multiplexed to simultaneously target three genes. The CRISPR-edited cells can be cryopreserved and rethawed with high viability for future studies. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Thawing of natural killer cells Basic Protocol 2: Ex vivo expansion of natural killer cells Basic Protocol 3: Cryopreservation of expanded natural killer cells Basic Protocol 4: Characterization of natural killer cells: Flow cytometry and surface marker analysis Basic Protocol 5: Cytotoxicity and degranulation assays Basic Protocol 6: Preparation of homology-directed repair templates Basic Protocol 7: Nucleofection of CRISPR-Cas9 ribonucleoproteins Basic Protocol 8: Genotyping of gene-edited natural killer cells Basic Protocol 9: Phenotyping of gene-edited natural killer cells.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing
Gene Knockout Techniques
Humans
Immunotherapy, Adoptive
Killer Cells, Natural

@article {pmid34471126,
year = {2021},
author = {Tong, Y and Jørgensen, TS and Whitford, CM and Weber, T and Lee, SY},
title = {A versatile genetic engineering toolkit for E. coli based on CRISPR-prime editing.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {5206},
pmid = {34471126},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Bacterial ; Escherichia coli/*genetics ; Gene Editing/*methods ; Genetic Engineering/*methods ; Genome, Bacterial ; Plasmids ; RNA, Guide/genetics ; },
abstract = {CRISPR base editing is a powerful method to engineer bacterial genomes. However, it restricts editing to single-nucleotide substitutions. Here, to address this challenge, we adapt a CRISPR-Prime Editing-based, DSB-free, versatile, and single-nucleotide resolution genetic manipulation toolkit for prokaryotes. It can introduce substitutions, deletions, insertions, and the combination thereof, both in plasmids and the chromosome of E. coli with high fidelity. Notably, under optimal conditions, the efficiency of 1-bp deletions reach up to 40%. Moreover, deletions of up to 97 bp and insertions up to 33 bp were successful with the toolkit in E. coli, however, efficiencies dropped sharply with increased fragment sizes. With a second guide RNA, our toolkit can achieve multiplexed editing albeit with low efficiency. Here we report not only a useful addition to the genome engineering arsenal for E. coli, but also a potential basis for the development of similar toolkits for other bacteria.},
}

*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
DNA, Bacterial
Escherichia coli/*genetics
Gene Editing/*methods
Genetic Engineering/*methods
Genome, Bacterial
Plasmids
RNA, Guide/genetics

@article {pmid34445589,
year = {2021},
author = {Shaw, AM and Qasem, A and Naser, SA},
title = {Modulation of PTPN2/22 Function by Spermidine in CRISPR-Cas9-Edited T-Cells Associated with Crohn's Disease and Rheumatoid Arthritis.},
journal = {International journal of molecular sciences},
volume = {22},
number = {16},
pages = {},
pmid = {34445589},
issn = {1422-0067},
support = {2014-2020//Florida Legislative Grant/ ; },
mesh = {Arthritis, Rheumatoid/genetics ; *CRISPR-Cas Systems ; Crohn Disease/genetics ; Gene Expression Regulation, Leukemic/*drug effects ; Genetic Predisposition to Disease ; Humans ; Jurkat Cells ; Leukemia, T-Cell/drug therapy/genetics/*pathology ; Lymphocyte Activation ; *Polymorphism, Single Nucleotide ; Protein Tyrosine Phosphatase, Non-Receptor Type 2/antagonists & inhibitors/*genetics/metabolism ; Protein Tyrosine Phosphatase, Non-Receptor Type 22/antagonists & inhibitors/*genetics/metabolism ; Spermidine/*pharmacology ; },
abstract = {Crohn's Disease (CD) and Rheumatoid Arthritis (RA) share some single nucleotide polymorphisms (SNPs) in protein tyrosine phosphatase non-receptor types 2 and 22 (PTPN2/22). Recently, we reported that clinical samples from CD and RA patients associated with PTPN2:rs478582 or PTPN22:rs2476601 genotypes were linked to overactive immune response and exacerbation of inflammation. Here, we investigated in vitro the effects of these SNPs in Jurkat T-cells using CRISPR-Cas9. All cells were evaluated for PTPN22/22 loss of function and effects on cell response. We measured gene expression via RT-qPCR and cytokines by ELISA. We also measured cell proliferation using a BrdU labeling proliferation ELISA, and T-cell activation using CD-25 fluorescent immunostaining. In PTPN2 SNP-edited cells, PTPN2 expression decreased by 3.2-fold, and proliferation increased by 10.2-fold compared to control. Likewise, expression of PTPN22 decreased by 2.4-fold and proliferation increased by 8.4-fold in PTPN22 SNP-edited cells. IFN-γ and TNF-α secretions increased in both edited cell lines. CD25 expression (cell activation) was 80.32% in PTPN2 SNP-edited cells and 85.82% in PTPN22 SNP-edited cells compared to 70.48% in unedited Jurkat T-cells. Treatment of PTPN2 and PTPN22-edited cells with a maximum 20 μM spermidine restored PTPN2/22 expression and cell response including cell proliferation, activation, and cytokines secretion. Most importantly, the effect of spermidine on edited cells restored normal expression and secretion of IFN-γ and TNF-α. The data clearly demonstrated that edited SNPs in PTPN2 or PTPN22 were associated with reduced gene expression, which resulted in an increase in cell proliferation and activation and overactive immune response. The data validated our earlier observations in CD and RA clinical samples. Surprisingly, spermidine restored PTPN2/22 expression in edited Jurkat T-cells and the consequent beneficial effect on cell response and inflammation. The study supports the use of polyamines dietary supplements for management of CD and in RA patients.},
}

Arthritis, Rheumatoid/genetics
*CRISPR-Cas Systems
Crohn Disease/genetics
Gene Expression Regulation, Leukemic/*drug effects
Genetic Predisposition to Disease
Humans
Jurkat Cells
Leukemia, T-Cell/drug therapy/genetics/*pathology
Lymphocyte Activation
*Polymorphism, Single Nucleotide
Protein Tyrosine Phosphatase, Non-Receptor Type 2/antagonists & inhibitors/*genetics/metabolism
Protein Tyrosine Phosphatase, Non-Receptor Type 22/antagonists & inhibitors/*genetics/metabolism
Spermidine/*pharmacology

@article {pmid34341582,
year = {2021},
author = {Gemberling, MP and Siklenka, K and Rodriguez, E and Tonn-Eisinger, KR and Barrera, A and Liu, F and Kantor, A and Li, L and Cigliola, V and Hazlett, MF and Williams, CA and Bartelt, LC and Madigan, VJ and Bodle, JC and Daniels, H and Rouse, DC and Hilton, IB and Asokan, A and Ciofani, M and Poss, KD and Reddy, TE and West, AE and Gersbach, CA},
title = {Transgenic mice for in vivo epigenome editing with CRISPR-based systems.},
journal = {Nature methods},
volume = {18},
number = {8},
pages = {965-974},
pmid = {34341582},
issn = {1548-7105},
support = {R01 DA036865/DA/NIDA NIH HHS/United States ; R33 DA041878/DA/NIDA NIH HHS/United States ; U01 AI146356/AI/NIAID NIH HHS/United States ; UG3 AR075336/AR/NIAMS NIH HHS/United States ; R01 GM115474/GM/NIGMS NIH HHS/United States ; R01 DA047115/DA/NIDA NIH HHS/United States ; UM1 HG009428/HG/NHGRI NIH HHS/United States ; },
mesh = {Animals ; Brain/metabolism ; *CRISPR-Cas Systems ; *Epigenesis, Genetic ; *Epigenome ; Female ; Fibroblasts/metabolism ; Gene Editing/*methods ; *Gene Expression Regulation ; Humans ; Liver/metabolism ; Male ; Mice ; Mice, Transgenic ; T-Lymphocytes/metabolism ; },
abstract = {CRISPR-Cas9 technologies have dramatically increased the ease of targeting DNA sequences in the genomes of living systems. The fusion of chromatin-modifying domains to nuclease-deactivated Cas9 (dCas9) has enabled targeted epigenome editing in both cultured cells and animal models. However, delivering large dCas9 fusion proteins to target cells and tissues is an obstacle to the widespread adoption of these tools for in vivo studies. Here, we describe the generation and characterization of two conditional transgenic mouse lines for epigenome editing, Rosa26:LSL-dCas9-p300 for gene activation and Rosa26:LSL-dCas9-KRAB for gene repression. By targeting the guide RNAs to transcriptional start sites or distal enhancer elements, we demonstrate regulation of target genes and corresponding changes to epigenetic states and downstream phenotypes in the brain and liver in vivo, and in T cells and fibroblasts ex vivo. These mouse lines are convenient and valuable tools for facile, temporally controlled, and tissue-restricted epigenome editing and manipulation of gene expression in vivo.},
}

Animals
Brain/metabolism
*CRISPR-Cas Systems
*Epigenesis, Genetic
*Epigenome
Female
Fibroblasts/metabolism
Gene Editing/*methods
*Gene Expression Regulation
Humans
Liver/metabolism
Male
Mice
Mice, Transgenic
T-Lymphocytes/metabolism

@article {pmid34286737,
year = {2021},
author = {Fan, Z and Ding, Y and Yao, B and Wang, J and Zhang, K},
title = {Electrochemiluminescence platform for transcription factor diagnosis by using CRISPR-Cas12a trans-cleavage activity.},
journal = {Chemical communications (Cambridge, England)},
volume = {57},
number = {65},
pages = {8015-8018},
doi = {10.1039/d1cc03071j},
pmid = {34286737},
issn = {1364-548X},
mesh = {Bacterial Proteins/chemistry ; Biosensing Techniques/*methods ; CRISPR-Associated Proteins/chemistry ; *CRISPR-Cas Systems ; DNA/chemistry ; Electrochemical Techniques/methods ; Endodeoxyribonucleases/chemistry ; Gold/chemistry ; Graphite/chemistry ; Immobilized Nucleic Acids/chemistry ; Limit of Detection ; Luminescence ; Luminescent Agents/*chemistry ; Luminescent Measurements/methods ; Metal Nanoparticles/chemistry ; NF-kappa B p50 Subunit/*analysis ; Nucleic Acid Conformation ; Organometallic Compounds/chemistry ; },
abstract = {Herein, we exploited the double-stranded DNA (dsDNA) binding property of transcription factor (TF), combined with the trans cleavage characteristic of CRISPR-Cas12a, for the detection of NF-κB p50.},
}

Bacterial Proteins/chemistry
Biosensing Techniques/*methods
CRISPR-Associated Proteins/chemistry
*CRISPR-Cas Systems
DNA/chemistry
Electrochemical Techniques/methods
Endodeoxyribonucleases/chemistry
Gold/chemistry
Graphite/chemistry
Immobilized Nucleic Acids/chemistry
Limit of Detection
Luminescence
Luminescent Agents/*chemistry
Luminescent Measurements/methods
Metal Nanoparticles/chemistry
NF-kappa B p50 Subunit/*analysis
Nucleic Acid Conformation
Organometallic Compounds/chemistry

@article {pmid34234315,
year = {2021},
author = {Kwisda, K and Cantz, T and Hoppe, N},
title = {Regulatory and intellectual property conundrums surrounding xenotransplantation.},
journal = {Nature biotechnology},
volume = {39},
number = {7},
pages = {796-798},
pmid = {34234315},
issn = {1546-1696},
mesh = {Animals ; Biotechnology/*legislation & jurisprudence ; CRISPR-Cas Systems ; European Union ; Genetic Engineering ; Humans ; *Intellectual Property ; Organisms, Genetically Modified ; Swine/genetics ; Transplantation, Heterologous/*legislation & jurisprudence ; World Health Organization ; },
}

Animals
Biotechnology/*legislation & jurisprudence
CRISPR-Cas Systems
European Union
Genetic Engineering
Humans
*Intellectual Property
Organisms, Genetically Modified
Swine/genetics
Transplantation, Heterologous/*legislation & jurisprudence
World Health Organization

@article {pmid34157315,
year = {2021},
author = {Zamaraev, AV and Volik, PI and Sukhikh, GT and Kopeina, GS and Zhivotovsky, B},
title = {Long non-coding RNAs: A view to kill ovarian cancer.},
journal = {Biochimica et biophysica acta. Reviews on cancer},
volume = {1876},
number = {1},
pages = {188584},
doi = {10.1016/j.bbcan.2021.188584},
pmid = {34157315},
issn = {1879-2561},
mesh = {Animals ; Antineoplastic Agents/therapeutic use ; Biomarkers, Tumor/genetics/*metabolism ; CRISPR-Cas Systems ; Drug Resistance, Neoplasm ; Female ; Gene Editing ; Gene Expression Regulation, Neoplastic ; Genetic Therapy ; Humans ; Oligonucleotides, Antisense/therapeutic use ; Ovarian Neoplasms/genetics/*metabolism/pathology/therapy ; RNA, Long Noncoding/genetics/*metabolism ; Signal Transduction ; },
abstract = {An emerging role of long non-coding RNAs (lncRNAs) in tumor progression has been revealed in the last decade. Through interactions with nucleic acids and proteins, lncRNAs could act as enhancers, scaffolds or decoys for a number of oncoproteins and tumor suppressors. The aberrant lncRNA expression or mutations are often associated with changes in a variety of cellular processes, including proliferation, stress response and cell death. Here, we will focus on the tumor-associated lncRNAs in ovarian cancer according to their contribution to cancer hallmarks, such as intense proliferation, cell death resistance, altered energy metabolism, invasion and metastasis, and immune evasion. Moreover, the potential clinical implications of lncRNAs and their significance for the diagnosis, prognosis and therapy of ovarian cancer will be discussed.},
}

Animals
Antineoplastic Agents/therapeutic use
Biomarkers, Tumor/genetics/*metabolism
CRISPR-Cas Systems
Drug Resistance, Neoplasm
Female
Gene Editing
Gene Expression Regulation, Neoplastic
Genetic Therapy
Humans
Oligonucleotides, Antisense/therapeutic use
Ovarian Neoplasms/genetics/*metabolism/pathology/therapy
RNA, Long Noncoding/genetics/*metabolism
Signal Transduction

@article {pmid33875479,
year = {2021},
author = {Ouellette, SP and Blay, EA and Hatch, ND and Fisher-Marvin, LA},
title = {CRISPR Interference To Inducibly Repress Gene Expression in Chlamydia trachomatis.},
journal = {Infection and immunity},
volume = {89},
number = {7},
pages = {e0010821},
pmid = {33875479},
issn = {1098-5522},
support = {R21 AI141933/AI/NIAID NIH HHS/United States ; },
mesh = {Binding Sites ; *CRISPR-Cas Systems ; Chlamydia Infections/microbiology ; Chlamydia trachomatis/*genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; *Gene Expression Regulation, Bacterial ; Gene Targeting ; Plasmids/genetics ; RNA, Guide ; Ribosomes/metabolism ; },
abstract = {The ability to inducibly repress gene expression is critical to the study of organisms, like Chlamydia, with reduced genomes in which the majority of genes are likely to be essential. We recently described the feasibility of a CRISPR interference (CRISPRi) system to inducibly repress gene expression in Chlamydia trachomatis. However, the initial system suffered from some drawbacks, primarily leaky expression of the anhydrotetracycline (aTc)-inducible dCas9 ortholog and plasmid instability, which prevented population-wide studies (e.g., transcript analyses) of the effects of knockdown. Here, we describe various modifications to the original system that have allowed us to measure gene expression changes within a transformed population of C. trachomatis serovar L2. These modifications include (i) a change in the vector backbone, (ii) the introduction of a weaker ribosome binding site driving dCas9 translation, and (iii) the addition of a degradation tag to dCas9 itself. With these changes, we demonstrate the ability to inducibly repress a target gene sequence, as measured by the absence of protein by immunofluorescence analysis and by decreased transcript levels. Importantly, the expression of dCas9 alone (i.e., without a guide RNA [gRNA]) had minimal impact on chlamydial growth or development. We also describe complementation of the knockdown effect by introducing a transcriptional fusion of the target gene 3' to dCas9. Finally, we demonstrate the functionality of a second CRISPRi system based on a dCas12 system that expands the number of potential chromosomal targets. These tools should provide the ability to study essential gene function in Chlamydia.},
}

Binding Sites
*CRISPR-Cas Systems
Chlamydia Infections/microbiology
Chlamydia trachomatis/*genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
*Gene Editing
*Gene Expression Regulation, Bacterial
Gene Targeting
Plasmids/genetics
RNA, Guide
Ribosomes/metabolism

@article {pmid33725516,
year = {2021},
author = {Xu, Q and Huang, L and Xing, J and Zhang, J and Li, H and Liu, L and Hu, C and Liao, M and Yue, J and Qi, W},
title = {Japanese encephalitis virus manipulates lysosomes membrane for RNA replication and utilizes autophagy components for intracellular growth.},
journal = {Veterinary microbiology},
volume = {255},
number = {},
pages = {109025},
doi = {10.1016/j.vetmic.2021.109025},
pmid = {33725516},
issn = {1873-2542},
mesh = {Animals ; Antimalarials/pharmacology ; Autophagy ; CRISPR-Cas Systems ; Cell Line ; Cell Membrane/*physiology ; Chloroquine/pharmacology ; Cricetinae ; Encephalitis Virus, Japanese/*physiology ; Gene Deletion ; Gene Expression Regulation, Viral/drug effects ; Humans ; Lysosomes/*physiology ; Protein Transport ; RNA, Viral/genetics/*metabolism ; Viral Proteins/genetics/metabolism ; Virus Replication/*physiology ; },
abstract = {Japanese encephalitis virus is absolutely dependent on their host cells and has evolved various strategies to manipulate the cellular secretory pathways for viral replication. However, how cellular secretory pathways are hijacked, and the origin of the viral vesicles remains elusive during JEV replication. Here we show how JEV manipulates multiple components of the cellular secretory pathway, including autophagic machinery, to generate a superior environment for genome replication. We utilized double-strand RNA antibodies to label JEV RNA complex seeking the viral replication compartments and found that JEV genome replication takes place in lysosomes (LAMP1), not in autophagosomes (LC3). Subsequently, in situ hybridization results showed that viral RNAs (vRNAs) of JEV strongly colocalized with LAMP1. What surprised us was that JEV vRNAs markedly colocalized with LC3, indicating that autophagy plays an active role in JEV replication. Interestingly, we found that JEV utilized autophagic components for intracellular growth in an autophagy-dependent manner and the fusion of autophagosome-lysosome plays a positive role in JEV post-RNA replication processes. Collectively, our findings demonstrate that JEV can manipulate cellular secretory pathway to form genome replication organelles and exploit autophagy components for intracellular growth, providing new insights into the life cycle of JEV and uncovering an attractive target for antiviral drugs.},
}

Animals
Antimalarials/pharmacology
Autophagy
CRISPR-Cas Systems
Cell Line
Cell Membrane/*physiology
Chloroquine/pharmacology
Cricetinae
Encephalitis Virus, Japanese/*physiology
Gene Deletion
Gene Expression Regulation, Viral/drug effects
Humans
Lysosomes/*physiology
Protein Transport
RNA, Viral/genetics/*metabolism
Viral Proteins/genetics/metabolism
Virus Replication/*physiology

@article {pmid33711567,
year = {2021},
author = {Wu, X and Wu, H and Wang, H and Luo, L and Wang, J and Wu, B and He, Q and Cao, G and Lei, Y and Chen, X and Dai, J},
title = {A new strategy to develop pseudorabies virus-based bivalent vaccine with high immunogenicity of porcine circovirus type 2.},
journal = {Veterinary microbiology},
volume = {255},
number = {},
pages = {109022},
doi = {10.1016/j.vetmic.2021.109022},
pmid = {33711567},
issn = {1873-2542},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Circovirus/*immunology ; Female ; HEK293 Cells ; *Herpesvirus 1, Suid ; Humans ; Mice ; Mice, Inbred BALB C ; Plasmids ; Pseudorabies/*prevention & control ; Pseudorabies Vaccines/*immunology ; Swine ; Swine Diseases/blood/*prevention & control ; Viral Proteins/immunology ; },
abstract = {Herpesvirus based multivalent vaccines have been extensively studied, whereas few of them have been successfully used in clinic and animal husbandry industry due to the low expression of foreign immunogens in herpesvirus. In this study, we developed a new strategy to construct herpesvirus based bivalent vaccine with high-level expression of foreign immunogen, by which the ORF2 gene encoding the major antigen protein Cap of porcine circovirus type 2 (PCV2), was highly expressed in pseudorabies virus (PRV). To obtain the high expression of PCV2 immunogen, tandem repeats of PCV2 ORF2 gene were firstly linked by protein quantitation ratioing (PQR) linker to reach equal expression of each ORF2 gene. Then, the multiple copies of ORF2 gene were respectively inserted into the gE and gG sites of PRV using CRISPR/Cas9 system, in which the expression of ORF2 gene was driven by endogenous strong promoters of PRV. Through this way, the highest yield of Cap protein was achieved in two copies of quadruple ORF2 gene insertion. Finally, in mice and pigs immunized with the bivalent vaccine candidate, we detected high titer of specific antibodies for PRV and neutralized antibodies for PCV2, and observed protective effect of the bivalent vaccine candidate against PRV challenge in immunized pigs, suggesting a potential clinical application of the bivalent vaccine candidate we constructed. Together, our strategy could be extensively applied to the generation of other multivalent vaccines, and will pave the way to construct herpesvirus based multivalent vaccines to effectively reduce the cost of vaccine.},
}

Animals
CRISPR-Cas Systems
Cell Line
Circovirus/*immunology
Female
HEK293 Cells
*Herpesvirus 1, Suid
Humans
Mice
Mice, Inbred BALB C
Plasmids
Pseudorabies/*prevention & control
Pseudorabies Vaccines/*immunology
Swine
Swine Diseases/blood/*prevention & control
Viral Proteins/immunology

@article {pmid33705684,
year = {2021},
author = {Wang, G and Wen, B and Ren, X and Li, E and Zhang, Y and Guo, M and Xu, Y and Whitsett, JA and Kalin, TV and Kalinichenko, VV},
title = {Generation of Pulmonary Endothelial Progenitor Cells for Cell-based Therapy Using Interspecies Mouse-Rat Chimeras.},
journal = {American journal of respiratory and critical care medicine},
volume = {204},
number = {3},
pages = {326-338},
doi = {10.1164/rccm.202003-0758OC},
pmid = {33705684},
issn = {1535-4970},
mesh = {Animals ; Animals, Newborn ; CRISPR-Cas Systems ; Chimera ; Disease Models, Animal ; Embryonic Stem Cells/*cytology/metabolism ; Endothelial Progenitor Cells/*cytology/metabolism/transplantation ; Forkhead Transcription Factors/genetics ; Humans ; Induced Pluripotent Stem Cells/*cytology/metabolism ; Infant, Newborn ; Mice ; Persistent Fetal Circulation Syndrome/metabolism/pathology/*therapy ; Pluripotent Stem Cells ; RNA-Seq ; Rats ; Single-Cell Analysis ; *Stem Cell Transplantation ; },
abstract = {Rationale: Although pulmonary endothelial progenitor cells (EPCs) hold promise for cell-based therapies for neonatal pulmonary disorders, whether EPCs can be derived from pluripotent embryonic stem cells (ESCs) or induced pluripotent stem cells remains unknown.Objectives: To investigate the heterogeneity of pulmonary EPCs and derive functional EPCs from pluripotent ESCs.Methods: Single-cell RNA sequencing of neonatal human and mouse lung was used to identify the heterogeneity of pulmonary EPCs. CRISPR/Cas9 gene editing was used to genetically label and purify mouse pulmonary EPCs. Functional properties of the EPCs were assessed after cell transplantation into neonatal mice with S52F Foxf1 mutation, a mouse model of alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). Interspecies mouse-rat chimeras were produced through blastocyst complementation to generate EPCs from pluripotent ESCs for cell therapy in ACDMPV mice.Measurements and Main Results: We identified a unique population of EPCs, FOXF1+cKIT+ EPCs, as a subset of recently described general capillary cells (gCAPs) expressing SMAD7, ZBTB20, NFIA, and DLL4 but lacking mature arterial, venous, and lymphatic markers. FOXF1+cKIT+ gCAPs are reduced in ACDMPV, and their transcriptomic signature is conserved in mouse and human lungs. After cell transplantation into the neonatal circulation of ACDMPV mice, FOXF1+cKIT+ gCAPs engraft into the pulmonary vasculature, stimulate angiogenesis, improve oxygenation, and prevent alveolar simplification. FOXF1+cKIT+ gCAPs, produced from ESCs in interspecies chimeras, are fully competent to stimulate neonatal lung angiogenesis and alveolarization in ACDMPV mice.Conclusions: Cell-based therapy using donor or ESC/induced pluripotent stem cell-derived FOXF1+cKIT+ endothelial progenitors may be considered for treatment of human ACDMPV.},
}

Animals
Animals, Newborn
CRISPR-Cas Systems
Chimera
Disease Models, Animal
Embryonic Stem Cells/*cytology/metabolism
Endothelial Progenitor Cells/*cytology/metabolism/transplantation
Forkhead Transcription Factors/genetics
Humans
Induced Pluripotent Stem Cells/*cytology/metabolism
Infant, Newborn
Mice
Persistent Fetal Circulation Syndrome/metabolism/pathology/*therapy
Pluripotent Stem Cells
RNA-Seq
Rats
Single-Cell Analysis
*Stem Cell Transplantation

@article {pmid33411694,
year = {2021},
author = {Schmid, RS and Deng, X and Panikker, P and Msackyi, M and Breton, C and Wilson, JM},
title = {CRISPR/Cas9 directed to the Ube3a antisense transcript improves Angelman syndrome phenotype in mice.},
journal = {The Journal of clinical investigation},
volume = {131},
number = {5},
pages = {},
pmid = {33411694},
issn = {1558-8238},
mesh = {Angelman Syndrome/genetics/*metabolism/*therapy ; Animals ; Brain/*metabolism ; *CRISPR-Cas Systems ; *Gene Editing ; Humans ; Mice ; RNA, Antisense/genetics/*metabolism ; Ubiquitin-Protein Ligases/genetics/*metabolism ; },
abstract = {Gene editing holds the potential to correct mutations and cure devastating genetic disorders. The technology has not yet proven efficacious for therapeutic use in CNS diseases with ubiquitous neuronal defects. Angelman syndrome (AS), a severe neurodevelopmental disorder, is caused by a lack of maternal expression of the UBE3A gene. Because of genomic imprinting, only neurons are affected. One therapeutic approach focuses on the intact paternal UBE3A copy in patients with AS that is silenced by an antisense transcript (UBE3A-ATS). We show here that gene editing of Ube3a-ATS in the mouse brain resulted in the formation of base pair insertions/deletions (indels) in neurons and the subsequent unsilencing of the paternal Ube3a allele in neurons, which partially corrected the behavioral phenotype of a murine AS model. This study provides compelling evidence to further investigate editing of the homologous region of the human UBE3A-ATS because this may provide a lasting therapeutic effect for patients with AS.},
}

Angelman Syndrome/genetics/*metabolism/*therapy
Animals
Brain/*metabolism
*CRISPR-Cas Systems
*Gene Editing
Humans
Mice
RNA, Antisense/genetics/*metabolism
Ubiquitin-Protein Ligases/genetics/*metabolism

@article {pmid33357766,
year = {2019},
author = {Moscoso, CG and Steer, CJ},
title = {Liver targeted gene therapy: Insights into emerging therapies.},
journal = {Drug discovery today. Technologies},
volume = {34},
number = {},
pages = {9-19},
doi = {10.1016/j.ddtec.2020.11.001},
pmid = {33357766},
issn = {1740-6749},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; DNA Transposable Elements/genetics ; Disease Models, Animal ; Gene Editing/*methods/trends ; Genetic Therapy/*methods/trends ; Genetic Vectors/administration & dosage/genetics ; Humans ; Liver/metabolism/pathology ; Liver Diseases/genetics/metabolism/pathology/*therapy ; Metabolic Diseases/genetics/metabolism/pathology/*therapy ; Transposases/genetics ; },
abstract = {The large number of monogenic metabolic disorders originating in the liver poses a unique opportunity for development of gene therapy modalities to pursue curative approaches. Various disorders have been successfully treated via liver-directed gene therapy, though most of the advances have been in animal models, with only limited success in clinical trials. Pre-clinical data in animals using non-viral approaches, including the Sleeping Beauty transposon system, are discussed. The various advances with viral vectors for liver-directed gene therapy are also a focus of this review, including retroviral, adenoviral, recombinant adeno-associated viral, and SV40 vectors. Genome editing techniques, including zinc finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short palindromic repeats (CRISPR), are also described. Further, the various controversies in the field with regards to somatic vs. germline editing using CRISPR in humans are explored, while also highlighting the myriad of preclinical advances. Lastly, newer technologies are reviewed, including base editing and prime editing, which use CRISPR with exciting adjunctive properties to avoid double-stranded breaks and thus the recruitment of endogenous repair mechanisms. While encouraging results have been achieved recently, there are still significant challenges to overcome prior to the broad use of vector-based and genome editing techniques in the clinical arena. As these technologies mature, the promise of a cure for many disabling inherited metabolic disorders is within reach, and urgently needed.},
}

Animals
CRISPR-Cas Systems/genetics
DNA Transposable Elements/genetics
Disease Models, Animal
Gene Editing/*methods/trends
Genetic Therapy/*methods/trends
Genetic Vectors/administration & dosage/genetics
Humans
Liver/metabolism/pathology
Liver Diseases/genetics/metabolism/pathology/*therapy
Metabolic Diseases/genetics/metabolism/pathology/*therapy
Transposases/genetics

@article {pmid33021790,
year = {2020},
author = {Grant, CV and Cai, S and Risinger, AL and Liang, H and O'Keefe, BR and Doench, JG and Cichewicz, RH and Mooberry, SL},
title = {CRISPR-Cas9 Genome-Wide Knockout Screen Identifies Mechanism of Selective Activity of Dehydrofalcarinol in Mesenchymal Stem-like Triple-Negative Breast Cancer Cells.},
journal = {Journal of natural products},
volume = {83},
number = {10},
pages = {3080-3092},
pmid = {33021790},
issn = {1520-6025},
support = {U01 CA182740/CA/NCI NIH HHS/United States ; },
mesh = {17-Hydroxysteroid Dehydrogenases/drug effects/genetics ; Aldehyde Oxidoreductases/drug effects/genetics ; Antineoplastic Agents, Phytogenic/*pharmacology ; Breast Neoplasms/*drug therapy ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Female ; Gene Knockout Techniques/*methods ; Humans ; Molecular Structure ; Neoplastic Stem Cells/*drug effects ; PPAR gamma/agonists ; RNA, Small Interfering/pharmacology ; Triple Negative Breast Neoplasms/*drug therapy ; },
abstract = {There are no targeted therapies available for triple-negative breast cancers (TNBCs) in part because they represent a heterogeneous group of tumors with diverse oncogenic drivers. Our goal is to identify targeted therapies for subtypes of these cancers using a mechanism-blind screen of natural product extract libraries. An extract from Desmanthodium guatemalense was 4-fold more potent for cytotoxicity against MDA-MB-231 cells, which represent the mesenchymal stem-like (MSL) subtype, as compared to cells of other TNBC subtypes. Bioassay-guided fractionation led to the isolation of six polyacetylenes, and subsequent investigations of plant sources known to produce polyacetylenes yielded six additional structurally related compounds. A subset of these compounds retained selective cytotoxic effects in MSL subtype cells. Studies suggest that these selective effects do not appear to be due to PPARγ agonist activities that have previously been reported for polyacetylenes. A CRISPR-Cas9-mediated gene knockout screen was employed to identify the mechanism of selective cytotoxic activity of the most potent and selective compound, dehydrofalcarinol (1a). This genomic screen identified HSD17B11, the gene encoding the enzyme 17β-hydroxysteroid dehydrogenase type 11, as a mediator of the selective cytotoxic effects of 1a in MDA-MB-231 cells that express high levels of this protein. The Project Achilles cancer dependency database further identified a subset of Ewing sarcoma cell lines as highly dependent on HSD17B11 expression, and it was found these were also highly sensitive to 1a. This report demonstrates the value of CRISPR-Cas9 genome-wide screens to identify the mechanisms underlying the selective activities of natural products.},
}

17-Hydroxysteroid Dehydrogenases/drug effects/genetics
Aldehyde Oxidoreductases/drug effects/genetics
Antineoplastic Agents, Phytogenic/*pharmacology
Breast Neoplasms/*drug therapy
*CRISPR-Cas Systems
Cell Line, Tumor
Female
Gene Knockout Techniques/*methods
Humans
Molecular Structure
Neoplastic Stem Cells/*drug effects
PPAR gamma/agonists
RNA, Small Interfering/pharmacology
Triple Negative Breast Neoplasms/*drug therapy

@article {pmid32841375,
year = {2021},
author = {Ge, L and Kang, J and Dong, X and Luan, D and Su, G and Li, G and Zhang, Y and Quan, F},
title = {Myostatin site-directed mutation and simultaneous PPARγ site-directed knockin in bovine genome.},
journal = {Journal of cellular physiology},
volume = {236},
number = {4},
pages = {2592-2605},
doi = {10.1002/jcp.30017},
pmid = {32841375},
issn = {1097-4652},
mesh = {Adipogenesis ; Animals ; CRISPR-Associated Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Cattle ; Cell Line ; Cell Proliferation ; Cell Transdifferentiation ; Clustered Regularly Interspaced Short Palindromic Repeats ; Feasibility Studies ; *Gene Editing ; Gene Expression Regulation, Developmental ; *Gene Knock-In Techniques ; Muscle Development ; *Mutagenesis, Site-Directed ; *Mutation ; Myoblasts/*metabolism ; Myostatin/*genetics/metabolism ; Nuclear Transfer Techniques ; PPAR gamma/*genetics/metabolism ; },
abstract = {Most studies on the acquisition of advantageous traits in transgenic animals only focus on monogenic traits. In practical applications, transgenic animals need to possess multiple advantages. Therefore, multiple genes need to be edited simultaneously. CRISPR/Cas9 technology has been widely used in many research fields. However, few studies on endogenous gene mutation and simultaneous exogenous gene insertion performed via CRISPR/Cas9 technology are available. In this study, the CRISPR/Cas9 technology was used to achieve myostatin (MSTN) point mutation and simultaneous peroxisome proliferator-activated receptor-γ (PPARγ) site-directed knockin in the bovine genome. The feasibility of this gene editing strategy was verified on a myoblast model. The same gene editing strategy was used to construct a mutant myoblast model with MSTN mutation and simultaneous PPARγ knockin. Quantitative reverse-transcription polymerase chain reaction, immunofluorescence staining, and western blot analyses were used to detect the expression levels of MSTN and PPARγ in the mutant myoblast. Results showed that this strategy can inhibit the expression of MSTN and promote the expression of PPARγ. The cell counting kit-8 cell proliferation analysis, 5-ethynyl-2'-deoxyuridine cell proliferation analysis, myotube fusion index statistics, oil red O staining, and triglyceride content detection revealed that the proliferation, myogenic differentiation, and adipogenic transdifferentiation abilities of the mutant myoblasts were higher than those of the wild myoblasts. Finally, transgenic bovine embryos were obtained via somatic cell nuclear transfer. This study provides a breeding material and technical strategy to breed high-quality bovine and a gene editing method to realize the mutation of endogenous genes and simultaneous insertion of exogenous genes in genomes.},
}

Adipogenesis
Animals
CRISPR-Associated Proteins/genetics/metabolism
*CRISPR-Cas Systems
Cattle
Cell Line
Cell Proliferation
Cell Transdifferentiation
Clustered Regularly Interspaced Short Palindromic Repeats
Feasibility Studies
*Gene Editing
Gene Expression Regulation, Developmental
*Gene Knock-In Techniques
Muscle Development
*Mutagenesis, Site-Directed
*Mutation
Myoblasts/*metabolism
Myostatin/*genetics/metabolism
Nuclear Transfer Techniques
PPAR gamma/*genetics/metabolism

@article {pmid34534948,
year = {2021},
author = {van Dongen, JE and Berendsen, JTW and Eijkel, JCT and Segerink, LI},
title = {A CRISPR/Cas12a-assisted in vitro diagnostic tool for identification and quantification of single CpG methylation sites.},
journal = {Biosensors & bioelectronics},
volume = {194},
number = {},
pages = {113624},
doi = {10.1016/j.bios.2021.113624},
pmid = {34534948},
issn = {1873-4235},
abstract = {The excellent specificity and selectivity of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/associated nuclease (Cas) is determined by CRISPR RNA's (crRNA's) interchangeable spacer sequence, as well as the position and number of mismatches between target sequence and the crRNA sequence. Some diseases are characterized by epigenetic alterations rather than nucleotide changes, and are therefore unsuitable for CRISPR-assisted sensing methods. Here we demonstrate an in vitro diagnostic tool to discriminate single CpG site methylation in DNA by the use of methylation-sensitive restriction enzymes (MSREs) followed by Cas12a-assisted sensing. Non-methylated sequences are digested by MSREs, resulting in fragmentation of the target sequence that influences the R-loop formation between crRNA and target DNA. We show that fragment size, fragmentation position and number of fragments influence the subsequent collateral trans-cleavage activity towards single stranded DNA (ssDNA), enabling deducting the methylation position from the cleavage activity. Utilizing MSREs in combination with Cas12a, single CpG site methylation levels of a cancer gene are determined. The modularity of both Cas12a and MSREs provides a high level of versatility to the Cas12a-MSRE combined sensing method, which opens the possibility to easily and rapidly study single CpG methylation sites for disease detection.},
}

@article {pmid34533605,
year = {2021},
author = {Pulido-Quetglas, C and Johnson, R},
title = {Designing libraries for pooled CRISPR functional screens of long noncoding RNAs.},
journal = {Mammalian genome : official journal of the International Mammalian Genome Society},
volume = {},
number = {},
pages = {},
pmid = {34533605},
issn = {1432-1777},
support = {4534-08-2018//helmut horten stiftung, swiss cancer research foundation/ ; 18/FRL/6194//science foundation ireland through future research leaders award/ ; },
abstract = {Human and other genomes encode tens of thousands of long noncoding RNAs (lncRNAs), the vast majority of which remain uncharacterised. High-throughput functional screening methods, notably those based on pooled CRISPR-Cas perturbations, promise to unlock the biological significance and biomedical potential of lncRNAs. Such screens are based on libraries of single guide RNAs (sgRNAs) whose design is critical for success. Few off-the-shelf libraries are presently available, and lncRNAs tend to have cell-type-specific expression profiles, meaning that library design remains in the hands of researchers. Here we introduce the topic of pooled CRISPR screens for lncRNAs and guide readers through the three key steps of library design: accurate annotation of transcript structures, curation of optimal candidate sets, and design of sgRNAs. This review is a starting point and reference for researchers seeking to design custom CRISPR screening libraries for lncRNAs.},
}

@article {pmid34529907,
year = {2021},
author = {Zeng, XX and Zeng, J and Zhu, B},
title = {Future generation of combined multimodal approach to treat brain glioblastoma multiforme and potential impact on micturition control.},
journal = {Reviews in the neurosciences},
volume = {},
number = {},
pages = {},
doi = {10.1515/revneuro-2021-0068},
pmid = {34529907},
issn = {2191-0200},
abstract = {Glioblastoma remains lethal even when treated with standard therapy. This review aims to outline the recent development of various advanced therapeutics for glioblastoma and briefly discuss the potential impact of glioblastoma and some of its therapeutic approaches on the neurological function micturition control. Although immunotherapy led to success in treating hematological malignancies, but no similar success occurred in treatment for brain glioblastoma. Neither regenerative medicine nor stem cell therapy led to astounding success in glioblastoma. However, CRISPR Cas system holds potential in multiple applications due to its capacity to knock-in and knock-out genes, modify immune cells and cell receptors, which will enable it to address clinical challenges in immunotherapy such as CAR-T and regenerative therapy for brain glioblastoma, improving the precision and safety of these approaches. The studies mentioned in this review could indicate that glioblastoma is a malignant disease with multiple sophisticated barriers to be overcome and more challenges might arise in the attempt of researchers to yield a successful cure. A multimodal approach of future generation of refined and safe therapeutics derived from CRISPR Cas therapeutics, immunotherapy, and regenerative therapeutics mentioned in this review might prolong survival or even contribute towards a potential cure for glioblastoma.},
}

@article {pmid34437126,
year = {2021},
author = {Gaudet, RG and Zhu, S and Halder, A and Kim, BH and Bradfield, CJ and Huang, S and Xu, D and Mamiñska, A and Nguyen, TN and Lazarou, M and Karatekin, E and Gupta, K and MacMicking, JD},
title = {A human apolipoprotein L with detergent-like activity kills intracellular pathogens.},
journal = {Science (New York, N.Y.)},
volume = {373},
number = {6552},
pages = {},
pmid = {34437126},
issn = {1095-9203},
support = {R01 AI068041/AI/NIAID NIH HHS/United States ; R01 AI108834/AI/NIAID NIH HHS/United States ; R01 NS113236/NS/NINDS NIH HHS/United States ; },
mesh = {Apolipoproteins L/chemistry/genetics/*metabolism ; Bacterial Outer Membrane/metabolism ; Bacteriolysis ; CRISPR-Cas Systems ; Cell Membrane/chemistry/*metabolism/ultrastructure ; Cell Membrane Permeability ; Cells, Cultured ; Cytosol/*microbiology ; Detergents/metabolism ; GTP-Binding Proteins/metabolism ; Gene Editing ; Gram-Negative Bacteria/immunology/pathogenicity/*physiology/ultrastructure ; Humans ; Immunity, Innate ; Interferon-gamma/*immunology ; Lipoproteins/chemistry ; Microbial Viability ; O Antigens/metabolism ; Protein Domains ; Salmonella typhimurium/immunology/pathogenicity/physiology/ultrastructure ; Solubility ; },
abstract = {Activation of cell-autonomous defense by the immune cytokine interferon-γ (IFN-γ) is critical to the control of life-threatening infections in humans. IFN-γ induces the expression of hundreds of host proteins in all nucleated cells and tissues, yet many of these proteins remain uncharacterized. We screened 19,050 human genes by CRISPR-Cas9 mutagenesis and identified IFN-γ-induced apolipoprotein L3 (APOL3) as a potent bactericidal agent protecting multiple non-immune barrier cell types against infection. Canonical apolipoproteins typically solubilize mammalian lipids for extracellular transport; APOL3 instead targeted cytosol-invasive bacteria to dissolve their anionic membranes into human-bacterial lipoprotein nanodiscs detected by native mass spectrometry and visualized by single-particle cryo-electron microscopy. Thus, humans have harnessed the detergent-like properties of extracellular apolipoproteins to fashion an intracellular lysin, thereby endowing resident nonimmune cells with a mechanism to achieve sterilizing immunity.},
}

Apolipoproteins L/chemistry/genetics/*metabolism
Bacterial Outer Membrane/metabolism
Bacteriolysis
CRISPR-Cas Systems
Cell Membrane/chemistry/*metabolism/ultrastructure
Cell Membrane Permeability
Cells, Cultured
Cytosol/*microbiology
Detergents/metabolism
GTP-Binding Proteins/metabolism
Gene Editing
Gram-Negative Bacteria/immunology/pathogenicity/*physiology/ultrastructure
Humans
Immunity, Innate
Interferon-gamma/*immunology
Lipoproteins/chemistry
Microbial Viability
O Antigens/metabolism
Protein Domains
Salmonella typhimurium/immunology/pathogenicity/physiology/ultrastructure
Solubility

@article {pmid34413309,
year = {2021},
author = {Moreb, EA and Lynch, MD},
title = {Genome dependent Cas9/gRNA search time underlies sequence dependent gRNA activity.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {5034},
pmid = {34413309},
issn = {2041-1723},
mesh = {Animals ; *CRISPR-Cas Systems ; Computational Biology/*methods ; Gene Editing/*methods ; Humans ; *RNA, Guide ; Retrospective Studies ; Species Specificity ; },
abstract = {CRISPR-Cas9 is a powerful DNA editing tool. A gRNA directs Cas9 to cleave any DNA sequence with a PAM. However, some gRNA sequences mediate cleavage at higher efficiencies than others. To understand this, numerous studies have screened large gRNA libraries and developed algorithms to predict gRNA sequence dependent activity. These algorithms do not predict other datasets as well as their training dataset and do not predict well between species. Here, to better understand these discrepancies, we retrospectively examine sequence features that impact gRNA activity in 44 published data sets. We find strong evidence that gRNA sequence dependent activity is largely influenced by the ability of the Cas9/gRNA complex to find the target site rather than activity at the target site and that this drives sequence dependent differences in gRNA activity between different species. This understanding will help guide future work to understand Cas9 activity as well as efforts to identify optimal gRNAs and improve Cas9 variants.},
}

Animals
*CRISPR-Cas Systems
Computational Biology/*methods
Gene Editing/*methods
Humans
*RNA, Guide
Retrospective Studies
Species Specificity

@article {pmid34321217,
year = {2021},
author = {Carullo, NVN and Hinds, JE and Revanna, JS and Tuscher, JJ and Bauman, AJ and Day, JJ},
title = {A Cre-Dependent CRISPR/dCas9 System for Gene Expression Regulation in Neurons.},
journal = {eNeuro},
volume = {8},
number = {4},
pages = {},
pmid = {34321217},
issn = {2373-2822},
support = {K99 DA034681/DA/NIDA NIH HHS/United States ; R00 DA034681/DA/NIDA NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Expression Regulation ; HEK293 Cells ; Humans ; Integrases ; Neurons ; Rats ; },
abstract = {Site-specific genetic and epigenetic targeting of distinct cell populations is a central goal in molecular neuroscience and is crucial to understand the gene regulatory mechanisms that underlie complex phenotypes and behaviors. While recent technological advances have enabled unprecedented control over gene expression, many of these approaches are focused on selected model organisms and/or require labor-intensive customization for different applications. The simplicity and modularity of clustered regularly interspaced short palindromic repeats (CRISPR)-based systems have transformed genome editing and expanded the gene regulatory toolbox. However, there are few available tools for cell-selective CRISPR regulation in neurons. We designed, validated, and optimized CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) systems for Cre recombinase-dependent gene regulation. Unexpectedly, CRISPRa systems based on a traditional double-floxed inverted open reading frame (DIO) strategy exhibited leaky target gene induction even without Cre. Therefore, we developed an intron-containing Cre-dependent CRISPRa system (SVI-DIO-dCas9-VPR) that alleviated leaky gene induction and outperformed the traditional DIO system at endogenous genes in HEK293T cells and rat primary neuron cultures. Using gene-specific CRISPR sgRNAs, we demonstrate that SVI-DIO-dCas9-VPR can activate numerous rat or human genes (GRM2, Tent5b, Fos, Sstr2, and Gadd45b) in a Cre-specific manner. To illustrate the versatility of this tool, we created a parallel CRISPRi construct that successfully inhibited expression from a luciferase reporter in HEK293T cells only in the presence of Cre. These results provide a robust framework for Cre-dependent CRISPR-dCas9 approaches across different model systems, and enable cell-specific targeting when combined with common Cre driver lines or Cre delivery via viral vectors.},
}

Animals
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Gene Expression Regulation
HEK293 Cells
Humans
Integrases
Neurons
Rats

@article {pmid34285418,
year = {2021},
author = {Paulusma, CC and Bosma, PJ},
title = {Therapeutic base editing in the adult liver.},
journal = {Nature reviews. Gastroenterology & hepatology},
volume = {18},
number = {9},
pages = {597-598},
pmid = {34285418},
issn = {1759-5053},
mesh = {Adult ; *CRISPR-Cas Systems ; *Gene Editing ; Humans ; Liver ; },
}

Adult
*CRISPR-Cas Systems
*Gene Editing
Humans
Liver

@article {pmid34059824,
year = {2021},
author = {Mohassel, P and Donkervoort, S and Lone, MA and Nalls, M and Gable, K and Gupta, SD and Foley, AR and Hu, Y and Saute, JAM and Moreira, AL and Kok, F and Introna, A and Logroscino, G and Grunseich, C and Nickolls, AR and Pourshafie, N and Neuhaus, SB and Saade, D and Gangfuß, A and Kölbel, H and Piccus, Z and Le Pichon, CE and Fiorillo, C and Ly, CV and Töpf, A and Brady, L and Specht, S and Zidell, A and Pedro, H and Mittelmann, E and Thomas, FP and Chao, KR and Konersman, CG and Cho, MT and Brandt, T and Straub, V and Connolly, AM and Schara, U and Roos, A and Tarnopolsky, M and Höke, A and Brown, RH and Lee, CH and Hornemann, T and Dunn, TM and Bönnemann, CG},
title = {Childhood amyotrophic lateral sclerosis caused by excess sphingolipid synthesis.},
journal = {Nature medicine},
volume = {27},
number = {7},
pages = {1197-1204},
pmid = {34059824},
issn = {1546-170X},
support = {K08 NS10762//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; R01 NS072446/NS/NINDS NIH HHS/United States ; grant #31003A_179371//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; grant # W81XWH-20-1-0219//United States Department of Defense | United States Army | Army Medical Command | Congressionally Directed Medical Research Programs (CDMRP)/ ; R01 NS072446/NS/NINDS NIH HHS/United States ; },
mesh = {Adolescent ; Adult ; Alleles ; Amino Acid Sequence ; Amyotrophic Lateral Sclerosis/enzymology/genetics/*metabolism ; CRISPR-Cas Systems ; Child ; Female ; Genes, Dominant ; HEK293 Cells ; Humans ; Male ; Middle Aged ; Mutation ; Serine C-Palmitoyltransferase/genetics/metabolism ; Sphingolipids/*biosynthesis ; Young Adult ; },
abstract = {Amyotrophic lateral sclerosis (ALS) is a progressive, neurodegenerative disease of the lower and upper motor neurons with sporadic or hereditary occurrence. Age of onset, pattern of motor neuron degeneration and disease progression vary widely among individuals with ALS. Various cellular processes may drive ALS pathomechanisms, but a monogenic direct metabolic disturbance has not been causally linked to ALS. Here we show SPTLC1 variants that result in unrestrained sphingoid base synthesis cause a monogenic form of ALS. We identified four specific, dominantly acting SPTLC1 variants in seven families manifesting as childhood-onset ALS. These variants disrupt the normal homeostatic regulation of serine palmitoyltransferase (SPT) by ORMDL proteins, resulting in unregulated SPT activity and elevated levels of canonical SPT products. Notably, this is in contrast with SPTLC1 variants that shift SPT amino acid usage from serine to alanine, result in elevated levels of deoxysphingolipids and manifest with the alternate phenotype of hereditary sensory and autonomic neuropathy. We custom designed small interfering RNAs that selectively target the SPTLC1 ALS allele for degradation, leave the normal allele intact and normalize sphingolipid levels in vitro. The role of primary metabolic disturbances in ALS has been elusive; this study defines excess sphingolipid biosynthesis as a fundamental metabolic mechanism for motor neuron disease.},
}

Adolescent
Adult
Alleles
Amino Acid Sequence
Amyotrophic Lateral Sclerosis/enzymology/genetics/*metabolism
CRISPR-Cas Systems
Child
Female
Genes, Dominant
HEK293 Cells
Humans
Male
Middle Aged
Mutation
Serine C-Palmitoyltransferase/genetics/metabolism
Sphingolipids/*biosynthesis
Young Adult

@article {pmid34023780,
year = {2021},
author = {Homma, Y and Fukuda, M},
title = {Knockout analysis of Rab6 effector proteins revealed the role of VPS52 in the secretory pathway.},
journal = {Biochemical and biophysical research communications},
volume = {561},
number = {},
pages = {151-157},
doi = {10.1016/j.bbrc.2021.05.009},
pmid = {34023780},
issn = {1090-2104},
mesh = {Animals ; CRISPR-Cas Systems ; Cells, Cultured ; Dogs ; Gene Knockdown Techniques/*methods ; Golgi Apparatus ; Humans ; Intracellular Membranes ; Lysosomes/*metabolism ; Protein Transport ; Secretory Pathway/*physiology ; Vesicular Transport Proteins/antagonists & inhibitors/genetics/*metabolism ; rab GTP-Binding Proteins/*metabolism ; },
abstract = {Rab small GTPases regulate intracellular membrane trafficking by interacting with specific binding proteins called Rab effectors. Although Rab6 is implicated in basement membrane formation and secretory cargo trafficking, its precise regulatory mechanisms have remained largely unknown. In the present study we established five knockout cell lines for candidate Rab6 effectors and discovered that knockout of VPS52, a subunit of the GARP complex, resulted in attenuated secretion and lysosomal accumulation of secretory cargos, the same as Rab6-knockout does. We also evaluated the functional importance of the previously uncharacterized C-terminal region of VPS52 for restoring these phenotypes, as well as for the sorting of lysosomal proteins. Our findings suggest that VPS52 is an effector protein that is responsible for the Rab6-dependent secretory cargo trafficking.},
}

Animals
CRISPR-Cas Systems
Cells, Cultured
Dogs
Gene Knockdown Techniques/*methods
Golgi Apparatus
Humans
Intracellular Membranes
Lysosomes/*metabolism
Protein Transport
Secretory Pathway/*physiology
Vesicular Transport Proteins/antagonists & inhibitors/genetics/*metabolism
rab GTP-Binding Proteins/*metabolism

@article {pmid34023776,
year = {2021},
author = {Kang, JY and Park, JW and Hwang, Y and Hahm, JY and Park, J and Park, KS and Seo, SB},
title = {The H3K4 methyltransferase SETD1A is required for proliferation of non-small cell lung cancer cells by promoting S-phase progression.},
journal = {Biochemical and biophysical research communications},
volume = {561},
number = {},
pages = {120-127},
doi = {10.1016/j.bbrc.2021.05.026},
pmid = {34023776},
issn = {1090-2104},
mesh = {CRISPR-Cas Systems ; Carcinoma, Non-Small-Cell Lung/*enzymology/genetics/metabolism/*pathology ; Cell Cycle Proteins/metabolism ; Cell Line, Tumor ; Cell Proliferation ; Cells, Cultured ; Computational Biology/methods ; DNA Replication ; Databases, Genetic ; Gene Expression Regulation, Neoplastic ; Histone-Lysine N-Methyltransferase/antagonists & inhibitors/genetics/*metabolism ; Humans ; Lung Neoplasms/*enzymology/genetics/metabolism/*pathology ; Protein-Serine-Threonine Kinases/metabolism ; S Phase ; },
abstract = {Epigenetic dysregulation has been strongly implicated in carcinogenesis and is one of the mechanisms that contribute to the development of lung cancer. Using genome-wide CRISPR/Cas9 library screening, we showed SET domain-containing protein 1A (SETD1A) is an essential epigenetic modifier of the proliferation of NSCLC H1299 cells. Depletion of SETD1A strikingly inhibited the proliferation of NSCLC cells. IHC staining and bioinformatics showed that SETD1A is upregulated in lung cancer. Kaplan-Meier survival analysis indicated that high expression of SETD1A is associated with poor prognosis of patients with NSCLC. We revealed that loss of SETD1A inhibits DNA replication and induces replication stress accompanied by impaired fork progression. In addition, transcription of CDC7 and TOP1, which are involved in replication origin activation and fork progression, respectively, was significantly reduced by knockdown of SETD1A. Taken together, these findings demonstrated SETD1A is a critical epigenetic modifier of NSCLC cell proliferation by promoting the transcription of a subset of DNA replication-associated genes.},
}

CRISPR-Cas Systems
Carcinoma, Non-Small-Cell Lung/*enzymology/genetics/metabolism/*pathology
Cell Cycle Proteins/metabolism
Cell Line, Tumor
Cell Proliferation
Cells, Cultured
Computational Biology/methods
DNA Replication
Databases, Genetic
Gene Expression Regulation, Neoplastic
Histone-Lysine N-Methyltransferase/antagonists & inhibitors/genetics/*metabolism
Humans
Lung Neoplasms/*enzymology/genetics/metabolism/*pathology
Protein-Serine-Threonine Kinases/metabolism
S Phase

@article {pmid33716172,
year = {2021},
author = {Fan, HH and Lee, KH and Chen, YT and Lin, LJ and Yang, TL and Lin, SW and Yu, IS},
title = {Wdhd1 is essential for early mouse embryogenesis.},
journal = {Biochimica et biophysica acta. Molecular cell research},
volume = {1868},
number = {6},
pages = {119011},
doi = {10.1016/j.bbamcr.2021.119011},
pmid = {33716172},
issn = {1879-2596},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Culture Techniques ; Cell Line ; Cell Proliferation ; DNA-Binding Proteins/*genetics/*metabolism ; *Embryonic Development ; Fertility ; Gastrulation ; Gene Editing ; Loss of Function Mutation ; Mice ; },
abstract = {WD repeat and HMG-box DNA binding protein 1 (Wdhd1) is the mouse ortholog of budding yeast Chromosome Transmission Fidelity 4 (CTF4), the protein product of which integrates the MCM2-7 helicase and DNA polymerase α/primase complex to initiate DNA replication. Previous work in fruit flies, Xenopus egg extracts, and human cell lines suggest that Wdhd1 is required for efficient DNA synthesis. However, rigorous in vivo functional studies on Wdhd1 in mammals are unavailable. In the present study, we have successfully generated a Wdhd1 null allele in mice through CRISPR/Cas9-mediated genome editing to investigate the role of Wdhd1 in embryogenesis in vivo. We characterized Wdhd1 expression using quantitative reverse-transcription polymerase chain reaction, and assessed embryonic cell proliferation by histology in both pre- and peri-implantation embryos. While Wdhd1 heterozygous mutant mice were grossly normal and fertile, we observed a reduction in cell proliferation by the gastrulation stage in Wdhd1 homozygous null mutant embryos which severely hampered their growth and viability. These results indicate that Wdhd1 plays a major role in cell proliferation during embryogenesis in mice.},
}

Animals
CRISPR-Cas Systems
Cell Culture Techniques
Cell Line
Cell Proliferation
DNA-Binding Proteins/*genetics/*metabolism
*Embryonic Development
Fertility
Gastrulation
Gene Editing
Loss of Function Mutation
Mice

@article {pmid33688075,
year = {2021},
author = {Gandhi, S and Li, Y and Tang, W and Christensen, JB and Urrutia, HA and Vieceli, FM and Piacentino, ML and Bronner, ME},
title = {A single-plasmid approach for genome editing coupled with long-term lineage analysis in chick embryos.},
journal = {Development (Cambridge, England)},
volume = {148},
number = {7},
pages = {},
pmid = {33688075},
issn = {1477-9129},
support = {K99 DE029240/DE/NIDCR NIH HHS/United States ; R01 DE027568/DE/NIDCR NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Chick Embryo ; Gene Editing/*methods ; Gene Expression Regulation, Developmental ; Gene Knockout Techniques/methods ; Neural Crest/metabolism ; PAX6 Transcription Factor/genetics ; PAX7 Transcription Factor ; Plasmids/*genetics ; RNA, Guide/genetics ; SOXE Transcription Factors/genetics ; },
abstract = {An important strategy for establishing mechanisms of gene function during development is through mutation of individual genes and analysis of subsequent effects on cell behavior. Here, we present a single-plasmid approach for genome editing in chick embryos to study experimentally perturbed cells in an otherwise normal embryonic environment. To achieve this, we have engineered a plasmid that encodes Cas9 protein, gene-specific guide RNA (gRNA), and a fluorescent marker within the same construct. Using transfection- and electroporation-based approaches, we show that this construct can be used to perturb gene function in early embryos as well as human cell lines. Importantly, insertion of this cistronic construct into replication-incompetent avian retroviruses allowed us to couple gene knockouts with long-term lineage analysis. We demonstrate the application of our newly engineered constructs and viruses by perturbing β-catenin in vitro and Sox10, Pax6 and Pax7 in the neural crest, retina, and neural tube and segmental plate in vivo, respectively. Together, this approach enables genes of interest to be knocked out in identifiable cells in living embryos and can be broadly applied to numerous genes in different embryonic tissues.},
}

Animals
*CRISPR-Cas Systems
Chick Embryo
Gene Editing/*methods
Gene Expression Regulation, Developmental
Gene Knockout Techniques/methods
Neural Crest/metabolism
PAX6 Transcription Factor/genetics
PAX7 Transcription Factor
Plasmids/*genetics
RNA, Guide/genetics
SOXE Transcription Factors/genetics

@article {pmid33185489,
year = {2020},
author = {Maroufi, F and Maali, A and Abdollahpour-Alitappeh, M and Ahmadi, MH and Azad, M},
title = {CRISPR-mediated modification of DNA methylation pattern in the new era of cancer therapy.},
journal = {Epigenomics},
volume = {12},
number = {20},
pages = {1845-1859},
doi = {10.2217/epi-2020-0110},
pmid = {33185489},
issn = {1750-192X},
mesh = {CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; *DNA Methylation ; Epigenesis, Genetic ; Epigenome ; *Gene Editing ; Humans ; Neoplasms/genetics/*therapy ; },
abstract = {In the last 2 decades, a wide variety of studies have been conducted on epigenetics and its role in various cancers. A major mechanism of epigenetic regulation is DNA methylation, including aberrant DNA methylation variations such as hypermethylation and hypomethylation in the promoters of critical genes, which are commonly detected in tumors and mark the early stages of cancer development. Therefore, epigenetic therapy has been of special importance in the last decade for cancer treatment. In epigenetic therapy, all efforts are made to modulate gene expression to the normal status. Importantly, recent studies have shown that epigenetic therapy is focusing on the new gene editing technology, CRISPR-Cas9. This tool was found to be able to effectively modulate gene expression and alter almost any sequence in the genome of cells, resulting in events such as a change in acetylation, methylation, or histone modifications. Of note, the CRISPR-Cas9 system can be used for the treatment of cancers caused by epigenetic alterations. The CRISPR-Cas9 system has greater advantages than other available methods, including potent activity, easy design and high velocity as well as the ability to target any DNA or RNA site. In this review, we described epigenetic modulators, which can be used in the CRISPR-Cas9 system, as well as their functions in gene expression alterations that lead to cancer initiation and progression. In addition, we surveyed various species of CRISPR-dead Cas9 (dCas9) systems, a mutant version of Cas9 with no endonuclease activity. Such systems are applicable in epigenetic therapy for gene expression modulation through chemical group editing on nucleosomes and chromatin remodeling, which finally return the cell to the normal status and prevent cancer progression.},
}

CRISPR-Associated Protein 9
*CRISPR-Cas Systems
*DNA Methylation
Epigenesis, Genetic
Epigenome
*Gene Editing
Humans
Neoplasms/genetics/*therapy

@article {pmid31035284,
year = {2019},
author = {Turan, S and Boerstler, T and Kavyanifar, A and Loskarn, S and Reis, A and Winner, B and Lie, DC},
title = {A novel human stem cell model for Coffin-Siris syndrome-like syndrome reveals the importance of SOX11 dosage for neuronal differentiation and survival.},
journal = {Human molecular genetics},
volume = {28},
number = {15},
pages = {2589-2599},
doi = {10.1093/hmg/ddz089},
pmid = {31035284},
issn = {1460-2083},
mesh = {Abnormalities, Multiple/genetics/metabolism ; CRISPR-Cas Systems ; Cell Differentiation ; *Cell Line ; Cell Proliferation ; Face/abnormalities ; *Gene Dosage ; Gene Editing ; Gene Expression Regulation ; Hand Deformities, Congenital/genetics/metabolism ; Haploinsufficiency ; Human Embryonic Stem Cells/metabolism/*physiology ; Humans ; Intellectual Disability/genetics/metabolism ; Micrognathism/genetics/metabolism ; *Models, Biological ; Neck/abnormalities ; Neural Stem Cells ; Neurodevelopmental Disorders/genetics/*metabolism ; SOXC Transcription Factors/*genetics ; },
abstract = {The SOXC transcription factors Sox4, Sox11 and Sox12, are critical neurodevelopmental regulators that are thought to function in a highly redundant fashion. Surprisingly, heterozygous missense mutations or deletions of SOX11 were recently detected in patients with Coffin-Siris syndrome-like syndrome (CSSLS), a neurodevelopmental disorder associated with intellectual disability, demonstrating that in humans SOX11 haploinsufficiency cannot be compensated and raising the question of the function of SOX11 in human neurodevelopment. Here, we describe the generation of SOX11+/- heterozygous human embryonic stem cell (hESC) lines by CRISPR/Cas9 genome engineering. SOX11 haploinsufficiency impaired the generation of neurons and resulted in a proliferation/differentiation imbalance of neural precursor cells and enhanced neuronal cell death. Using the SOX11+/- hESC model we provide for the first time experimental evidence that SOX11 haploinsufficiency is sufficient to impair key processes of human neurodevelopment, giving a first insight into the pathophysiology of CSSLS and SOX11 function in human neurodevelopment.},
}

Abnormalities, Multiple/genetics/metabolism
CRISPR-Cas Systems
Cell Differentiation
*Cell Line
Cell Proliferation
Face/abnormalities
*Gene Dosage
Gene Editing
Gene Expression Regulation
Hand Deformities, Congenital/genetics/metabolism
Haploinsufficiency
Human Embryonic Stem Cells/metabolism/*physiology
Humans
Intellectual Disability/genetics/metabolism
Micrognathism/genetics/metabolism
*Models, Biological
Neck/abnormalities
Neural Stem Cells
Neurodevelopmental Disorders/genetics/*metabolism
SOXC Transcription Factors/*genetics

@article {pmid34524446,
year = {2021},
author = {Hacker, L and Dorn, A and Enderle, J and Puchta, H},
title = {The repair of topoisomerase 2 cleavage complexes in Arabidopsis.},
journal = {The Plant cell},
volume = {},
number = {},
pages = {},
doi = {10.1093/plcell/koab228},
pmid = {34524446},
issn = {1532-298X},
abstract = {DNA-protein crosslinks (DPCs) and DNA double-stranded breaks (DSBs), including those produced by stalled topoisomerase 2 cleavage complexes (TOP2ccs), must be repaired to ensure genome stability. The basic mechanisms of TOP2cc repair have been characterized in other eukaryotes, but we lack information for plants. Using CRISPR/Cas-induced mutants, we show that Arabidopsis thaliana has two main TOP2cc repair pathways: one is defined by TYROSYL-DNA-PHOSPHODIESTERASE 2 (TDP2), which hydrolyzes TOP2-DNA linkages, the other by the DNA-dependent protease WSS1A (a homolog of human SPARTAN/yeast weak suppressor of smt3 [Wss1]), which also functions in DPC repair. TDP1 and TDP2 function non-redundantly in TOP1cc repair, indicating that they act specifically on their respective stalled cleavage complexes. The nuclease METHYL METHANESULFONATE AND UV SENSITIVE PROTEIN 81 (MUS81) plays a major role in global DPC repair and a minor role in TOP2cc repair. DSBs arise as intermediates of TOP2cc repair and are repaired by classical and alternative non-homologous end joining (NHEJ) pathways. Double mutant analysis indicates that "clean" DNA ends caused by TDP2 hydrolysis are mainly religated by classical NHEJ, which helps avoid mutation. In contrast, the mutagenic alternative NHEJ pathway mainly processes non-ligateable DNA ends. Thus, TDP2 promotes maintenance of plant genome integrity by error-free repair of TOP2cc.},
}

@article {pmid34524200,
year = {2021},
author = {León, Y and Faherty, CS},
title = {Bacteriophages against enteropathogens: rediscovery and refinement of novel antimicrobial therapeutics.},
journal = {Current opinion in infectious diseases},
volume = {34},
number = {5},
pages = {491-499},
pmid = {34524200},
issn = {1473-6527},
abstract = {PURPOSE OF REVIEW: Alarming rates of antibiotic resistance in bacteria and gastrointestinal dysbiosis associated with traditional antimicrobial therapy have led to renewed interests in developing bacteriophages as novel therapeutics. In this review, we highlight some of the recent advances in bacteriophage therapeutic development targeting important enteropathogens of the gastrointestinal tract.

RECENT FINDINGS: Bacteriophages are viruses that infect bacteria, either to utilize the bacterial machinery to produce new progeny or stably integrate into the bacterial chromosome to ensure maintenance of the viral genome. With recent advances in synthetic biology and the discovery of CRISPR-Cas systems used by bacteria to protect against bacteriophages, novel molecular applications are taking us beyond the discovery of bacteriophages and toward innovative applications, including the targeting of bacterial virulence factors, the use of temperate bacteriophages, and the production of bacteriophage proteins as antimicrobial agents. These technologies offer promise to target enteropathogens without disrupting the healthy microbiota of the gastrointestinal tract. Moreover, the use of nanoparticle technology and other modifications are helping researchers circumvent the harsh gastrointestinal conditions that could limit the efficacy of bacteriophages against enteric pathogens.

SUMMARY: This era of discovery and development offers significant potential to modify bacteriophages and overcome the global impact of enteropathogens.},
}

@article {pmid34522785,
year = {2021},
author = {Rao, GS and Jiang, W and Mahfouz, M},
title = {Synthetic directed evolution in plants: unlocking trait engineering and improvement.},
journal = {Synthetic biology (Oxford, England)},
volume = {6},
number = {1},
pages = {ysab025},
doi = {10.1093/synbio/ysab025},
pmid = {34522785},
issn = {2397-7000},
abstract = {Genetic variation accelerates adaptation and resilience and enables the survival of species in their changing environment. Increasing the genetic diversity of crop species is essential to improve their yield and enhance food security. Synthetic directed evolution (SDE) employs localized sequence diversification (LSD) of gene sequence and selection pressure to evolve gene variants with better fitness, improved properties and desired phenotypes. Recently, CRISPR-Cas-dependent and -independent technologies have been applied for LSD to mediate synthetic evolution in diverse species, including plants. SDE holds excellent promise to discover, accelerate and expand the range of traits of the value in crop species. Here, we highlight the efficient SDE approaches for the LSD of plant genes, selection strategies and critical traits for targeted improvement. We discuss the potential of emerging technologies, including CRISPR-Cas base editing, retron editing, EvolvR and prime editing, to establish efficient SDE in plants. Moreover, we cover CRISPR-Cas-independent technologies, including T7 polymerase editor for continuous evolution. We highlight the key challenges and potential solutions of applying SDE technologies to improve the plant traits of the value.},
}

@article {pmid34521192,
year = {2021},
author = {Wang, C and Han, C and Du, X and Guo, W},
title = {Versatile CRISPR-Cas12a-Based Biosensing Platform Modulated with Programmable Entropy-Driven Dynamic DNA Networks.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.1c01597},
pmid = {34521192},
issn = {1520-6882},
abstract = {In addition to their roles as revolutionary genome engineering tools, CRISPR-Cas systems are also highly promising candidates in the construction of biosensing systems and diagnostic devices, which have attracted significant attention recently. However, the CRISPR-Cas system cannot be directly applied in the sensing of non-nucleic acid targets, and the needs of synthesizing and storing different vulnerable guide RNA for different targets also increase the application and storage costs of relevant biosensing systems, and therefore restrict their widespread applications. To tackle these barriers, in this work, a versatile CRISPR-Cas12a-based biosensing platform was developed through the introduction of an enzyme-free and robust DNA reaction network, the entropy-driven dynamic DNA network. By programming the sequences of the system, the entropy-driven catalysis-based dynamic DNA network can respond to different types of targets, such as nucleic acids or proteins, and then activate the CRISPR-Cas12a to generate amplified signals. As a proof of concept, both nucleic acid targets (a DNA target with random sequence, T, and an RNA target, microRNA-21 (miR-21)) and a non-nucleic acid target (a protein target, thrombin) were chosen as model analytes to address the feasibility of the designed sensing platform, with detection limits at the pM level for the nucleic acid analytes (7.4 pM for the DNA target T and 25.5 pM for miR-21) and 0.4 nM for thrombin. In addition, the detection of miR-21 or thrombin in human serum samples further demonstrated the applicability of the proposed biosensing platform in real sample analysis.},
}

@article {pmid34517842,
year = {2021},
author = {Luo, Y and Na, R and Nowak, JS and Qiu, Y and Lu, QS and Yang, C and Marsolais, F and Tian, L},
title = {Development of a Csy4-processed guide RNA delivery system with soybean-infecting virus ALSV for genome editing.},
journal = {BMC plant biology},
volume = {21},
number = {1},
pages = {419},
pmid = {34517842},
issn = {1471-2229},
mesh = {CRISPR-Cas Systems/*genetics ; Crops, Agricultural/genetics/virology ; Gene Editing/*methods ; Gene Expression Regulation, Plant ; Gene Expression Regulation, Viral ; Genome, Plant ; Host-Pathogen Interactions/*genetics ; Mutagenesis ; Plant Viruses/*genetics ; RNA, Guide ; RNA, Plant ; RNA, Viral ; Soybeans/*genetics/*virology ; Virus Diseases/*genetics ; },
abstract = {BACKGROUND: A key issue for implementation of CRISPR-Cas9 genome editing for plant trait improvement and gene function analysis is to efficiently deliver the components, including guide RNAs (gRNAs) and Cas9, into plants. Plant virus-based gRNA delivery strategy has proven to be an important tool for genome editing. However, its application in soybean which is an important crop has not been reported yet. ALSV (apple latent spherical virus) is highly infectious virus and could be explored for delivering elements for genome editing.

RESULTS: To develop a ALSV-based gRNA delivery system, the Cas9-based Csy4-processed ALSV Carry (CCAC) system was developed. In this system, we engineered the soybean-infecting ALSV to carry and deliver gRNA(s). The endoribonuclease Csy4 effectively releases gRNAs that function efficiently in Cas9-mediated genome editing. Genome editing of endogenous phytoene desaturase (PDS) loci and exogenous 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) sequence in Nicotiana. benthamiana (N. benthamiana) through CCAC was confirmed using Sanger sequencing. Furthermore, CCAC-induced mutagenesis in two soybean endogenous GW2 paralogs was detected.

CONCLUSIONS: With the aid of the CCAC system, the target-specific gRNA(s) can be easily manipulated and efficiently delivered into soybean plant cells by viral infection. This is the first virus-based gRNA delivery system for soybean for genome editing and can be used for gene function study and trait improvement.},
}

CRISPR-Cas Systems/*genetics
Crops, Agricultural/genetics/virology
Gene Editing/*methods
Gene Expression Regulation, Plant
Gene Expression Regulation, Viral
Genome, Plant
Host-Pathogen Interactions/*genetics
Mutagenesis
Plant Viruses/*genetics
RNA, Guide
RNA, Plant
RNA, Viral
Soybeans/*genetics/*virology
Virus Diseases/*genetics

@article {pmid34489579,
year = {2021},
author = {van der Oost, J and Fresco, LO},
title = {Waive CRISPR patents to meet food needs in low-income countries.},
journal = {Nature},
volume = {597},
number = {7875},
pages = {178},
doi = {10.1038/d41586-021-02397-7},
pmid = {34489579},
issn = {1476-4687},
mesh = {*CRISPR-Cas Systems ; Developing Countries/*economics ; *Food Supply/methods/standards ; Gene Editing/economics/*legislation & jurisprudence ; Humans ; Licensure/economics/*legislation & jurisprudence ; Patents as Topic/*legislation & jurisprudence ; Plant Breeding/economics/legislation & jurisprudence/methods ; Poverty ; },
}

*CRISPR-Cas Systems
Developing Countries/*economics
*Food Supply/methods/standards
Gene Editing/economics/*legislation & jurisprudence
Humans
Licensure/economics/*legislation & jurisprudence
Patents as Topic/*legislation & jurisprudence
Plant Breeding/economics/legislation & jurisprudence/methods
Poverty

@article {pmid34433009,
year = {2021},
author = {Socha, M and Sowińska-Seidler, A and Melo, US and Kragesteen, BK and Franke, M and Heinrich, V and Schöpflin, R and Nagel, I and Gruchy, N and Mundlos, S and Sreenivasan, VKA and López, C and Vingron, M and Bukowska-Olech, E and Spielmann, M and Jamsheer, A},
title = {Position effects at the FGF8 locus are associated with femoral hypoplasia.},
journal = {American journal of human genetics},
volume = {108},
number = {9},
pages = {1725-1734},
doi = {10.1016/j.ajhg.2021.08.001},
pmid = {34433009},
issn = {1537-6605},
mesh = {Adolescent ; Alleles ; Animals ; CRISPR-Cas Systems ; Child, Preschool ; Chromatin/chemistry/metabolism ; *Chromosome Duplication ; Chromosomes, Human, Pair 10/*chemistry/metabolism ; *DNA Copy Number Variations ; Enhancer Elements, Genetic ; Family ; Female ; Femur/abnormalities/diagnostic imaging/metabolism ; Fibroblast Growth Factor 8/*genetics/metabolism ; Gene Editing ; Heterozygote ; Humans ; Infant ; Lower Extremity Deformities, Congenital/diagnostic imaging/*genetics/metabolism/pathology ; Male ; Mice ; Mice, Transgenic ; Pedigree ; Phenotype ; },
abstract = {Copy-number variations (CNVs) are a common cause of congenital limb malformations and are interpreted primarily on the basis of their effect on gene dosage. However, recent studies show that CNVs also influence the 3D genome chromatin organization. The functional interpretation of whether a phenotype is the result of gene dosage or a regulatory position effect remains challenging. Here, we report on two unrelated families with individuals affected by bilateral hypoplasia of the femoral bones, both harboring de novo duplications on chromosome 10q24.32. The ∼0.5 Mb duplications include FGF8, a key regulator of limb development and several limb enhancer elements. To functionally characterize these variants, we analyzed the local chromatin architecture in the affected individuals' cells and re-engineered the duplications in mice by using CRISPR-Cas9 genome editing. We found that the duplications were associated with ectopic chromatin contacts and increased FGF8 expression. Transgenic mice carrying the heterozygous tandem duplication including Fgf8 exhibited proximal shortening of the limbs, resembling the human phenotype. To evaluate whether the phenotype was a result of gene dosage, we generated another transgenic mice line, carrying the duplication on one allele and a concurrent Fgf8 deletion on the other allele, as a control. Surprisingly, the same malformations were observed. Capture Hi-C experiments revealed ectopic interaction with the duplicated region and Fgf8, indicating a position effect. In summary, we show that duplications at the FGF8 locus are associated with femoral hypoplasia and that the phenotype is most likely the result of position effects altering FGF8 expression rather than gene dosage effects.},
}

Adolescent
Alleles
Animals
CRISPR-Cas Systems
Child, Preschool
Chromatin/chemistry/metabolism
*Chromosome Duplication
Chromosomes, Human, Pair 10/*chemistry/metabolism
*DNA Copy Number Variations
Enhancer Elements, Genetic
Family
Female
Femur/abnormalities/diagnostic imaging/metabolism
Fibroblast Growth Factor 8/*genetics/metabolism
Gene Editing
Heterozygote
Humans
Infant
Lower Extremity Deformities, Congenital/diagnostic imaging/*genetics/metabolism/pathology
Male
Mice
Mice, Transgenic
Pedigree
Phenotype

@article {pmid34411650,
year = {2021},
author = {Malik, D and Mahendiratta, S and Kaur, H and Medhi, B},
title = {Futuristic approach to cancer treatment.},
journal = {Gene},
volume = {805},
number = {},
pages = {145906},
doi = {10.1016/j.gene.2021.145906},
pmid = {34411650},
issn = {1879-0038},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/methods/trends ; Genetic Therapy/methods ; Humans ; Neoplasms/*genetics/*therapy ; Precision Medicine/methods/*trends ; },
abstract = {Cancer is becoming one of the deadliest disease in both developed as well as developing countries and continuous effort is being made to find innovative therapies for myriad types of cancers that afflict the human body. Therapeutic options for cancer have grown exponentially over the time but we are quite a way off from finding a magic bullet that can help cure cancer and based on the current evidence we may never find a catch all cure ever and it becomes crucial that we keep on innovating and find multiple ways to attack the menace of this dreaded disease. Many patients suffer recurrence of disease and require second-line or in some cases more than two lines of treatment. In this review article we have discussed the available therapies along with the newer advancements that have been made in cancer therapy. Latest developments in treatment of various cancers that have been discussed include gene editing using CRISPR/Cas9, theranostics, viral mediated therapy, artificial intelligence, tumor infiltrating lymphocyte therapy, etc.},
}

CRISPR-Cas Systems/genetics
Gene Editing/methods/trends
Genetic Therapy/methods
Humans
Neoplasms/*genetics/*therapy
Precision Medicine/methods/*trends

@article {pmid33763075,
year = {2021},
author = {Luff, DH and Wojdyla, K and Oxley, D and Chessa, T and Hudson, K and Hawkins, PT and Stephens, LR and Barry, ST and Okkenhaug, K},
title = {PI3Kδ Forms Distinct Multiprotein Complexes at the TCR Signalosome in Naïve and Differentiated CD4+ T Cells.},
journal = {Frontiers in immunology},
volume = {12},
number = {},
pages = {631271},
pmid = {33763075},
issn = {1664-3224},
support = {/WT_/Wellcome Trust/United Kingdom ; BBS/E/B/000L0799/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; MR/R000409/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; CD4-Positive T-Lymphocytes/classification/drug effects/*immunology ; CRISPR-Cas Systems ; Cell Differentiation/*immunology ; Class I Phosphatidylinositol 3-Kinases/*genetics/*immunology ; Gene Editing ; Gene Knockout Techniques ; Interleukin-2/pharmacology ; Lymphocyte Activation/immunology ; Mice ; Mice, Inbred C57BL ; Multiprotein Complexes/*biosynthesis/*immunology ; Receptors, Antigen, T-Cell/genetics/*immunology ; Signal Transduction ; Specific Pathogen-Free Organisms ; },
abstract = {Phosphoinositide 3-kinases (PI3Ks) play a central role in adaptive immunity by transducing signals from the T cell antigen receptor (TCR) via production of PIP3. PI3Kδ is a heterodimer composed of a p110δ catalytic subunit associated with a p85α or p85β regulatory subunit and is preferentially engaged by the TCR upon T cell activation. The molecular mechanisms leading to PI3Kδ recruitment and activation at the TCR signalosome remain unclear. In this study, we have used quantitative mass spectrometry, biochemical approaches and CRISPR-Cas9 gene editing to uncover the p110δ interactome in primary CD4+ T cells. Moreover, we have determined how the PI3Kδ interactome changes upon the differentiation of small naïve T cells into T cell blasts expanded in the presence of IL-2. Our interactomic analyses identified multiple constitutive and inducible PI3Kδ-interacting proteins, some of which were common to naïve and previously-activated T cells. Our data reveals that PI3Kδ rapidly interacts with as many as seven adaptor proteins upon TCR engagement, including the Gab-family proteins, GAB2 and GAB3, a CD5-CBL signalosome and the transmembrane proteins ICOS and TRIM. Our results also suggest that PI3Kδ pre-forms complexes with the adaptors SH3KBP1 and CRKL in resting cells that could facilitate the localization and activation of p110δ at the plasma membrane by forming ternary complexes during early TCR signalling. Furthermore, we identify interactions that were not previously known to occur in CD4+ T cells, involving BCAP, GAB3, IQGAP3 and JAML. We used CRISPR-Cas9-mediated gene knockout in primary T cells to confirm that BCAP is a positive regulator of PI3K-AKT signalling in CD4+ T cell blasts. Overall, our results provide evidence for a large protein network that regulates the recruitment and activation of PI3Kδ in T cells. Finally, this work shows how the PI3Kδ interactome is remodeled as CD4+ T cells differentiate from naïve T cells to activated T cell blasts. These activated T cells upregulate additional PI3Kδ adaptor proteins, including BCAP, GAB2, IQGAP3 and ICOS. This rewiring of TCR-PI3K signalling that occurs upon T cell differentiation may serve to reduce the threshold of activation and diversify the inputs for the PI3K pathway in effector T cells.},
}

Animals
CD4-Positive T-Lymphocytes/classification/drug effects/*immunology
CRISPR-Cas Systems
Cell Differentiation/*immunology
Class I Phosphatidylinositol 3-Kinases/*genetics/*immunology
Gene Editing
Gene Knockout Techniques
Interleukin-2/pharmacology
Lymphocyte Activation/immunology
Mice
Mice, Inbred C57BL
Multiprotein Complexes/*biosynthesis/*immunology
Receptors, Antigen, T-Cell/genetics/*immunology
Signal Transduction
Specific Pathogen-Free Organisms

@article {pmid33740450,
year = {2021},
author = {Zoloth, L},
title = {The ethical scientist in a time of uncertainty.},
journal = {Cell},
volume = {184},
number = {6},
pages = {1430-1439},
doi = {10.1016/j.cell.2021.02.051},
pmid = {33740450},
issn = {1097-4172},
mesh = {CRISPR-Cas Systems/genetics ; Epigenesis, Genetic ; *Ethics, Research ; Genetics ; Humans ; Malaria/genetics/prevention & control ; *Research Personnel ; Risk ; *Uncertainty ; },
abstract = {All of science takes place amidst a world shaken by uncertainty, social and political upheaval, and challenges to truthful testimony. Just at the moment in which increasing control over biology has been theorized, our social world has become increasingly contentious and its values more divisive. Using the example of gene drives for malaria control to explore the problem of deep uncertainty in biomedical research, I argue that profound uncertainty is an essential feature. Applying the language and presumptions of the discipline of philosophical ethics, I describe three types of uncertainty that raise ethical challenges in scientific research. Rather than mitigate these challenges with excessive precautions and limits on progress, I suggest that researchers can cultivate classic values of veracity, courage, humility, and fidelity in their research allowing science to proceed ethically under conditions of deep uncertainty.},
}

CRISPR-Cas Systems/genetics
Epigenesis, Genetic
*Ethics, Research
Genetics
Humans
Malaria/genetics/prevention & control
*Research Personnel
Risk
*Uncertainty

@article {pmid33452424,
year = {2021},
author = {Aprilyanto, V and Aditama, R and Tanjung, ZA and Utomo, C and Liwang, T},
title = {CROP: a CRISPR/Cas9 guide selection program based on mapping guide variants.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {1504},
pmid = {33452424},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems/genetics ; Computational Biology/*methods ; Gene Editing/*methods ; Genetic Engineering/*methods ; Genetic Variation/genetics ; Genome/genetics ; Models, Theoretical ; RNA, Guide/genetics ; Software ; },
abstract = {The off-target effect, in which DNA cleavage was conducted outside the targeted region, is a major problem which limits the applications of CRISPR/Cas9 genome editing system. CRISPR Off-target Predictor (CROP) is standalone program developed to address this problem by predicting off-target propensity of guide RNAs and thereby allowing the user to select the optimum guides. The approach used by CROP involves generating substitution, deletion and insertion combinations which are then mapped into the reference genome. Based on these mapped variants, scoring and alignment are conducted and then reported as a table comprising the off-target propensity of all guide RNAs from a given gene sequence. The Python script for this program is freely available from: https://github.com/vaprilyanto/crop .},
}

CRISPR-Cas Systems/genetics
Computational Biology/*methods
Gene Editing/*methods
Genetic Engineering/*methods
Genetic Variation/genetics
Genome/genetics
Models, Theoretical
RNA, Guide/genetics
Software

@article {pmid33336604,
year = {2021},
author = {Luo, N and Li, J and Chen, Y and Xu, Y and Wei, Y and Lu, J and Dong, R},
title = {Hepatic stellate cell reprogramming via exosome-mediated CRISPR/dCas9-VP64 delivery.},
journal = {Drug delivery},
volume = {28},
number = {1},
pages = {10-18},
pmid = {33336604},
issn = {1521-0464},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Line ; Drug Carriers ; Exosomes/*physiology ; Gene Editing/*methods ; Hepatic Stellate Cells/*physiology ; Humans ; Mice ; Mice, Inbred C57BL ; },
abstract = {Hepatic stellate cells (HSCs) play a crucial role in the progression of liver fibrosis, which can be considered as the specific therapeutic target of anti-fibrotic treatment. Targeted induction of HSCs to hepatocytes via delivery of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (dCas9) system holds promise for hepatic fibrosis treatment. Our study here revealed that CRISPR/dCas9-VP64 system encapsulated in AML12 cell-derived exosomes could efficiently and successfully be delivered into the HSCs. In turn, the CRISPR/dCas9-VP64 system loaded in the exosomes can be efficiently released into the HSCs. As a proof-of-concept study, gRNA against hepatocyte nuclear factor 4α (HNF4α) together with the delivery of CRISPR/dCas9-VP64 system induced the HSCs to hepatocyte-like phenotype. In conclusion, our study here revealed that CRISPR/dCas9-VP64 system encapsulated in AML12 cell-derived exosomes could be functional in HSCs, emerging as a gene therapy strategy for hepatic fibrosis.},
}

Animals
CRISPR-Cas Systems/*genetics
Cell Line
Drug Carriers
Exosomes/*physiology
Gene Editing/*methods
Hepatic Stellate Cells/*physiology
Humans
Mice
Mice, Inbred C57BL

@article {pmid34520771,
year = {2021},
author = {Mirzaei, S and Paskeh, MDA and Hashemi, F and Zabolian, A and Hashemi, M and Entezari, M and Tabari, T and Ashrafizadeh, M and Raee, P and Aghamiri, S and Aref, AR and Leong, HC and Kumar, AP and Samarghandian, S and Zarrabi, A and Hushmandi, K},
title = {Long non-coding RNAs as new players in bladder cancer: Lessons from pre-clinical and clinical studies.},
journal = {Life sciences},
volume = {},
number = {},
pages = {119948},
doi = {10.1016/j.lfs.2021.119948},
pmid = {34520771},
issn = {1879-0631},
abstract = {The clinical management of bladder cancer (BC) has become an increasing challenge due to high incidence rate of BC, malignant behavior of cancer cells and drug resistance. The non-coding RNAs are considered as key factors involved in BC progression. The long non-coding RNAs (lncRNAs) are RNA molecules and do not encode proteins. They have more than 200 nucleotides in length and affect gene expression at epigenetic, transcriptional and post-transcriptional phases. The lncRNAs demonstrate abnormal expression in BC cells and tissues. The present aims to identifying lncRNAs with tumor-suppressor and tumor-promoting roles, and evaluating their roles as regulatory of growth and migration. Apoptosis, glycolysis and EMT are tightly regulated by lncRNAs in BC. Response of BC cells to cisplatin, doxorubicin and gemcitabine chemotherapy is modulated by lncRNAs. LncRNAs regulate immune cell infiltration in tumor microenvironment and affect response of BC cells to immunotherapy. Besides, lncRNAs are able to regulate microRNAs, STAT3, Wnt, PTEN and PI3K/Akt pathways in affecting both proliferation and migration of BC cells. Noteworthy, anti-tumor compounds and genetic tools such as siRNA, shRNA and CRISPR/Cas systems can regulate lncRNA expression in BC. Finally, lncRNAs and exosomal lncRNAs can be considered as potential diagnostic and prognostic tools in BC.},
}

@article {pmid34519839,
year = {2021},
author = {Kruglov, O and Johnson, LDS and Minic, A and Jordan, K and Uger, RA and Wong, M and Sievers, EL and Shou, Y and Akilov, OE},
title = {The pivotal role of cytotoxic NK cells in mediating the therapeutic effect of anti-CD47 therapy in mycosis fungoides.},
journal = {Cancer immunology, immunotherapy : CII},
volume = {},
number = {},
pages = {},
pmid = {34519839},
issn = {1432-0851},
abstract = {CD47 is frequently overexpressed on tumor cells and is an attractive therapeutic target. The mechanism by which anti-CD47 immunotherapy eliminates cutaneous lymphoma has not been explored. We utilized CRISPR/Cas-9 CD47 knock-out, depletion of NK cells, and mice genetically deficient in IFN-γ to elucidate the mechanism of anti-CD47 therapy in a murine model of cutaneous T cell lymphoma (CTCL). CD47 was found to be a crucial factor for tumor progression since CD47 KO CTCL exhibited a delay in tumor growth. The treatment of CD47 WT murine CTCL with anti-CD47 antibodies led to a significant reduction in tumor burden as early as four days after the first treatment and accompanied by an increased percentage of cytotoxic NK cells at the tumor site. The depletion of NK cells resulted in marked attenuation of the anti-tumor effect of anti-CD47. Notably, the treatment of CD47 WT tumors in IFN-γ KO mice with anti-CD47 antibodies was efficient, demonstrating that IFN-γ was not required to mediate anti-CD47 therapy. We were able to potentiate the therapeutic effect of anti-CD47 therapy by IFN-α. That combination resulted in an increased number of cytotoxic CD107a + IFN-γ-NK1.1 cells and intermediate CD62L + NKG2a-NK1.1. Correlative data from a clinical trial (clinicaltrials.gov, NCT02890368) in patients with CTCL utilizing SIRPαFc to block CD47 confirmed our in vivo observations.},
}

@article {pmid34518669,
year = {2021},
author = {Molla, KA and Sretenovic, S and Bansal, KC and Qi, Y},
title = {Precise plant genome editing using base editors and prime editors.},
journal = {Nature plants},
volume = {},
number = {},
pages = {},
pmid = {34518669},
issn = {2055-0278},
support = {IOS-1758745//NSF | BIO | Division of Integrative Organismal Systems (IOS)/ ; IOS-1758745//NSF | BIO | Division of Integrative Organismal Systems (IOS)/ ; },
abstract = {The development of CRISPR-Cas systems has sparked a genome editing revolution in plant genetics and breeding. These sequence-specific RNA-guided nucleases can induce DNA double-stranded breaks, resulting in mutations by imprecise non-homologous end joining (NHEJ) repair or precise DNA sequence replacement by homology-directed repair (HDR). However, HDR is highly inefficient in many plant species, which has greatly limited precise genome editing in plants. To fill the vital gap in precision editing, base editing and prime editing technologies have recently been developed and demonstrated in numerous plant species. These technologies, which are mainly based on Cas9 nickases, can introduce precise changes into the target genome at a single-base resolution. This Review provides a timely overview of the current status of base editors and prime editors in plants, covering both technological developments and biological applications.},
}

@article {pmid34517762,
year = {2021},
author = {Wang, L and Sola, I and Enjuanes, L and Zuñiga, S},
title = {MOV10 Helicase Interacts with Coronavirus Nucleocapsid Protein and Has Antiviral Activity.},
journal = {mBio},
volume = {},
number = {},
pages = {e0131621},
doi = {10.1128/mBio.01316-21},
pmid = {34517762},
issn = {2150-7511},
abstract = {Coronaviruses (CoVs) are emergent pathogens that may cause life-threatening respiratory diseases in humans. Understanding of CoV-host interactions may help to identify novel therapeutic targets. MOV10 is an RNA helicase involved in different steps of cellular RNA metabolism. Both MOV10 antiviral and proviral activities have been described in a limited number of viruses, but this protein has not been previously associated with CoVs. We found that during Middle East respiratory syndrome coronavirus (MERS-CoV) infection, MOV10 aggregated in cytoplasmic structures colocalizing with viral nucleocapsid (N) protein. MOV10-N interaction was confirmed by endogenous MOV10 coimmunoprecipitation, and the presence of other cellular proteins was also detected in MOV10 complexes. MOV10 silencing significantly increased both N protein accumulation and virus titer, with no changes in the accumulation of viral RNAs. Moreover, MOV10 overexpression caused a 10-fold decrease in viral titers. These data indicated that MOV10 has antiviral activity during MERS-CoV infection. We postulated that this activity could be mediated by viral RNA sequestration, and in fact, RNA immunoprecipitation data showed the presence of viral RNAs in the MOV10 cytoplasmic complexes. Expression of wild-type MOV10 or of a MOV10 mutant without helicase activity in MOV10 knockout cell lines, developed by CRISPR-Cas technology, indicated that the helicase activity of MOV10 was required for its antiviral effect. Interestingly MOV10-N interaction was conserved in other mildly or highly pathogenic human CoVs, including the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), although MOV10 antiviral activity was found only in highly pathogenic CoVs, suggesting a potential role of MOV10 in the modulation of human CoVs pathogenesis. IMPORTANCE Coronaviruses (CoVs) are emerging pathogens causing life-threatening diseases in humans. Knowledge of virus-host interactions and viral subversion mechanisms of host pathways is required for the development of effective countermeasures against CoVs. The interaction between cellular RNA helicase MOV10 and nucleocapsid (N) protein from several human CoVs is shown. Using MERS-CoV as a model, we demonstrate that MOV10 has antiviral function, requiring its helicase activity, most likely mediated by viral RNA sequestration in cytoplasmic ribonucleoprotein structures. Furthermore, we found that MOV10 antiviral activity may act only in highly pathogenic human CoVs, suggesting a role for MOV10 in modulating CoVs pathogenesis. The present study uncovers a complex network of viral and cellular RNAs and proteins interaction modulating the antiviral response against CoVs.},
}

@article {pmid34496881,
year = {2021},
author = {Liang, J and Teng, P and Xiao, W and He, G and Song, Q and Zhang, Y and Peng, B and Li, G and Hu, L and Cao, D and Tang, Y},
title = {Application of the amplification-free SERS-based CRISPR/Cas12a platform in the identification of SARS-CoV-2 from clinical samples.},
journal = {Journal of nanobiotechnology},
volume = {19},
number = {1},
pages = {273},
pmid = {34496881},
issn = {1477-3155},
mesh = {COVID-19/diagnosis/virology ; CRISPR-Cas Systems/*genetics ; Gene Expression Regulation, Fungal ; Genes, Viral ; Humans ; *Nucleic Acid Amplification Techniques ; RNA, Viral/analysis ; SARS-CoV-2/*genetics/*isolation & purification ; Sensitivity and Specificity ; *Spectrum Analysis, Raman ; },
abstract = {The control of contagious or refractory diseases requires early, rapid diagnostic assays that are simple, fast, and easy-to-use. Here, easy-to-implement CRISPR/Cas12a-based diagnostic platform through Raman transducer generated by Raman enhancement effect, term as SERS-CRISPR (S-CRISPR), are described. The S-CRISPR uses high-activity noble metallic nanoscopic materials to increase the sensitivity in the detection of nucleic acids, without amplification. This amplification-free platform, which can be performed within 30-40 min of incubation time, is then used for detection of SARS-CoV-2 derived nucleic acids in RNA extracts obtained from nasopharyngeal swab specimens (n = 112). Compared with the quantitative reverse transcription polymerase chain reaction (RT-qPCR), the sensitivity and specificity of S-CRISPR reaches 87.50% and 100%, respectively. In general, the S-CRISPR can rapidly identify the RNA of SARS-CoV-2 RNA without amplification and is a potential strategy for nucleic acid point of care test (POCT).},
}

COVID-19/diagnosis/virology
CRISPR-Cas Systems/*genetics
Gene Expression Regulation, Fungal
Genes, Viral
Humans
*Nucleic Acid Amplification Techniques
RNA, Viral/analysis
SARS-CoV-2/*genetics/*isolation & purification
Sensitivity and Specificity
*Spectrum Analysis, Raman

@article {pmid34475598,
year = {2021},
author = {},
title = {Kazuto Kato: the ethics of editing humanity.},
journal = {Bulletin of the World Health Organization},
volume = {99},
number = {9},
pages = {616-617},
doi = {10.2471/BLT.21.030921},
pmid = {34475598},
issn = {1564-0604},
mesh = {*Bioethical Issues/history ; Biotechnology/*ethics ; CRISPR-Cas Systems ; Ethical Theory ; *Ethics, Medical/history ; Gene Editing/*ethics/history ; History, 21st Century ; Human Characteristics ; Humans ; },
abstract = {Gary Humphreys talks to Kazuto Kato about the ethical and societal challenges posed by biotechnologies that allow for the editing of the human genome.},
}

*Bioethical Issues/history
Biotechnology/*ethics
CRISPR-Cas Systems
Ethical Theory
*Ethics, Medical/history
Gene Editing/*ethics/history
History, 21st Century
Human Characteristics
Humans

@article {pmid34445407,
year = {2021},
author = {Prieto-Bermejo, R and Romo-González, M and Pérez-Fernández, A and García-Macías, MC and Sánchez-Bernal, C and García-Tuñón, I and Sánchez-Yagüe, J and Sánchez-Martín, M and Hernández-Hernández, Á},
title = {Granuloma Formation in a Cyba-Deficient Model of Chronic Granulomatous Disease Is Associated with Myeloid Hyperplasia and the Exhaustion of B-Cell Lineage.},
journal = {International journal of molecular sciences},
volume = {22},
number = {16},
pages = {},
pmid = {34445407},
issn = {1422-0067},
support = {SA077P20//Consejería de Educación, Junta de Castilla y León/ ; },
mesh = {Animals ; B-Lymphocytes/*metabolism ; CRISPR-Cas Systems ; Cell Lineage ; Cytochrome b Group/*genetics ; Disease Models, Animal ; Female ; Gene Knockdown Techniques ; Granulomatous Disease, Chronic/genetics/immunology/*pathology ; Humans ; Hyperplasia ; Male ; Mice ; Myeloid Cells/immunology/*pathology ; NADPH Oxidases/*genetics ; },
abstract = {Haematopoiesis is a paradigm of cell differentiation because of the wide variety and overwhelming number of mature blood cells produced daily. Under stress conditions, the organism must adapt to a boosted demand for blood cells. Chronic granulomatous disease (CGD) is a genetic disease caused by inactivating mutations that affect the phagocyte oxidase. Besides a defective innate immune system, CGD patients suffer from recurrent hyper-inflammation episodes, circumstances upon which they must face emergency haematopoiesis. The targeting of Cybb and Ncf1 genes have produced CGD animal models that are a useful surrogate when studying the pathophysiology and treatment of this disease. Here, we show that Cyba-/- mice spontaneously develop granuloma and, therefore, constitute a CGD animal model to complement the existing Cybb-/- and Ncf1-/- models. More importantly, we have analysed haematopoiesis in granuloma-bearing Cyba-/- mice. These animals showed a significant loss of weight, developed remarkable splenomegaly, bone marrow myeloid hyperplasia, and signs of anaemia. Haematological analyses showed a sharped decrease of B-cells and a striking development of myeloid cells in all compartments. Collectively, our results show that granuloma inflammatory lesions dramatically change haematopoiesis homeostasis. Consequently, we suggest that besides their defective innate immunity, the alteration of haematopoiesis homeostasis upon granuloma may contribute to the dismal outcome of CGD.},
}

Animals
B-Lymphocytes/*metabolism
CRISPR-Cas Systems
Cell Lineage
Cytochrome b Group/*genetics
Disease Models, Animal
Female
Gene Knockdown Techniques
Granulomatous Disease, Chronic/genetics/immunology/*pathology
Humans
Hyperplasia
Male
Mice
Myeloid Cells/immunology/*pathology
NADPH Oxidases/*genetics

@article {pmid34445221,
year = {2021},
author = {Abashkin, DA and Kurishev, AO and Karpov, DS and Golimbet, VE},
title = {Cellular Models in Schizophrenia Research.},
journal = {International journal of molecular sciences},
volume = {22},
number = {16},
pages = {},
pmid = {34445221},
issn = {1422-0067},
support = {21-15-00124//Russian Science Foundation/ ; },
mesh = {Biomedical Research ; *CRISPR-Cas Systems ; *Gene Editing ; *Gene Expression Regulation ; Genome-Wide Association Study ; Humans ; *Models, Neurological ; *Schizophrenia/genetics/metabolism ; },
abstract = {Schizophrenia (SZ) is a prevalent functional psychosis characterized by clinical behavioural symptoms and underlying abnormalities in brain function. Genome-wide association studies (GWAS) of schizophrenia have revealed many loci that do not directly identify processes disturbed in the disease. For this reason, the development of cellular models containing SZ-associated variations has become a focus in the post-GWAS research era. The application of revolutionary clustered regularly interspaced palindromic repeats CRISPR/Cas9 gene-editing tools, along with recently developed technologies for cultivating brain organoids in vitro, have opened new perspectives for the construction of these models. In general, cellular models are intended to unravel particular biological phenomena. They can provide the missing link between schizophrenia-related phenotypic features (such as transcriptional dysregulation, oxidative stress and synaptic dysregulation) and data from pathomorphological, electrophysiological and behavioural studies. The objectives of this review are the systematization and classification of cellular models of schizophrenia, based on their complexity and validity for understanding schizophrenia-related phenotypes.},
}

Biomedical Research
*CRISPR-Cas Systems
*Gene Editing
*Gene Expression Regulation
Genome-Wide Association Study
Humans
*Models, Neurological
*Schizophrenia/genetics/metabolism

@article {pmid34365302,
year = {2021},
author = {Ryu, AJ and Jeong, BR and Kang, NK and Jeon, S and Sohn, MG and Yun, HJ and Lim, JM and Jeong, SW and Park, YI and Jeong, WJ and Park, S and Chang, YK and Jeong, KJ},
title = {Safe-Harboring based novel genetic toolkit for Nannochloropsis salina CCMP1776: Efficient overexpression of transgene via CRISPR/Cas9-Mediated Knock-in at the transcriptional hotspot.},
journal = {Bioresource technology},
volume = {340},
number = {},
pages = {125676},
doi = {10.1016/j.biortech.2021.125676},
pmid = {34365302},
issn = {1873-2976},
mesh = {*CRISPR-Cas Systems/genetics ; Fatty Acid Desaturases/genetics/metabolism ; Genetic Engineering ; *Stramenopiles/genetics/metabolism ; Transgenes ; },
abstract = {Transgene expression in microalgae can be hampered by transgene silencing and unstable expression due to position effects. To overcome this, "safe harboring" transgene expression system was established for Nannochloropsis. Initially, transformants were obtained expressing a sfGFP reporter, followed by screening for high expression of sfGFP with fluorescence-activated cell sorter (FACS). 'T1' transcriptional hotspot was identified from a mutant showing best expression of sfGFP, but did not affect growth or lipid contents. By using a Cas9 editor strain, FAD12 gene, encoding Δ12-fatty acid desaturase (FAD12), was successfully knocked-in at the T1 locus, resulting in significantly higher expression of FAD12 than those of random integration. Importantly, the "safe harbored" FAD12 transformants showed four-fold higher production of linoleic acid (LA), the product of FAD12, leading to 1.5-fold increase in eicosapentaenoic acid (EPA). This safe harboring principle provide excellent proof of the concept for successful genetic/metabolic engineering of microalgae and other organisms.},
}

*CRISPR-Cas Systems/genetics
Fatty Acid Desaturases/genetics/metabolism
Genetic Engineering
*Stramenopiles/genetics/metabolism
Transgenes

@article {pmid34360895,
year = {2021},
author = {Matboli, M and Kamel, MM and Essawy, N and Bekhit, MM and Abdulrahman, B and Mohamed, GF and Eissa, S},
title = {Identification of Novel Insulin Resistance Related ceRNA Network in T2DM and Its Potential Editing by CRISPR/Cas9.},
journal = {International journal of molecular sciences},
volume = {22},
number = {15},
pages = {},
pmid = {34360895},
issn = {1422-0067},
support = {Egyptian Academy of Scientific Research and Technology, JESOR//Egyptian Academy of Scientific Research and Technology, JESOR/ ; },
mesh = {Adult ; Aged ; Aged, 80 and over ; *CRISPR-Cas Systems ; Case-Control Studies ; Cell Line ; Computational Biology/*methods ; Diabetes Mellitus, Type 2/blood/*genetics ; Female ; Gene Editing/*methods ; *Gene Expression ; Gene Regulatory Networks ; Hospitals, University ; Humans ; Insulin Resistance/*genetics ; Lymphocytes/metabolism ; Male ; MicroRNAs/*genetics ; Middle Aged ; RNA, Long Noncoding/*genetics ; RNA, Messenger/*genetics ; },
abstract = {BACKGROUND: Type 2 diabetes mellitus is one of the leading causes of morbidity and mortality worldwide and is derived from an accumulation of genetic and epigenetic changes. In this study, we aimed to construct Insilco, a competing endogenous RNA (ceRNA) network linked to the pathogenesis of insulin resistance followed by its experimental validation in patients', matched control and cell line samples, as well as to evaluate the efficacy of CRISPR/Cas9 as a potential therapeutic strategy to modulate the expression of this deregulated network. By applying bioinformatics tools through a two-step process, we identified and verified a ceRNA network panel of mRNAs, miRNAs and lncRNA related to insulin resistance, Then validated the expression in clinical samples (123 patients and 106 controls) and some of matched cell line samples using real time PCR. Next, two guide RNAs were designed to target the sequence flanking LncRNA/miRNAs interaction by CRISPER/Cas9 in cell culture. Gene editing tool efficacy was assessed by measuring the network downstream proteins GLUT4 and mTOR via immunofluorescence.

RESULTS: LncRNA-RP11-773H22.4, together with RET, IGF1R and mTOR mRNAs, showed significant upregulation in T2DM compared with matched controls, while miRNA (i.e., miR-3163 and miR-1) and mRNA (i.e., GLUT4 and AKT2) expression displayed marked downregulation in diabetic samples. CRISPR/Cas9 successfully knocked out LncRNA-RP11-773H22.4, as evidenced by the reversal of the gene expression of the identified network at RNA and protein levels to the normal expression pattern after gene editing.

CONCLUSIONS: The present study provides the significance of this ceRNA based network and its related target genes panel both in the pathogenesis of insulin resistance and as a therapeutic target for gene editing in T2DM.},
}

Adult
Aged
Aged, 80 and over
*CRISPR-Cas Systems
Case-Control Studies
Cell Line
Computational Biology/*methods
Diabetes Mellitus, Type 2/blood/*genetics
Female
Gene Editing/*methods
*Gene Expression
Gene Regulatory Networks
Hospitals, University
Humans
Insulin Resistance/*genetics
Lymphocytes/metabolism
Male
MicroRNAs/*genetics
Middle Aged
RNA, Long Noncoding/*genetics
RNA, Messenger/*genetics

@article {pmid34192988,
year = {2021},
author = {Arlt, B and Mastrobuoni, G and Wuenschel, J and Astrahantseff, K and Eggert, A and Kempa, S and Deubzer, HE},
title = {Inhibiting PHGDH with NCT-503 reroutes glucose-derived carbons into the TCA cycle, independently of its on-target effect.},
journal = {Journal of enzyme inhibition and medicinal chemistry},
volume = {36},
number = {1},
pages = {1282-1289},
pmid = {34192988},
issn = {1475-6374},
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Citric Acid Cycle/drug effects ; Enzyme Inhibitors/*pharmacology ; Gas Chromatography-Mass Spectrometry/methods ; Glucose/metabolism ; Humans ; Metabolomics ; Phosphoglycerate Dehydrogenase/*antagonists & inhibitors/genetics ; },
abstract = {The small-molecule inhibitor of phosphoglycerate dehydrogenase, NCT-503, reduces incorporation of glucose-derived carbons into serine in vitro. Here we describe an off-target effect of NCT-503 in neuroblastoma cell lines expressing divergent phosphoglycerate dehydrogenase (PHGDH) levels and single-cell clones with CRISPR-Cas9-directed PHGDH knockout or their respective wildtype controls. NCT-503 treatment strongly reduced synthesis of glucose-derived citrate in all cell models investigated compared to the inactive drug control and independent of PHGDH expression level. Incorporation of glucose-derived carbons entering the TCA cycle via pyruvate carboxylase was enhanced by NCT-503 treatment. The activity of citrate synthase was not altered by NCT-503 treatment. We also detected no change in the thermal stabilisation of citrate synthase in cellular thermal shift assays from NCT-503-treated cells. Thus, the direct cause of the observed off-target effect remains enigmatic. Our findings highlight off-target potential within a metabolic assessment of carbon usage in cells treated with the small-molecule inhibitor, NCT-503.},
}

CRISPR-Cas Systems
Cell Line, Tumor
Cell Proliferation/drug effects
Citric Acid Cycle/drug effects
Enzyme Inhibitors/*pharmacology
Gas Chromatography-Mass Spectrometry/methods
Glucose/metabolism
Humans
Metabolomics
Phosphoglycerate Dehydrogenase/*antagonists & inhibitors/genetics

@article {pmid33825445,
year = {2021},
author = {Matsumoto, D and Nomura, W},
title = {Molecular Switch Engineering for Precise Genome Editing.},
journal = {Bioconjugate chemistry},
volume = {32},
number = {4},
pages = {639-648},
doi = {10.1021/acs.bioconjchem.1c00088},
pmid = {33825445},
issn = {1520-4812},
mesh = {CRISPR-Cas Systems ; DNA/genetics ; *Gene Editing ; *Genetic Engineering ; Nucleic Acid Hybridization ; Proteolysis ; RNA/genetics ; },
abstract = {Genome editing technology commenced in 1996 with the discovery of the first zinc-finger nuclease. Application of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) associated protein 9 (Cas9) technology to genome editing of mammalian cells allowed researchers to use genome editing more easily and cost-effectively. However, one of the technological problems that remains to be solved is "off-target effects", which are unexpected mutations in nontarget DNA. One significant improvement in genome editing technology has been achieved with molecular/protein engineering. The key to this engineering is a "switch" to control function. In this review, we discuss recent efforts to design novel "switching" systems for precise editing using genome editing tools.},
}

CRISPR-Cas Systems
DNA/genetics
*Gene Editing
*Genetic Engineering
Nucleic Acid Hybridization
Proteolysis
RNA/genetics

@article {pmid33636132,
year = {2021},
author = {Huang, H and Zhou, P and Wei, J and Long, L and Shi, H and Dhungana, Y and Chapman, NM and Fu, G and Saravia, J and Raynor, JL and Liu, S and Palacios, G and Wang, YD and Qian, C and Yu, J and Chi, H},
title = {In vivo CRISPR screening reveals nutrient signaling processes underpinning CD8+ T cell fate decisions.},
journal = {Cell},
volume = {184},
number = {5},
pages = {1245-1261.e21},
pmid = {33636132},
issn = {1097-4172},
support = {R01 AI131703/AI/NIAID NIH HHS/United States ; R01 CA250533/CA/NCI NIH HHS/United States ; R01 AI150514/AI/NIAID NIH HHS/United States ; R37 AI105887/AI/NIAID NIH HHS/United States ; R01 CA176624/CA/NCI NIH HHS/United States ; R01 CA221290/CA/NCI NIH HHS/United States ; R01 AI140761/AI/NIAID NIH HHS/United States ; R01 AI105887/AI/NIAID NIH HHS/United States ; R01 GM134382/GM/NIGMS NIH HHS/United States ; R01 AI150241/AI/NIAID NIH HHS/United States ; },
mesh = {Amino Acid Transport Systems/metabolism ; Amino Acids/*metabolism ; Animals ; CD8-Positive T-Lymphocytes/*cytology/immunology ; CRISPR-Cas Systems ; Cell Cycle ; Cell Differentiation ; Disease Models, Animal ; Female ; Gene Knock-In Techniques ; *Immunologic Memory ; Lymphocytic Choriomeningitis/immunology ; Male ; Mice ; Mice, Transgenic ; Precursor Cells, T-Lymphoid/cytology ; *Signal Transduction ; },
abstract = {How early events in effector T cell (TEFF) subsets tune memory T cell (TMEM) responses remains incompletely understood. Here, we systematically investigated metabolic factors in fate determination of TEFF and TMEM cells using in vivo pooled CRISPR screening, focusing on negative regulators of TMEM responses. We found that amino acid transporters Slc7a1 and Slc38a2 dampened the magnitude of TMEM differentiation, in part through modulating mTORC1 signaling. By integrating genetic and systems approaches, we identified cellular and metabolic heterogeneity among TEFF cells, with terminal effector differentiation associated with establishment of metabolic quiescence and exit from the cell cycle. Importantly, Pofut1 (protein-O-fucosyltransferase-1) linked GDP-fucose availability to downstream Notch-Rbpj signaling, and perturbation of this nutrient signaling axis blocked terminal effector differentiation but drove context-dependent TEFF proliferation and TMEM development. Our study establishes that nutrient uptake and signaling are key determinants of T cell fate and shape the quantity and quality of TMEM responses.},
}

Amino Acid Transport Systems/metabolism
Amino Acids/*metabolism
Animals
CD8-Positive T-Lymphocytes/*cytology/immunology
CRISPR-Cas Systems
Cell Cycle
Cell Differentiation
Disease Models, Animal
Female
Gene Knock-In Techniques
*Immunologic Memory
Lymphocytic Choriomeningitis/immunology
Male
Mice
Mice, Transgenic
Precursor Cells, T-Lymphoid/cytology
*Signal Transduction

@article {pmid33636129,
year = {2021},
author = {Chen, Z and Arai, E and Khan, O and Zhang, Z and Ngiow, SF and He, Y and Huang, H and Manne, S and Cao, Z and Baxter, AE and Cai, Z and Freilich, E and Ali, MA and Giles, JR and Wu, JE and Greenplate, AR and Hakeem, MA and Chen, Q and Kurachi, M and Nzingha, K and Ekshyyan, V and Mathew, D and Wen, Z and Speck, NA and Battle, A and Berger, SL and Wherry, EJ and Shi, J},
title = {In vivo CD8+ T cell CRISPR screening reveals control by Fli1 in infection and cancer.},
journal = {Cell},
volume = {184},
number = {5},
pages = {1262-1280.e22},
pmid = {33636129},
issn = {1097-4172},
support = {P01 CA210944/CA/NCI NIH HHS/United States ; R01 HG010480/HG/NHGRI NIH HHS/United States ; T32 CA009140/CA/NCI NIH HHS/United States ; U19 AI082630/AI/NIAID NIH HHS/United States ; P01 AI112521/AI/NIAID NIH HHS/United States ; R01 AI115712/AI/NIAID NIH HHS/United States ; P01 AI108545/AI/NIAID NIH HHS/United States ; R01 AI105343/AI/NIAID NIH HHS/United States ; R01 MH109905/MH/NIMH NIH HHS/United States ; U19 AI117950/AI/NIAID NIH HHS/United States ; R01 AI155577/AI/NIAID NIH HHS/United States ; F99 CA234842/CA/NCI NIH HHS/United States ; U19 AI149680/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; CD8-Positive T-Lymphocytes/*cytology/immunology/metabolism ; CRISPR-Cas Systems ; Cell Differentiation ; Chronic Disease ; Core Binding Factor Alpha 3 Subunit/metabolism ; Epigenesis, Genetic ; Gene Regulatory Networks ; Infections/immunology ; Mice ; Neoplasms/immunology ; Proto-Oncogene Protein c-fli-1/*metabolism ; },
abstract = {Improving effector activity of antigen-specific T cells is a major goal in cancer immunotherapy. Despite the identification of several effector T cell (TEFF)-driving transcription factors (TFs), the transcriptional coordination of TEFF biology remains poorly understood. We developed an in vivo T cell CRISPR screening platform and identified a key mechanism restraining TEFF biology through the ETS family TF, Fli1. Genetic deletion of Fli1 enhanced TEFF responses without compromising memory or exhaustion precursors. Fli1 restrained TEFF lineage differentiation by binding to cis-regulatory elements of effector-associated genes. Loss of Fli1 increased chromatin accessibility at ETS:RUNX motifs, allowing more efficient Runx3-driven TEFF biology. CD8+ T cells lacking Fli1 provided substantially better protection against multiple infections and tumors. These data indicate that Fli1 safeguards the developing CD8+ T cell transcriptional landscape from excessive ETS:RUNX-driven TEFF cell differentiation. Moreover, genetic deletion of Fli1 improves TEFF differentiation and protective immunity in infections and cancer.},
}

Animals
CD8-Positive T-Lymphocytes/*cytology/immunology/metabolism
CRISPR-Cas Systems
Cell Differentiation
Chronic Disease
Core Binding Factor Alpha 3 Subunit/metabolism
Epigenesis, Genetic
Gene Regulatory Networks
Infections/immunology
Mice
Neoplasms/immunology
Proto-Oncogene Protein c-fli-1/*metabolism

@article {pmid33539781,
year = {2021},
author = {Yu, H and Lin, T and Meng, X and Du, H and Zhang, J and Liu, G and Chen, M and Jing, Y and Kou, L and Li, X and Gao, Q and Liang, Y and Liu, X and Fan, Z and Liang, Y and Cheng, Z and Chen, M and Tian, Z and Wang, Y and Chu, C and Zuo, J and Wan, J and Qian, Q and Han, B and Zuccolo, A and Wing, RA and Gao, C and Liang, C and Li, J},
title = {A route to de novo domestication of wild allotetraploid rice.},
journal = {Cell},
volume = {184},
number = {5},
pages = {1156-1170.e14},
doi = {10.1016/j.cell.2021.01.013},
pmid = {33539781},
issn = {1097-4172},
mesh = {CRISPR-Cas Systems ; Crops, Agricultural/*genetics ; *Domestication ; Food Security ; Gene Editing ; Genetic Variation ; Genome, Plant ; Oryza/classification/*genetics ; Polyploidy ; },
abstract = {Cultivated rice varieties are all diploid, and polyploidization of rice has long been desired because of its advantages in genome buffering, vigorousness, and environmental robustness. However, a workable route remains elusive. Here, we describe a practical strategy, namely de novo domestication of wild allotetraploid rice. By screening allotetraploid wild rice inventory, we identified one genotype of Oryza alta (CCDD), polyploid rice 1 (PPR1), and established two important resources for its de novo domestication: (1) an efficient tissue culture, transformation, and genome editing system and (2) a high-quality genome assembly discriminated into two subgenomes of 12 chromosomes apiece. With these resources, we show that six agronomically important traits could be rapidly improved by editing O. alta homologs of the genes controlling these traits in diploid rice. Our results demonstrate the possibility that de novo domesticated allotetraploid rice can be developed into a new staple cereal to strengthen world food security.},
}

CRISPR-Cas Systems
Crops, Agricultural/*genetics
*Domestication
Food Security
Gene Editing
Genetic Variation
Genome, Plant
Oryza/classification/*genetics
Polyploidy

@article {pmid33393280,
year = {2021},
author = {Wang, W and Liang, Z and Ma, P and Zhao, Q and Dai, M and Zhu, J and Han, X and Xu, H and Chang, Q and Zhen, Y},
title = {Application of CRISPR/Cas9 System to Reverse ABC-Mediated Multidrug Resistance.},
journal = {Bioconjugate chemistry},
volume = {32},
number = {1},
pages = {73-81},
doi = {10.1021/acs.bioconjchem.0c00627},
pmid = {33393280},
issn = {1520-4812},
mesh = {ATP-Binding Cassette Transporters/*physiology ; Animals ; Antineoplastic Agents/pharmacology ; *CRISPR-Cas Systems ; Drug Resistance, Multiple/drug effects/genetics/*physiology ; Gene Editing ; Humans ; },
abstract = {Multidrug resistance (MDR) is the main obstacle in cancer chemotherapy. ATP-binding cassette (ABC) transporters can transport a wide range of antitumor drugs out of cells, which is the most common reason in the development of resistance to drugs. Currently, various therapeutic strategies are used to reverse MDR, among which CRISPR/Cas9 gene editing technique is expected to be an effective way. Here, we reviewed the research progress of reversing ABC-mediated drug resistance by CRISPR/Cas9 system.},
}

ATP-Binding Cassette Transporters/*physiology
Animals
Antineoplastic Agents/pharmacology
*CRISPR-Cas Systems
Drug Resistance, Multiple/drug effects/genetics/*physiology
Gene Editing
Humans

@article {pmid33181064,
year = {2020},
author = {Gaudelli, NM and Komor, AC},
title = {Celebrating Rosalind Franklin's Centennial with a Nobel Win for Doudna and Charpentier.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {12},
pages = {2519-2520},
pmid = {33181064},
issn = {1525-0024},
mesh = {COVID-19/*epidemiology/virology ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems ; *Chemistry ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Female ; *Gene Editing ; Humans ; *Nobel Prize ; *Pandemics ; },
}

COVID-19/*epidemiology/virology
CRISPR-Associated Protein 9/genetics
CRISPR-Cas Systems
*Chemistry
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Female
*Gene Editing
Humans
*Nobel Prize
*Pandemics

@article {pmid33172906,
year = {2020},
author = {Lach, FP and Singh, S and Rickman, KA and Ruiz, PD and Noonan, RJ and Hymes, KB and DeLacure, MD and Kennedy, JA and Chandrasekharappa, SC and Smogorzewska, A},
title = {Esophageal cancer as initial presentation of Fanconi anemia in patients with a hypomorphic FANCA variant.},
journal = {Cold Spring Harbor molecular case studies},
volume = {6},
number = {6},
pages = {},
pmid = {33172906},
issn = {2373-2873},
support = {UL1 TR000043/TR/NCATS NIH HHS/United States ; TL1 TR002386/TR/NCATS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; R01 CA204127/CA/NCI NIH HHS/United States ; UL1 TR001866/TR/NCATS NIH HHS/United States ; R01 HL120922/HL/NHLBI NIH HHS/United States ; T32 GM007739/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Chromosome Breakage ; DNA ; DNA Repair ; Esophageal Neoplasms/diagnosis/*genetics/therapy ; Esophageal Squamous Cell Carcinoma/diagnosis/genetics/metabolism ; Fanconi Anemia/diagnosis/*genetics/therapy ; Fanconi Anemia Complementation Group A Protein/*genetics ; Fibroblasts/metabolism ; Gene Editing ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; Middle Aged ; Mutation ; },
abstract = {Fanconi anemia (FA) is a clinically heterogenous and genetically diverse disease with 22 known complementation groups (FA-A to FA-W), resulting from the inability to repair DNA interstrand cross-links. This rare disorder is characterized by congenital defects, bone marrow failure, and cancer predisposition. FANCA is the most commonly mutated gene in FA and a variety of mostly private mutations have been documented, including small and large indels and point and splicing variants. Genotype-phenotype associations in FA are complex, and a relationship between particular FANCA variants and the observed cellular phenotype or illness severity remains unclear. In this study, we describe two siblings with compound heterozygous FANCA variants (c.3788_3790delTCT and c.4199G > A) who both presented with esophageal squamous cell carcinoma at the age of 51. The proband came to medical attention when he developed pancytopenia after a single cycle of low-dose chemotherapy including platinum-based therapy. Other than a minor thumb abnormality, neither patient had prior findings to suggest FA, including normal blood counts and intact fertility. Patient fibroblasts from both siblings display increased chromosomal breakage and hypersensitivity to interstrand cross-linking agents as seen in typical FA. Based on our functional data demonstrating that the c.4199G > A/p.R1400H variant represents a hypomorphic FANCA allele, we conclude that the residual activity of the Fanconi anemia repair pathway accounts for lack of spontaneous bone marrow failure or infertility with the late presentation of malignancy as the initial disease manifestation. This and similar cases of adult-onset esophageal cancer stress the need for chromosome breakage testing in patients with early onset of aerodigestive tract squamous cell carcinomas before platinum-based therapy is initiated.},
}

CRISPR-Cas Systems
Chromosome Breakage
DNA
DNA Repair
Esophageal Neoplasms/diagnosis/*genetics/therapy
Esophageal Squamous Cell Carcinoma/diagnosis/genetics/metabolism
Fanconi Anemia/diagnosis/*genetics/therapy
Fanconi Anemia Complementation Group A Protein/*genetics
Fibroblasts/metabolism
Gene Editing
Gene Expression Regulation, Neoplastic
Humans
Male
Middle Aged
Mutation

@article {pmid33171139,
year = {2020},
author = {Marsh, S and Hanson, B and Wood, MJA and Varela, MA and Roberts, TC},
title = {Application of CRISPR-Cas9-Mediated Genome Editing for the Treatment of Myotonic Dystrophy Type 1.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {12},
pages = {2527-2539},
pmid = {33171139},
issn = {1525-0024},
support = {MR/P01741X/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Dependovirus/genetics ; Disease Models, Animal ; Gene Editing/*methods ; Genetic Therapy/*methods ; Genetic Vectors/administration & dosage ; Humans ; Mice ; Myotonic Dystrophy/genetics/*therapy ; Myotonin-Protein Kinase/genetics ; RNA, Guide/genetics ; Treatment Outcome ; Trinucleotide Repeat Expansion/genetics ; },
abstract = {Myotonic dystrophy type 1 (DM1) is a debilitating multisystemic disorder, caused by expansion of a CTG microsatellite repeat in the 3' untranslated region of the DMPK (dystrophia myotonica protein kinase) gene. To date, novel therapeutic approaches have focused on transient suppression of the mutant, repeat-expanded RNA. However, recent developments in the field of genome editing have raised the exciting possibility of inducing permanent correction of the DM1 genetic defect. Specifically, repurposing of the prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) system has enabled programmable, site-specific, and multiplex genome editing. CRISPR-based strategies for the treatment of DM1 can be applied either directly to patients, or indirectly through the ex vivo modification of patient-derived cells, and they include excision of the repeat expansion, insertion of synthetic polyadenylation signals upstream of the repeat, steric interference with RNA polymerase II procession through the repeat leading to transcriptional downregulation of DMPK, and direct RNA targeting of the mutant RNA species. Potential obstacles to such therapies are discussed, including the major challenge of Cas9 and guide RNA transgene/ribonuclear protein delivery, off-target gene editing, vector genome insertion at cut sites, on-target unintended mutagenesis (e.g., repeat inversion), pre-existing immunity to Cas9 or AAV antigens, immunogenicity, and Cas9 persistence.},
}

Animals
CRISPR-Associated Protein 9/*genetics
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Dependovirus/genetics
Disease Models, Animal
Gene Editing/*methods
Genetic Therapy/*methods
Genetic Vectors/administration & dosage
Humans
Mice
Myotonic Dystrophy/genetics/*therapy
Myotonin-Protein Kinase/genetics
RNA, Guide/genetics
Treatment Outcome
Trinucleotide Repeat Expansion/genetics

@article {pmid32882179,
year = {2020},
author = {Cheng, X},
title = {Valproic Acid Thermally Destabilizes and Inhibits SpyCas9 Activity.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {12},
pages = {2635-2641},
pmid = {32882179},
issn = {1525-0024},
mesh = {Bacterial Proteins/*chemistry/*metabolism ; CRISPR-Associated Protein 9/*chemistry/*metabolism ; CRISPR-Cas Systems ; Enzyme Activation/drug effects ; Enzyme Inhibitors/chemistry/*metabolism ; Gene Editing ; *Hot Temperature ; Indocyanine Green ; Mutation ; Protein Denaturation/drug effects ; Protein Stability/drug effects ; Streptococcus pyogenes/*enzymology ; Valproic Acid/*metabolism/*pharmacology ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system plays an important role in prokaryotic adaptive immunity. Due to its capacity for sequence-specific gene editing, CRISPR-Cas9 has become one of the most important tools widely used for genome editing in molecular biotechnology. However, its clinical application is currently limited by unwanted mutations at off-target sites. Various strategies have been developed for precise control of Cas9 in order to reduce these off-target effects, including chemical-based approaches. From a chemical screening, I observed that valproic acid (VPA) binds to and destabilizes Streptococcus pyogenes Cas9 (SpyCas9) protein in vitro, as well as in cells, while within its therapeutical concentration range under conditions of hyperthermia as demonstrated. Conditions were generated either by an external heat bag or in combination with the photothermal therapeutic agent indocyanine green activated by a near-infrared laser. Use of other histone deacetylase inhibitors failed, suggesting a histone deacetylase inhibition-independent function of VPA. Thus, this finding provides an uncomplicated thermotherapeutical approach for timely regulation of the activity of the CRISPR-Cas9 system at desired locations.},
}

Bacterial Proteins/*chemistry/*metabolism
CRISPR-Associated Protein 9/*chemistry/*metabolism
CRISPR-Cas Systems
Enzyme Activation/drug effects
Enzyme Inhibitors/chemistry/*metabolism
Gene Editing
*Hot Temperature
Indocyanine Green
Mutation
Protein Denaturation/drug effects
Protein Stability/drug effects
Streptococcus pyogenes/*enzymology
Valproic Acid/*metabolism/*pharmacology

@article {pmid32841372,
year = {2021},
author = {Khan, MHU and Hu, L and Zhu, M and Zhai, Y and Khan, SU and Ahmar, S and Amoo, O and Zhang, K and Fan, C and Zhou, Y},
title = {Targeted mutagenesis of EOD3 gene in Brassica napus L. regulates seed production.},
journal = {Journal of cellular physiology},
volume = {236},
number = {3},
pages = {1996-2007},
doi = {10.1002/jcp.29986},
pmid = {32841372},
issn = {1097-4652},
mesh = {Base Sequence ; Brassica napus/*genetics/*growth & development ; CRISPR-Cas Systems/genetics ; Cell Size ; Cloning, Molecular ; Cotyledon/anatomy & histology/growth & development ; Gene Editing ; Gene Expression Regulation, Plant ; *Genes, Plant ; Mutagenesis/*genetics ; Mutation/genetics ; Organ Size ; Phenotype ; Plant Proteins/*genetics/metabolism ; Plants, Genetically Modified ; RNA, Guide/genetics ; Seeds/*genetics/*growth & development ; Sequence Homology, Amino Acid ; },
abstract = {Seed size and number are central to the evolutionary fitness of plants and are also crucial for seed production of crops. However, the molecular mechanisms of seed production control are poorly understood in Brassica crops. Here, we report the gene cloning, expression analysis, and functional characterization of the EOD3/CYP78A6 gene in rapeseed. BnaEOD3 has four copies located in two subgenomes, which exhibited a steady higher expression during seed development with differential expression among copies. The targeted mutations of BnaEOD3 gene were efficiently generated by stable transformation of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat) vector. These mutations were stably transmitted to T1 and T2 generations and a large collection of homozygous mutants with combined loss-of-function alleles across four BnaEOD3 copies were created for phenotyping. All mutant T1 lines had shorter siliques, smaller seeds, and an increased number of seeds per silique, in which the quadrable mutants showed the most significant changes in these traits. Consequently, the seed weight per plant in the quadrable mutants increased by 13.9% on average compared with that of wild type, indicating that these BnaEOD3 copies have redundant functions in seed development in rapeseed. The phenotypes of the different allelic combinations of BnaEOD3 copies also revealed gene functional differentiation among the two subgenomes. Cytological observations indicated that the BnaEOD3 could act maternally to promote cotyledon cell expansion and proliferation to regulate seed growth in rapeseed. Collectively, our findings reveal the quantitative involvement of the different BnaEOD3 copies function in seed development, but also provided valuable resources for rapeseed breeding programs.},
}

Base Sequence
Brassica napus/*genetics/*growth & development
CRISPR-Cas Systems/genetics
Cell Size
Cloning, Molecular
Cotyledon/anatomy & histology/growth & development
Gene Editing
Gene Expression Regulation, Plant
*Genes, Plant
Mutagenesis/*genetics
Mutation/genetics
Organ Size
Phenotype
Plant Proteins/*genetics/metabolism
Plants, Genetically Modified
RNA, Guide/genetics
Seeds/*genetics/*growth & development
Sequence Homology, Amino Acid

@article {pmid32827460,
year = {2020},
author = {Di Roberto, RB and Castellanos-Rueda, R and Frey, S and Egli, D and Vazquez-Lombardi, R and Kapetanovic, E and Kucharczyk, J and Reddy, ST},
title = {A Functional Screening Strategy for Engineering Chimeric Antigen Receptors with Reduced On-Target, Off-Tumor Activation.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {12},
pages = {2564-2576},
pmid = {32827460},
issn = {1525-0024},
mesh = {Animals ; Antibody Affinity ; Antigens, Neoplasm/*immunology/metabolism ; Breast Neoplasms/*immunology/pathology ; CRISPR-Cas Systems ; Cell Engineering/*methods ; Coculture Techniques ; Female ; Gene Editing/methods ; HEK293 Cells ; High-Throughput Nucleotide Sequencing/methods ; Humans ; Immunotherapy, Adoptive/*methods ; Interleukin-2/genetics/metabolism ; MCF-7 Cells ; Mice ; Receptor, ErbB-2/*immunology/metabolism ; Receptors, Antigen, T-Cell, alpha-beta/*genetics/*immunology/metabolism ; Receptors, Chimeric Antigen/*genetics/*immunology/metabolism ; Single-Chain Antibodies/immunology ; },
abstract = {In recent years, chimeric antigen receptor (CAR) T cell cancer immunotherapies have advanced substantially in the clinic. However, challenges related to safety persist; one major concern occurs when CARs trigger a response to antigen present on healthy cells (on-target, off-tumor response). A strategy to ameliorate this relies on the complex relationship between receptor affinity and signaling, such that one can engineer a CAR that is only activated by tumor cells expressing high antigen levels. Here, we developed a CAR T cell display platform with stable genomic expression and rapid functional screening based on interleukin-2 signaling. Starting with a CAR with high affinity toward its target antigen, we combined CRISPR-Cas9 genome editing and deep mutational scanning to generate a library of antigen-binding domain variants. This library was subjected to multiple rounds of selection based on either antigen binding or cell signaling. Deep sequencing of the resulting libraries and a comparative analysis revealed the enrichment and depletion of specific variants from which we selected CARs that were selectively activated by tumor cells based on antigen expression levels. Our platform demonstrates how directed evolution based on functional screening and deep sequencing-guided selection can be combined to enhance the selectivity and safety of CARs.},
}

Animals
Antibody Affinity
Antigens, Neoplasm/*immunology/metabolism
Breast Neoplasms/*immunology/pathology
CRISPR-Cas Systems
Cell Engineering/*methods
Coculture Techniques
Female
Gene Editing/methods
HEK293 Cells
High-Throughput Nucleotide Sequencing/methods
Humans
Immunotherapy, Adoptive/*methods
Interleukin-2/genetics/metabolism
MCF-7 Cells
Mice
Receptor, ErbB-2/*immunology/metabolism
Receptors, Antigen, T-Cell, alpha-beta/*genetics/*immunology/metabolism
Receptors, Chimeric Antigen/*genetics/*immunology/metabolism
Single-Chain Antibodies/immunology

@article {pmid32822592,
year = {2020},
author = {Tran, NT and Graf, R and Wulf-Goldenberg, A and Stecklum, M and Strauß, G and Kühn, R and Kocks, C and Rajewsky, K and Chu, VT},
title = {CRISPR-Cas9-Mediated ELANE Mutation Correction in Hematopoietic Stem and Progenitor Cells to Treat Severe Congenital Neutropenia.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {12},
pages = {2621-2634},
pmid = {32822592},
issn = {1525-0024},
mesh = {Alleles ; Animals ; CRISPR-Cas Systems/*genetics ; Cell Differentiation/genetics ; Congenital Bone Marrow Failure Syndromes/genetics/pathology/*therapy ; Exons ; Genetic Therapy/*methods ; Granulocyte-Macrophage Colony-Stimulating Factor/genetics/metabolism ; HEK293 Cells ; Hematopoietic Stem Cell Transplantation/*methods ; Humans ; Interleukin-3/genetics/metabolism ; Leukocyte Elastase/*genetics ; Mice ; Mice, Transgenic ; *Mutation ; Neutropenia/*congenital/genetics/pathology/therapy ; Neutrophils/metabolism ; RNA, Guide/genetics ; Transfection ; Treatment Outcome ; },
abstract = {Severe congenital neutropenia (SCN) is a monogenic disorder. SCN patients are prone to recurrent life-threatening infections. The main causes of SCN are autosomal dominant mutations in the ELANE gene that lead to a block in neutrophil differentiation. In this study, we use CRISPR-Cas9 ribonucleoproteins and adeno-associated virus (AAV)6 as a donor template delivery system to repair the ELANEL172P mutation in SCN patient-derived hematopoietic stem and progenitor cells (HSPCs). We used a single guide RNA (sgRNA) specifically targeting the mutant allele, and an sgRNA targeting exon 4 of ELANE. Using the latter sgRNA, ∼34% of the known ELANE mutations can in principle be repaired. We achieved gene correction efficiencies of up to 40% (with sgELANE-ex4) and 56% (with sgELANE-L172P) in the SCN patient-derived HSPCs. Gene repair restored neutrophil differentiation in vitro and in vivo upon HSPC transplantation into humanized mice. Mature edited neutrophils expressed normal elastase levels and behaved normally in functional assays. Thus, we provide a proof of principle for using CRISPR-Cas9 to correct ELANE mutations in patient-derived HSPCs, which may translate into gene therapy for SCN.},
}

Alleles
Animals
CRISPR-Cas Systems/*genetics
Cell Differentiation/genetics
Congenital Bone Marrow Failure Syndromes/genetics/pathology/*therapy
Exons
Genetic Therapy/*methods
Granulocyte-Macrophage Colony-Stimulating Factor/genetics/metabolism
HEK293 Cells
Hematopoietic Stem Cell Transplantation/*methods
Humans
Interleukin-3/genetics/metabolism
Leukocyte Elastase/*genetics
Mice
Mice, Transgenic
*Mutation
Neutropenia/*congenital/genetics/pathology/therapy
Neutrophils/metabolism
RNA, Guide/genetics
Transfection
Treatment Outcome

@article {pmid32671729,
year = {2021},
author = {Liu, Z and Liao, Z and Chen, Y and Zhou, L and Huangting, W and Xiao, H},
title = {Research on CRISPR/system in major cancers and its potential in cancer treatments.},
journal = {Clinical & translational oncology : official publication of the Federation of Spanish Oncology Societies and of the National Cancer Institute of Mexico},
volume = {23},
number = {3},
pages = {425-433},
pmid = {32671729},
issn = {1699-3055},
support = {Grant no.30305030698//the National Key Specialty Construction Project of Clinical Pharmacy/ ; Grant no. 2009SZ0226//the Science & Technology Program of Sichuan Province/ ; 2014FZ0103//the Science & Technology Program of Sichuan Province/ ; 2015JQO027//the Science & Technology Program of Sichuan Province/ ; 2015ZR0160//the Science & Technology Program of Sichuan Province/ ; Grant no. 100491//the Health Department of Sichuan Province/ ; 120111//the Health Department of Sichuan Province/ ; 17ZD038//the Health Department of Sichuan Province/ ; Grant no. YB2019001//Sichuan Cancer Hospital/ ; Grant no.11PPYB010SF-289//Chengdu City Science and Technology Project/ ; Grant no. 30305030606//the Young Scholars Foundation of Sichuan Provincial People's Hospital/ ; 30305030859//the Young Scholars Foundation of Sichuan Provincial People's Hospital/ ; Grant No. 2019-801//The Cadre Health Care Research Project of Sichuan Province/ ; Grant No. 201905//Zambon Pharmaceutical Scientific Research Foundation of the Chengdu Pharmaceutical Association/ ; },
mesh = {Bacterial Proteins ; CRISPR-Associated Protein 9 ; CRISPR-Associated Proteins ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Colorectal Neoplasms/genetics/therapy ; Endodeoxyribonucleases ; Female ; Gene Editing/*methods ; Gene Knockout Techniques ; Genetic Therapy ; Humans ; Immunotherapy/methods ; Liver Neoplasms/genetics/therapy ; Lung Neoplasms/genetics/therapy ; Lymphoma/genetics/therapy ; Male ; Mutation ; Neoplasms/*genetics/*therapy ; Prostatic Neoplasms/genetics/therapy ; Research ; Uterine Cervical Neoplasms/genetics/therapy/virology ; },
abstract = {Cancer is a serious public health problem in the world and the prevention and control of cancer has become one of the health strategies of governments around the world. According to the data of the International Agency for Research on Cancer (IARC), about 8 million people die of cancer every year in the world. With the continuous progress of medical technology, there are many methods to treat cancer at present. However, many treatment methods have achieved different therapeutic effects, some of them have obvious toxic and side effects. Therefore, it is necessary to study simpler and more effective new therapies for alleviating pain and prolonging lifetime of patients. In this view, we focus on the application progress of CRISPR system in some major cancers and its potential in cancer treatments.},
}

Bacterial Proteins
CRISPR-Associated Protein 9
CRISPR-Associated Proteins
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Colorectal Neoplasms/genetics/therapy
Endodeoxyribonucleases
Female
Gene Editing/*methods
Gene Knockout Techniques
Genetic Therapy
Humans
Immunotherapy/methods
Liver Neoplasms/genetics/therapy
Lung Neoplasms/genetics/therapy
Lymphoma/genetics/therapy
Male
Mutation
Neoplasms/*genetics/*therapy
Prostatic Neoplasms/genetics/therapy
Research
Uterine Cervical Neoplasms/genetics/therapy/virology

@article {pmid32040336,
year = {2020},
author = {Carballar-Lejarazú, R and Kelsey, A and Pham, TB and Bennett, EP and James, AA},
title = {Digital droplet PCR and IDAA for the detection of CRISPR indel edits in the malaria species Anopheles stephensi.},
journal = {BioTechniques},
volume = {68},
number = {4},
pages = {172-179},
pmid = {32040336},
issn = {1940-9818},
mesh = {Animals ; Anopheles/*genetics ; CRISPR-Cas Systems/*genetics ; DNA End-Joining Repair/genetics ; Gene Editing ; INDEL Mutation/*genetics ; Malaria/transmission ; Mosquito Vectors/*genetics ; Mutagenesis, Insertional/genetics ; Polymerase Chain Reaction/*methods ; },
abstract = {CRISPR/Cas9 technology is a powerful tool for the design of gene-drive systems to control and/or modify mosquito vector populations; however, CRISPR/Cas9-mediated nonhomologous end joining mutations can have an important impact on generating alleles resistant to the drive and thus on drive efficiency. We demonstrate and compare the insertions or deletions (indels) detection capabilities of two techniques in the malaria vector mosquito Anopheles stephensi: Indel Detection by Amplicon Analysis (IDAA™) and Droplet Digital™ PCR (ddPCR™). Both techniques showed accuracy and reproducibility for indel frequencies across mosquito samples containing different ratios of indels of various sizes. Moreover, these techniques have advantages that make them potentially better suited for high-throughput nonhomologous end joining analysis in cage trials and contained field testing of gene-drive mosquitoes.},
}

Animals
Anopheles/*genetics
CRISPR-Cas Systems/*genetics
DNA End-Joining Repair/genetics
Gene Editing
INDEL Mutation/*genetics
Malaria/transmission
Mosquito Vectors/*genetics
Mutagenesis, Insertional/genetics
Polymerase Chain Reaction/*methods

@article {pmid32040335,
year = {2020},
author = {Her, NG and Babic, I and Yenugonda, VM and Kesari, S and Nurmemmedov, E},
title = {Cellular thermal shift analysis for interrogation of CRISPR-assisted proteomic changes.},
journal = {BioTechniques},
volume = {68},
number = {4},
pages = {180-184},
doi = {10.2144/btn-2019-0100},
pmid = {32040335},
issn = {1940-9818},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Line ; Gene Editing/*methods ; Humans ; Mutagenesis, Insertional/*genetics ; Proteome/analysis/*genetics ; Proteomics/*methods ; },
abstract = {CRISPR-Cas9 has proven to be a versatile tool for the discovery of essential genetic elements involved in various disease states. CRISPR-assisted dense mutagenesis focused on therapeutically challenging protein complexes allows us to systematically perturb protein-coding sequences in situ and correlate them with functional readouts. Such perturbations can mimic targeting by therapeutics and serve as a foundation for the discovery of highly specific modulators. However, translation of such genomics data has been challenging due to the missing link for proteomics under the physiological state of the cell. We present a method based on cellular thermal shift assays to easily interrogate proteomic shifts generated by CRISPR-assisted dense mutagenesis, as well as a case focused on NuRD epigenetic complex.},
}

CRISPR-Cas Systems/*genetics
Cell Line
Gene Editing/*methods
Humans
Mutagenesis, Insertional/*genetics
Proteome/analysis/*genetics
Proteomics/*methods

@article {pmid31973565,
year = {2020},
author = {Zhou, X},
title = {Empowering chimeric antigen receptor T-cell therapy with CRISPR.},
journal = {BioTechniques},
volume = {68},
number = {4},
pages = {169-171},
doi = {10.2144/btn-2019-0107},
pmid = {31973565},
issn = {1940-9818},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Humans ; *Immunotherapy, Adoptive ; *Receptors, Chimeric Antigen ; },
}

*CRISPR-Cas Systems
*Gene Editing
Humans
*Immunotherapy, Adoptive
*Receptors, Chimeric Antigen

@article {pmid31905010,
year = {2020},
author = {Zhang, K and Su, L and Wu, J},
title = {Recent Advances in Recombinant Protein Production by Bacillus subtilis.},
journal = {Annual review of food science and technology},
volume = {11},
number = {},
pages = {295-318},
doi = {10.1146/annurev-food-032519-051750},
pmid = {31905010},
issn = {1941-1421},
mesh = {Bacillus subtilis/*metabolism ; CRISPR-Cas Systems ; Gene Editing ; Proteomics ; Recombinant Proteins/biosynthesis ; },
abstract = {Bacillus subtilis has become a widely used microbial cell factory for the production of recombinant proteins, especially those associated with foods and food processing. Recent advances in genetic manipulation and proteomic analysis have been used to greatly improve protein production in B. subtilis. This review begins with a discussion of genome-editing technologies and application of the CRISPR-Cas9 system to B. subtilis. A summary of the characteristics of crucial legacy strains is followed by suggestions regarding the choice of origin strain for genetic manipulation. Finally, the review analyzes the genes and operons of B. subtilis that are important for the production of secretory proteins and provides suggestions and examples of how they can be altered to improve protein production. This review is intended to promote the engineering of this valuable microbial cell factory for better recombinant protein production.},
}

Bacillus subtilis/*metabolism
CRISPR-Cas Systems
Gene Editing
Proteomics
Recombinant Proteins/biosynthesis

@article {pmid34517092,
year = {2021},
author = {Savadi, S and Mangalassery, S and Sandesh, MS},
title = {Advances in genomics and genome editing for breeding next generation of fruit and nut crops.},
journal = {Genomics},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ygeno.2021.09.001},
pmid = {34517092},
issn = {1089-8646},
abstract = {Fruit tree crops are an essential part of the food production systems and are key to achieve food and nutrition security. Genetic improvement of fruit trees by conventional breeding has been slow due to the long juvenile phase. Advancements in genomics and molecular biology have paved the way for devising novel genetic improvement tools like genome editing, which can accelerate the breeding of these perennial crops to a great extent. In this article, advancements in genomics of fruit trees covering genome sequencing, transcriptome sequencing, genome editing technologies (GET), CRISPR-Cas system based genome editing, potential applications of CRISPR-Cas9 in fruit tree crops improvement, the factors influencing the CRISPR-Cas editing efficiency and the challenges for CRISPR-Cas9 applications in fruit tree crops improvement are reviewed. Besides, base editing, a recently emerging more precise editing system, and the future perspectives of genome editing in the improvement of fruit and nut crops are covered.},
}

@article {pmid34516103,
year = {2021},
author = {Xia, X and Ma, B and Zhang, T and Lu, Y and Khan, MR and Hu, Y and Lei, C and Deng, S and He, Q and He, G and Zhang, K and Deng, R},
title = {G-Quadruplex-Probing CRISPR-Cas12 Assay for Label-Free Analysis of Foodborne Pathogens and Their Colonization In Vivo.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.1c01061},
pmid = {34516103},
issn = {2379-3694},
abstract = {Foodborne pathogen infection is a key issue of food safety. Herein, we developed a label-free assay for Salmonella enterica (S. enterica) detection based on the G-quadruplex-probing CRISPR-Cas12 system (termed G-CRISPR-Cas), allowing highly sensitive detection of S. enterica and investigation of their colonization in chickens. The introduction of the G-quadruplex probe serving as the substrate of Cas 12a realized a label-free analysis for foodborne pathogens. Due to the amplification process induced by loop-mediated isothermal amplification (LAMP), G-CRISPR-Cas assay can detect S. enterica as low as 20 CFU. Specificity for pathogenic gene detection was guaranteed by the dual recognition process via LAMP primers and Cas 12a-guided RNA binding. The G-CRISPR-Cas assay was applied to explore S. enterica colonization in the intestinal tract and organs of chickens and showed the risk of S. enterica infection outside of the intestinal tract. The G-CRISPR-Cas assay is promising for on-site diagnosis of the infection or contamination of foodborne pathogens outside the laboratories, such as abattoirs and markets.},
}

@article {pmid34514290,
year = {2021},
author = {Jordan, WT and Currie, S and Schmitz, RJ},
title = {Multiplex genome editing in Arabidopsis thaliana using Mb3Cas12a.},
journal = {Plant direct},
volume = {5},
number = {9},
pages = {e344},
doi = {10.1002/pld3.344},
pmid = {34514290},
issn = {2475-4455},
abstract = {The use of CRISPR-Cas proteins for the creation of multiplex genome engineering represents an important avenue for crop improvement, and further improvements for creation of knock-in plant lines via CRISPR-based technologies may enable the high-throughput creation of designer alleles. To circumvent limitations of the commonly used CRISPR-Cas9 system for multiplex genome engineering, we explored the use of Moraxella bovoculi 3 Cas12a (Mb3Cas12a) for multiplex genome editing in Arabidopsis thaliana. We identified optimized cis-regulatory sequences for driving expression of single-transcript multiplex crRNA arrays in A. thaliana, resulting in stable germline transmission of Mb3Cas12a-edited alleles at multiple target sites. By utilizing this system, we demonstrate single-transcript multiplexed genome engineering using of up to 13 crRNA targets. We further show high target specificity of Mb3Cas12a-based genome editing via whole-genome sequencing. Taken together, our method provides a simplified platform for efficient multiplex genome engineering in plant-based systems.},
}

@article {pmid34514084,
year = {2021},
author = {Silva, FSR and Erdogmus, E and Shokr, A and Kandula, H and Thirumalaraju, P and Kanakasabapathy, MK and Hardie, JM and Pacheco, LGC and Li, JZ and Kuritzkes, DR and Shafiee, H},
title = {SARS-CoV-2 RNA Detection by a Cellphone-Based Amplification-Free System with CRISPR/CAS-Dependent Enzymatic (CASCADE) Assay.},
journal = {Advanced materials technologies},
volume = {},
number = {},
pages = {2100602},
doi = {10.1002/admt.202100602},
pmid = {34514084},
issn = {2365-709X},
abstract = {CRISPR (Clustered regularly interspaced short palindromic repeats)-based diagnostic technologies have emerged as a promising alternative to accelerate delivery of SARS-CoV-2 molecular detection at the point of need. However, efficient translation of CRISPR-diagnostic technologies to field application is still hampered by dependence on target amplification and by reliance on fluorescence-based results readout. Herein, an amplification-free CRISPR/Cas12a-based diagnostic technology for SARS-CoV-2 RNA detection is presented using a smartphone camera for results readout. This method, termed Cellphone-based amplification-free system with CRISPR/CAS-dependent enzymatic (CASCADE) assay, relies on mobile phone imaging of a catalase-generated gas bubble signal within a microfluidic channel and does not require any external hardware optical attachments. Upon specific detection of a SARS-CoV-2 reverse-transcribed DNA/RNA heteroduplex target (orf1ab) by the ribonucleoprotein complex, the transcleavage collateral activity of the Cas12a protein on a Catalase:ssDNA probe triggers the bubble signal on the system. High analytical sensitivity in signal detection without previous target amplification (down to 50 copies µL-1) is observed in spiked samples, in ≈71 min from sample input to results readout. With the aid of a smartphone vision tool, high accuracy (AUC = 1.0; CI: 0.715 - 1.00) is achieved when the CASCADE system is tested with nasopharyngeal swab samples of PCR-positive COVID-19 patients.},
}

@article {pmid34511901,
year = {2021},
author = {Piperno, A and Sciortino, MT and Giusto, E and Montesi, M and Panseri, S and Scala, A},
title = {Recent Advances and Challenges in Gene Delivery Mediated by Polyester-Based Nanoparticles.},
journal = {International journal of nanomedicine},
volume = {16},
number = {},
pages = {5981-6002},
doi = {10.2147/IJN.S321329},
pmid = {34511901},
issn = {1178-2013},
abstract = {Gene therapy is a promising approach for the treatment of several diseases, such as chronic or viral infections, inherited disorders, and cancer. The cellular internalization of exogenous nucleic acids (NA) requires efficient delivery vehicles to overcome their inherent pharmacokinetic drawbacks, e.g. electrostatic repulsions, enzymatic degradation, limited cellular uptake, fast clearance, etc. Nanotechnological advancements have enabled the use of polymer-based nanostructured biomaterials as safe and effective gene delivery systems, in addition to viral vector delivery methods. Among the plethora of polymeric nanoparticles (NPs), this review will provide a comprehensive and in-depth summary of the polyester-based nanovehicles, including poly(lactic-co-glycolic acid) (PLGA) and polylactic acid (PLA) NPs, used to deliver a variety of foreign NA, e.g. short interfering RNA (siRNA), messenger RNA (mRNA), and plasmid DNA (pDNA). The article will review the versatility of polyester-based nanocarriers including their recent application in the delivery of the clustered, regularly-interspaced, short palindromic repeats/Cas (CRISPR/Cas) genome editing system for treating gene-related diseases. The remaining challenges and future trend of the targeted delivery of this revolutionary genome-editing system will be discussed. Special attention will be given to the pivotal role of nanotechnology in tackling emerging infections such as coronavirus disease 2019 (COVID-19): ground-breaking mRNA vaccines delivered by NPs are currently used worldwide to fight the pandemic, pushing the boundaries of gene therapy.},
}

@article {pmid34509667,
year = {2021},
author = {Lee, BC and Lozano, RJ and Dunbar, CE},
title = {Understanding and Overcoming Adverse Consequences of Genome Editing on Hematopoietic Stem and Progenitor Cells.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2021.09.001},
pmid = {34509667},
issn = {1525-0024},
abstract = {Hematopoietic stem and progenitor cell (HSPC) gene therapies have recently moved beyond gene addition approaches to encompass targeted genome modification or correction, based on the development of zinc finger nucleases, TALENs and CRISPR/Cas technologies. Advances in ex vivo HSPC manipulation techniques have greatly improved HSPC susceptibility to genetic modification. Targeted gene editing techniques enable precise modifications at desired genomic sites. Numerous preclinical studies have already demonstrated the therapeutic potential of gene therapies based on targeted editing. However, several significant hurdles remain before broad clinical potential can be realized, related to adverse consequences of gene editing on HSPC function and genomic integrity. This review summarizes the status of HSPC gene editing, focusing on efficiency, genomic integrity, and long-term engraftment ability related to available genetic editing platforms and HSPC delivery methods. The response of long-term engrafting HSPCs to nuclease-mediated DNA breaks, with activation of p53, is a significant challenge, as are activation of innate and adaptive immune responses to editing components. Lastly, we propose alternative strategies that can overcome current hurdles to HSPC editing at various stages from cell collection to transplantation to facilitate successful clinical applications.},
}

@article {pmid34445569,
year = {2021},
author = {Sarkar, H and Toms, M and Moosajee, M},
title = {Involvement of Oxidative and Endoplasmic Reticulum Stress in RDH12-Related Retinopathies.},
journal = {International journal of molecular sciences},
volume = {22},
number = {16},
pages = {},
pmid = {34445569},
issn = {1422-0067},
support = {205174/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; //Retina UK/ ; },
mesh = {Alcohol Oxidoreductases/antagonists & inhibitors/genetics/*metabolism ; Animals ; *Apoptosis ; Autophagy ; CRISPR-Cas Systems ; *Endoplasmic Reticulum Stress ; HEK293 Cells ; Humans ; *Mutation ; *Oxidative Stress ; Retinal Diseases/etiology/metabolism/*pathology ; Zebrafish ; },
abstract = {Retinol dehydrogenase 12 (RDH12) is expressed in photoreceptor inner segments and catalyses the reduction of all-trans retinal (atRAL) to all-trans retinol (atROL), as part of the visual cycle. Mutations in RDH12 are primarily associated with autosomal recessive Leber congenital amaurosis. To further our understanding of the disease mechanisms, HEK-293 cell lines expressing wildtype (WT) and mutant RDH12 were created. The WT cells afforded protection from atRAL-induced toxicity and oxidative stress. Mutant RDH12 cells displayed reduced protein expression and activity, with an inability to protect cells from atRAL toxicity, inducing oxidative and endoplasmic reticulum (ER) stress, with upregulation of sXBP1, CHOP, and ATF4. Pregabalin, a retinal scavenger, attenuated atRAL-induced ER stress in the mutant RDH12 cell lines. A zebrafish rdh12 mutant model (rdh12u533 c.17_23del; p.(Val6AlafsTer5)) was generated through CRISPR-Cas9 gene editing. Mutant fish showed disrupted phagocytosis through transmission electron microscopy, with increased phagosome size at 12 months post-fertilisation. Rhodopsin mislocalisation and reduced expression of atg12 and sod2 indicated early signs of a rod-predominant degeneration. A lack of functional RDH12 results in ER and oxidative stress representing key pathways to be targeted for potential therapeutics.},
}

Alcohol Oxidoreductases/antagonists & inhibitors/genetics/*metabolism
Animals
*Apoptosis
Autophagy
CRISPR-Cas Systems
*Endoplasmic Reticulum Stress
HEK293 Cells
Humans
*Mutation
*Oxidative Stress
Retinal Diseases/etiology/metabolism/*pathology
Zebrafish

@article {pmid33830997,
year = {2021},
author = {Wallace, AD and Sasani, TA and Swanier, J and Gates, BL and Greenland, J and Pedersen, BS and Varley, KE and Quinlan, AR},
title = {CaBagE: A Cas9-based Background Elimination strategy for targeted, long-read DNA sequencing.},
journal = {PloS one},
volume = {16},
number = {4},
pages = {e0241253},
pmid = {33830997},
issn = {1932-6203},
support = {R01 GM124355/GM/NIGMS NIH HHS/United States ; R01 HG006693/HG/NHGRI NIH HHS/United States ; T32 HG008962/HG/NHGRI NIH HHS/United States ; },
mesh = {Amyotrophic Lateral Sclerosis/*genetics ; C9orf72 Protein/*genetics ; *CRISPR-Cas Systems ; *DNA Repeat Expansion ; *Genome, Human ; *High-Throughput Nucleotide Sequencing ; Humans ; *Microsatellite Repeats ; *Nanopores ; },
abstract = {A substantial fraction of the human genome is difficult to interrogate with short-read DNA sequencing technologies due to paralogy, complex haplotype structures, or tandem repeats. Long-read sequencing technologies, such as Oxford Nanopore's MinION, enable direct measurement of complex loci without introducing many of the biases inherent to short-read methods, though they suffer from relatively lower throughput. This limitation has motivated recent efforts to develop amplification-free strategies to target and enrich loci of interest for subsequent sequencing with long reads. Here, we present CaBagE, a method for target enrichment that is efficient and useful for sequencing large, structurally complex targets. The CaBagE method leverages the stable binding of Cas9 to its DNA target to protect desired fragments from digestion with exonuclease. Enriched DNA fragments are then sequenced with Oxford Nanopore's MinION long-read sequencing technology. Enrichment with CaBagE resulted in a median of 116X coverage (range 39-416) of target loci when tested on five genomic targets ranging from 4-20kb in length using healthy donor DNA. Four cancer gene targets were enriched in a single reaction and multiplexed on a single MinION flow cell. We further demonstrate the utility of CaBagE in two ALS patients with C9orf72 short tandem repeat expansions to produce genotype estimates commensurate with genotypes derived from repeat-primed PCR for each individual. With CaBagE there is a physical enrichment of on-target DNA in a given sample prior to sequencing. This feature allows adaptability across sequencing platforms and potential use as an enrichment strategy for applications beyond sequencing. CaBagE is a rapid enrichment method that can illuminate regions of the 'hidden genome' underlying human disease.},
}

Amyotrophic Lateral Sclerosis/*genetics
C9orf72 Protein/*genetics
*CRISPR-Cas Systems
*DNA Repeat Expansion
*Genome, Human
*High-Throughput Nucleotide Sequencing
Humans
*Microsatellite Repeats
*Nanopores

@article {pmid33667229,
year = {2021},
author = {Di Stazio, M and Foschi, N and Athanasakis, E and Gasparini, P and d'Adamo, AP},
title = {Systematic analysis of factors that improve homologous direct repair (HDR) efficiency in CRISPR/Cas9 technique.},
journal = {PloS one},
volume = {16},
number = {3},
pages = {e0247603},
pmid = {33667229},
issn = {1932-6203},
mesh = {*CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair/genetics ; DNA, Single-Stranded/genetics ; Gene Editing/*methods ; *Genome, Human ; HEK293 Cells ; Humans ; Mutation ; Nucleotides/genetics ; RNA, Guide/genetics ; Recombinational DNA Repair/*genetics ; Transfection ; Trinucleotide Repeats/genetics ; Tumor Necrosis Factor-alpha/*genetics ; },
abstract = {The CRISPR/Cas9 bacterial system has proven to be an powerful tool for genetic manipulation in several organisms, but the efficiency of sequence replacement by homologous direct repair (HDR) is substantially lower than random indel creation. Many studies focused on improving HDR efficiency using double sgRNA, cell synchronization cycle, and the delivery of single-stranded oligo DNA nucleotides (ssODN) with a rational design. In this study, we evaluate these three methods' synergistic effects to improve HDR efficiency. For our tests, we have chosen the TNFα gene (NM_000594) for its crucial role in various biological processes and diseases. For the first time, our results showed how the use of two sgRNA with asymmetric donor design and triple transfection events dramatically increase the HDR efficiency from an undetectable HDR event to 39% of HDR efficiency and provide a new strategy to facilitate CRISPR/Cas9-mediated human genome editing. Besides, we demonstrated that the TNFα locus could be edited with CRISPR/Cas9 methodology, an opportunity to safely correct, in the future, the specific mutations of each patient.},
}

*CRISPR-Cas Systems
DNA Breaks, Double-Stranded
DNA End-Joining Repair/genetics
DNA, Single-Stranded/genetics
Gene Editing/*methods
*Genome, Human
HEK293 Cells
Humans
Mutation
Nucleotides/genetics
RNA, Guide/genetics
Recombinational DNA Repair/*genetics
Transfection
Trinucleotide Repeats/genetics
Tumor Necrosis Factor-alpha/*genetics

@article {pmid33637689,
year = {2021},
author = {Baxter, PS and Márkus, NM and Dando, O and He, X and Al-Mubarak, BR and Qiu, J and Hardingham, GE},
title = {Targeted de-repression of neuronal Nrf2 inhibits α-synuclein accumulation.},
journal = {Cell death & disease},
volume = {12},
number = {2},
pages = {218},
pmid = {33637689},
issn = {2041-4889},
support = {/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; Astrocytes/metabolism/pathology ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Cell Death/drug effects ; Cells, Cultured ; Clustered Regularly Interspaced Short Palindromic Repeats ; Coculture Techniques ; Epigenetic Repression ; Female ; *Gene Targeting ; Hydroquinones/*pharmacology ; Lewy Body Disease/genetics/metabolism/pathology/*therapy ; Male ; Mice, Inbred C57BL ; Mice, Knockout ; NF-E2-Related Factor 2/*agonists/genetics/metabolism ; Neurons/drug effects/*metabolism/pathology ; Neuroprotective Agents/*pharmacology ; Prosencephalon/*drug effects/metabolism/pathology ; Proteostasis/drug effects ; alpha-Synuclein/genetics/*metabolism ; },
abstract = {Many neurodegenerative diseases are associated with neuronal misfolded protein accumulation, indicating a need for proteostasis-promoting strategies. Here we show that de-repressing the transcription factor Nrf2, epigenetically shut-off in early neuronal development, can prevent protein aggregate accumulation. Using a paradigm of α-synuclein accumulation and clearance, we find that the classical electrophilic Nrf2 activator tBHQ promotes endogenous Nrf2-dependent α-synuclein clearance in astrocytes, but not cortical neurons, which mount no Nrf2-dependent transcriptional response. Moreover, due to neuronal Nrf2 shut-off and consequent weak antioxidant defences, electrophilic tBHQ actually induces oxidative neurotoxicity, via Nrf2-independent Jun induction. However, we find that epigenetic de-repression of neuronal Nrf2 enables them to respond to Nrf2 activators to drive α-synuclein clearance. Moreover, activation of neuronal Nrf2 expression using gRNA-targeted dCas9-based transcriptional activation complexes is sufficient to trigger Nrf2-dependent α-synuclein clearance. Thus, targeting reversal of the developmental shut-off of Nrf2 in forebrain neurons may alter neurodegenerative disease trajectory by boosting proteostasis.},
}

Animals
Astrocytes/metabolism/pathology
CRISPR-Associated Protein 9/genetics/metabolism
*CRISPR-Cas Systems
Cell Death/drug effects
Cells, Cultured
Clustered Regularly Interspaced Short Palindromic Repeats
Coculture Techniques
Epigenetic Repression
Female
*Gene Targeting
Hydroquinones/*pharmacology
Lewy Body Disease/genetics/metabolism/pathology/*therapy
Male
Mice, Inbred C57BL
Mice, Knockout
NF-E2-Related Factor 2/*agonists/genetics/metabolism
Neurons/drug effects/*metabolism/pathology
Neuroprotective Agents/*pharmacology
Prosencephalon/*drug effects/metabolism/pathology
Proteostasis/drug effects
alpha-Synuclein/genetics/*metabolism

@article {pmid33328249,
year = {2021},
author = {Amici, DR and Jackson, JM and Truica, MI and Smith, RS and Abdulkadir, SA and Mendillo, ML},
title = {FIREWORKS: a bottom-up approach to integrative coessentiality network analysis.},
journal = {Life science alliance},
volume = {4},
number = {2},
pages = {},
pmid = {33328249},
issn = {2575-1077},
support = {PPO 16-135/HX/HSRD VA/United States ; R00 CA175293/CA/NCI NIH HHS/United States ; T32 GM008152/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Computational Biology/*methods ; *Gene Expression Regulation ; *Gene Regulatory Networks ; Gene Targeting ; Genetic Loci ; *Genomics/methods ; Humans ; Models, Biological ; *Software ; },
abstract = {Genetic coessentiality analysis, a computational approach which identifies genes sharing a common effect on cell fitness across large-scale screening datasets, has emerged as a powerful tool to identify functional relationships between human genes. However, widespread implementation of coessentiality to study individual genes and pathways is limited by systematic biases in existing coessentiality approaches and accessibility barriers for investigators without computational expertise. We created FIREWORKS, a method and interactive tool for the construction and statistical analysis of coessentiality networks centered around gene(s) provided by the user. FIREWORKS incorporates a novel bias reduction approach to reduce false discoveries, enables restriction of coessentiality analyses to custom subsets of cell lines, and integrates multiomic and drug-gene interaction datasets to investigate and target contextual gene essentiality. We demonstrate the broad utility of FIREWORKS through case vignettes investigating gene function and specialization, indirect therapeutic targeting of "undruggable" proteins, and context-specific rewiring of genetic networks.},
}

CRISPR-Cas Systems
Computational Biology/*methods
*Gene Expression Regulation
*Gene Regulatory Networks
Gene Targeting
Genetic Loci
*Genomics/methods
Humans
Models, Biological
*Software

@article {pmid32878767,
year = {2020},
author = {Lu, J and Chen, A and Ma, X and Shang, X and Zhang, Y and Guo, Y and Liu, M and Wang, X},
title = {Generation and Characterization of Cytochrome P450 2J3/10 CRISPR/Cas9 Knockout Rat Model.},
journal = {Drug metabolism and disposition: the biological fate of chemicals},
volume = {48},
number = {11},
pages = {1129-1136},
doi = {10.1124/dmd.120.000114},
pmid = {32878767},
issn = {1521-009X},
mesh = {Animals ; Astemizole/pharmacokinetics ; Biotransformation ; CRISPR-Cas Systems/genetics ; Cytochrome P-450 Enzyme System/genetics/*metabolism ; Drug Evaluation, Preclinical/methods ; Feasibility Studies ; Female ; Gene Knockdown Techniques ; Male ; Models, Animal ; Rats ; Rats, Transgenic ; },
abstract = {Cytochrome P450 2J2 (CYP2J2) enzyme attracts more attention because it not only metabolizes clinical drugs but also mediates the biotransformation of important endogenous substances and the regulation of physiologic function. Although CYP2J2 is very important, few animal models are available to study its function in vivo In particular, a CYP2J gene knockout (KO) rat model for drug metabolism and pharmacokinetics is not available. In this report, the CRISPR/Cas9 technology was used to delete rat CYP2J3/10, the orthologous genes of CYP2J2 in humans. The CYP2J3/10 KO rats were viable and fertile and showed no off-target effect. Compared with wild-type (WT) rats, the mRNA and protein expression of CYP2J3/10 in liver, small intestine, and heart of KO rats were completely absent. At the same time, CYP2J4 mRNA expression and protein expression were significantly decreased in these tissues. Further in vitro and in vivo metabolic studies of astemizole, a typical substrate of CYP2J, indicated that CYP2J was functionally inactive in KO rats. The heart function indexes of WT and KO rats were also measured and compared. The myocardial enzymes, including creatine kinase-muscle brain type (CK-MB), creatine kinase (CK), and CK-MB/CK ratio, of KO rats increased by nearly 140%, 80%, and 60%, respectively. In conclusion, this study successfully developed a new CYP2J3/10 KO rat model, which is a useful tool to study the function of CYP2J in drug metabolism and cardiovascular disease. SIGNIFICANCE STATEMENT: Human CYP2J2 is involved not only in clinical drug metabolism but also in the biotransformation of important endogenous substances. Therefore, it is very important to construct new animal models to study its function in vivo. This study successfully developed a new CYP2J knockout rat model by using CRISPR/Cas9 technology. This rat model provides a useful tool to study the role of CYP2J in drug metabolism and diseases.},
}

Animals
Astemizole/pharmacokinetics
Biotransformation
CRISPR-Cas Systems/genetics
Cytochrome P-450 Enzyme System/genetics/*metabolism
Drug Evaluation, Preclinical/methods
Feasibility Studies
Female
Gene Knockdown Techniques
Male
Models, Animal
Rats
Rats, Transgenic

@article {pmid32618081,
year = {2020},
author = {Guan, X and Zhang, H and Qin, H and Chen, C and Hu, Z and Tan, J and Zeng, L},
title = {CRISPR/Cas9-mediated whole genomic wide knockout screening identifies mitochondrial ribosomal proteins involving in oxygen-glucose deprivation/reperfusion resistance.},
journal = {Journal of cellular and molecular medicine},
volume = {24},
number = {16},
pages = {9313-9322},
pmid = {32618081},
issn = {1582-4934},
mesh = {*CRISPR-Cas Systems ; Gene Expression Regulation ; Glucose/*deficiency ; Humans ; Mitochondrial Proteins/*antagonists & inhibitors/genetics ; Neuroblastoma/genetics/metabolism/*pathology ; Oxidative Stress ; Oxygen/*metabolism ; Reperfusion Injury/*physiopathology ; Ribosomal Proteins/*antagonists & inhibitors/genetics ; Tumor Cells, Cultured ; },
abstract = {Recanalization therapy by intravenous thrombolysis or endovascular therapy is critical for the treatment of cerebral infarction. However, the recanalization treatment will also exacerbate acute brain injury and even severely threatens human life due to the reperfusion injury. So far, the underlying mechanisms for cerebral ischaemia-reperfusion injury are poorly understood and effective therapeutic interventions are yet to be discovered. Therefore, in the research, we subjected SK-N-BE(2) cells to oxygen-glucose deprivation/reperfusion (OGDR) insult and performed a pooled genome-wide CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) knockout screen to discover new potential therapeutic targets for cerebral ischaemia-reperfusion injury. We used Metascape to identify candidate genes which might involve in OGDR resistance. We found that the genes contributed to OGDR resistance were primarily involved in neutrophil degranulation, mitochondrial translation, and regulation of cysteine-type endopeptidase activity involved in apoptotic process and response to oxidative stress. We then knocked down some of the identified candidate genes individually. We demonstrated that MRPL19, MRPL32, MRPL52 and MRPL51 inhibition increased cell viability and attenuated OGDR-induced apoptosis. We also demonstrated that OGDR down-regulated the expression of MRPL19 and MRPL51 protein. Taken together, our data suggest that genome-scale screening with Cas9 is a reliable tool to analyse the cellular systems that respond to OGDR injury. MRPL19 and MRPL51 contribute to OGDR resistance and are supposed to be promising targets for the treatment of cerebral ischaemia-reperfusion damage.},
}

*CRISPR-Cas Systems
Gene Expression Regulation
Glucose/*deficiency
Humans
Mitochondrial Proteins/*antagonists & inhibitors/genetics
Neuroblastoma/genetics/metabolism/*pathology
Oxidative Stress
Oxygen/*metabolism
Reperfusion Injury/*physiopathology
Ribosomal Proteins/*antagonists & inhibitors/genetics
Tumor Cells, Cultured

@article {pmid32458463,
year = {2020},
author = {Tan, SI and Yu, PJ and Ng, IS},
title = {CRISPRi-mediated programming essential gene can as a Direct Enzymatic Performance Evaluation & Determination (DEPEND) system.},
journal = {Biotechnology and bioengineering},
volume = {117},
number = {9},
pages = {2842-2851},
doi = {10.1002/bit.27443},
pmid = {32458463},
issn = {1097-0290},
support = {MOST 108-2218-E-006-006//Ministry of Science and Technology, Taiwan/International ; MOST 108-2221-E-006-004-MY3//Ministry of Science and Technology, Taiwan/International ; MOST 108-2621-M-006-015//Ministry of Science and Technology, Taiwan/International ; },
mesh = {5-Aminolevulinate Synthetase/genetics/metabolism ; Biosensing Techniques/*methods ; CRISPR-Cas Systems/*genetics ; Carbonic Anhydrases/genetics/metabolism ; Escherichia coli/genetics ; Genes, Essential/*genetics ; RNA, Guide/genetics ; Recombinant Proteins/*genetics/metabolism ; },
abstract = {Harnessing enzyme expression for production of target chemicals is a critical and multifarious process, where screening of different genes by inspection of enzymatic activity plays an imperative role. Here, we conceived an idea to improve the time-consuming and labor-intensive process of enzyme screening. Controlling cell growth was achieved by the Cluster Regularly Interspaced Short Palindromic Repeat (CRISPRi) system with different single guide RNA targeting the essential gene can (CRISPRi::CA) that encodes a carbonic anhydrase for CO2 uptake. CRISPRi::CA comprises a whole-cell biosensor to monitor CO2 concentration, ranging from 1% to 5%. On the basis of CRISPRi::CA, an effective and simple Direct Enzymatic Performance Evaluation & Determination (DEPEND) system was developed by a single step of plasmid transformation for targeted enzymes. As a result, the activity of different carbonic anhydrases corresponded to the colony-forming units. Furthermore, the enzymatic performance of 5-aminolevulinic acid synthetase (ALAS), which converts glycine and succinate-CoA to release a molecule of CO2 , has also been distinguished, and the effect of the chaperone GroELS on ALAS enzyme folding was successfully identified in the DEPEND system. We provide a highly feasible, time-saving, and flexible technology for the screening and inspection of high-performance enzymes, which may accelerate protein engineering in the future.},
}

5-Aminolevulinate Synthetase/genetics/metabolism
Biosensing Techniques/*methods
CRISPR-Cas Systems/*genetics
Carbonic Anhydrases/genetics/metabolism
Escherichia coli/genetics
Genes, Essential/*genetics
RNA, Guide/genetics
Recombinant Proteins/*genetics/metabolism

@article {pmid32449788,
year = {2020},
author = {Zhang, F and Cheng, and Wang, S and Zhu, J},
title = {Crispr/Cas9-mediated cleavages facilitate homologous recombination during genetic engineering of a large chromosomal region.},
journal = {Biotechnology and bioengineering},
volume = {117},
number = {9},
pages = {2816-2826},
doi = {10.1002/bit.27441},
pmid = {32449788},
issn = {1097-0290},
support = {//Spokane County Health Sciences and Service Authority/International ; R01GM071725/NH/NIH HHS/United States ; R01GM071725/NH/NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; DNA Breaks, Double-Stranded ; Gene Editing/*methods ; Homologous Recombination/*genetics ; Humans ; Mice ; Plasmids/genetics ; },
abstract = {Homologous recombination over large genomic regions is difficult to achieve due to low efficiencies. Here, we report the successful engineering of a humanized mTert allele, hmTert, in the mouse genome by replacing an 18.1-kb genomic region around the mTert gene with a recombinant fragment of over 45.5 kb, using homologous recombination facilitated by the Crispr/Cas9 technology, in mouse embryonic stem cells (mESCs). In our experiments, with DNA double-strand breaks (DSBs) generated by Crispr/Cas9 system, the homologous recombination efficiency was up to 11% and 16% in two mESC lines TC1 and v6.5, respectively. Overall, we obtained a total of 27 mESC clones with heterozygous hmTert/mTert alleles and three clones with homozygous hmTert alleles. DSBs induced by Crispr/Cas9 cleavages also caused high rates of genomic DNA deletions and mutations at single-guide RNA target sites. Our results indicated that the Crispr/Cas9 system significantly increased the efficiency of homologous recombination-mediated gene editing over a large genomic region in mammalian cells, and also caused frequent mutations at unedited target sites. Overall, this strategy provides an efficient and feasible way for manipulating large chromosomal regions.},
}

Animals
CRISPR-Cas Systems/*genetics
DNA Breaks, Double-Stranded
Gene Editing/*methods
Homologous Recombination/*genetics
Humans
Mice
Plasmids/genetics

@article {pmid32437010,
year = {2020},
author = {Zhang, J and Hong, W and Guo, L and Wang, Y and Wang, Y},
title = {Enhancing plasmid transformation efficiency and enabling CRISPR-Cas9/Cpf1-based genome editing in Clostridium tyrobutyricum.},
journal = {Biotechnology and bioengineering},
volume = {117},
number = {9},
pages = {2911-2917},
doi = {10.1002/bit.27435},
pmid = {32437010},
issn = {1097-0290},
support = {2018-67021-27715//National Institute of Food and Agriculture/International ; ALA014-1017025//USDA-NIFA/International ; //Ocean University of China-Auburn University (OUC-AU) Grants/International ; //Auburn University Intramural Grants Program (IGP)/International ; //Alabama Agricultural Experiment Station/International ; },
mesh = {Batch Cell Culture Techniques ; Butyrates/metabolism ; CRISPR-Cas Systems/*genetics ; Clostridium tyrobutyricum/*genetics/metabolism ; Fermentation ; Gene Editing/*methods ; Plasmids/*genetics ; Transformation, Bacterial/*genetics ; },
abstract = {Clostridium tyrobutyricum ATCC 25755 is known as a natural hyper-butyrate producer with great potentials as an excellent platform to be engineered for valuable biochemical production from renewable resources. However, limited transformation efficiency and the lack of genetic manipulation tools have hampered the broader applications of this micro-organism. In this study, the effects of Type I restriction-modification system and native plasmid on conjugation efficiency of C. tyrobutyricum were investigated through gene deletion. The deletion of Type I restriction endonuclease resulted in a 3.7-fold increase in conjugation efficiency, while the additional elimination of the native plasmid further enhanced conjugation efficiency to 6.05 ± 0.75 × 103 CFU/ml-donor, which was 15.3-fold higher than the wild-type strain. Fermentation results indicated that the deletion of those two genetic elements did not significantly influence the end-products production in the resultant mutant ΔRMIΔNP. Thanks to the increased conjugation efficiency, the CRISPR-Cas9/Cpf1 systems, which previously could not be implemented in C. tyrobutyricum, were successfully employed for genome editing in ΔRMIΔNP with an efficiency of 12.5-25%. Altogether, approaches we developed herein offer valuable guidance for establishing efficient DNA transformation methods in nonmodel micro-organisms. The ΔRMIΔNP mutant can serve as a great chassis to be engineered for diverse valuable biofuel and biochemical production.},
}

Batch Cell Culture Techniques
Butyrates/metabolism
CRISPR-Cas Systems/*genetics
Clostridium tyrobutyricum/*genetics/metabolism
Fermentation
Gene Editing/*methods
Plasmids/*genetics
Transformation, Bacterial/*genetics

@article {pmid34506127,
year = {2021},
author = {Feng, W and Peng, H and Xu, J and Liu, Y and Pabbaraju, K and Tipples, G and Joyce, MA and Saffran, HA and Tyrrell, DL and Babiuk, S and Zhang, H and Le, XC},
title = {Integrating Reverse Transcription Recombinase Polymerase Amplification with CRISPR Technology for the One-Tube Assay of RNA.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.1c03456},
pmid = {34506127},
issn = {1520-6882},
abstract = {CRISPR-Cas systems integrated with nucleic acid amplification techniques improve both analytical specificity and sensitivity. We describe here issues and solutions for the successful integration of reverse transcription (RT), recombinase polymerase amplification (RPA), and CRISPR-Cas12a nuclease reactions into a single tube under an isothermal condition (40 °C). Specific detection of a few copies of a viral DNA sequence was achieved in less than 20 min. However, the sensitivity was orders of magnitude lower for the detection of viral RNA due to the slow initiation of RPA when the complementary DNA (cDNA) template remained hybridized to RNA. During the delay of RPA, the crRNA-Cas12a ribonucleoprotein (RNP) gradually lost its activity in the RPA solution, and nonspecific amplification reactions consumed the RPA reagents. We overcame these problems by taking advantage of the endoribonuclease function of RNase H to remove RNA from the RNA-cDNA hybrids and free the cDNA as template for the RPA reaction. As a consequence, we significantly enhanced the overall reaction rate of an integrated assay using RT-RPA and CRISPR-Cas12a for the detection of RNA. We showed successful detection of 200 or more copies of the S gene sequence of SARS-CoV-2 RNA within 5-30 min. We applied our one-tube assay to 46 upper respiratory swab samples for COVID-19 diagnosis, and the results from both fluorescence intensity measurements and end-point visualization were consistent with those of RT-qPCR analysis. The strategy and technique improve the sensitivity and speed of RT-RPA and CRISPR-Cas12a assays, potentially useful for both semi-quantitative and point-of-care analyses of RNA molecules.},
}

@article {pmid34505742,
year = {2021},
author = {Verosloff, MS and Shapiro, SJ and Hawkins, EM and Alpay, E and Verma, D and Stanfield, EG and Kreindler, L and Jain, S and McKay, B and Hubbell, SA and Hendriks, CG and Blizard, BA and Broughton, JP and Chen, JS},
title = {CRISPR-Cas enzymes: The toolkit revolutionizing diagnostics.},
journal = {Biotechnology journal},
volume = {},
number = {},
pages = {e2100304},
doi = {10.1002/biot.202100304},
pmid = {34505742},
issn = {1860-7314},
abstract = {The programmable nature of sequence-specific targeting by CRISPR-Cas nucleases has revolutionized a wide range of genomic applications and is now emerging as a method for nucleic acid detection. We explore how the diversity of CRISPR systems and their fundamental mechanisms have given rise to a wave of new methods for target recognition and readout. These cross-disciplinary advances found at the intersection of CRISPR biology and engineering have led to the ability to rapidly generate solutions for emerging global challenges like the COVID-19 pandemic. We further discuss the advances and potential for CRISPR-based detection to have an impact across a continuum of diagnostic applications. This article is protected by copyright. All rights reserved.},
}

@article {pmid34505269,
year = {2021},
author = {Stellon, D and Tran, MTN and Talbot, J and Chear, S and Khalid, MKNM and Pébay, A and Vickers, JC and King, AE and Hewitt, AW and Cook, AL},
title = {CRISPR/Cas-Mediated Knock-in of Genetically Encoded Fluorescent Biosensors into the AAVS1 Locus of Human-Induced Pluripotent Stem Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {},
number = {},
pages = {},
pmid = {34505269},
issn = {1940-6029},
abstract = {Genetically encoded fluorescent biosensors (GEFBs) enable researchers to visualize and quantify cellular processes in live cells. Induced pluripotent stem cells (iPSCs) can be genetically engineered to express GEFBs via integration into the Adeno-Associated Virus Integration Site 1 (AAVS1) safe harbor locus. This can be achieved using CRISPR/Cas ribonucleoprotein targeting to cause a double-strand break at the AAVS1 locus, which subsequently undergoes homology-directed repair (HDR) in the presence of a donor plasmid containing the GEFB sequence. We describe an optimized protocol for CRISPR/Cas-mediated knock-in of GEFBs into the AAVS1 locus of human iPSCs that allows puromycin selection and which exhibits negligible off-target editing. The resulting iPSC lines can be differentiated into cells of different lineages while retaining expression of the GEFB, enabling live-cell interrogation of cell pathway activities across a diversity of disease models.},
}

@article {pmid34505086,
year = {2021},
author = {Ghanta, KS and Ishidate, T and Mello, CC},
title = {Microinjection for precision genome editing in Caenorhabditis elegans.},
journal = {STAR protocols},
volume = {2},
number = {3},
pages = {100748},
doi = {10.1016/j.xpro.2021.100748},
pmid = {34505086},
issn = {2666-1667},
abstract = {In Caenorhabditis elegans, targeted genome editing techniques are now routinely used to generate germline edits. The remarkable ease of C. elegans germline editing is attributed to the syncytial nature of the pachytene ovary which is easily accessed by microinjection. This protocol describes the step-by-step details and troubleshooting tips for the entire CRISPR-Cas genome editing procedure, including gRNA design and microinjection of ribonucleoprotein complexes, followed by screening and genotyping in C. elegans, to help accessing this powerful genetic animal system. For complete details on the use and execution of this protocol, please refer to Ghanta and Mello (2020).},
}

@article {pmid34503839,
year = {2021},
author = {Toledo-Hernández, M and Lander, TA and Bao, C and Xie, K and Atta-Boateng, A and Wanger, TC},
title = {Genome-edited tree crops: mind the socioeconomic implementation gap.},
journal = {Trends in ecology & evolution},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tree.2021.08.007},
pmid = {34503839},
issn = {1872-8383},
abstract = {The discussion about CRISPR/Cas genome editing is focused mostly on technical aspects to improve productivity and climate resilience in major tree crops such as cocoa, coffee, and citrus. We suggest a solution to the largely ignored socioeconomic impacts for farmers, when new genome-edited varieties are introduced from the laboratory to the field.},
}

@article {pmid34502441,
year = {2021},
author = {Lyu, R and Ahmed, S and Fan, W and Yang, J and Wu, X and Zhou, W and Zhang, P and Yuan, L and Wang, H},
title = {Engineering Properties of Sweet Potato Starch for Industrial Applications by Biotechnological Techniques including Genome Editing.},
journal = {International journal of molecular sciences},
volume = {22},
number = {17},
pages = {},
doi = {10.3390/ijms22179533},
pmid = {34502441},
issn = {1422-0067},
support = {2018YFD1000705, 2019YFD1000703，2019YFD1000701-2，2019YFD1000704-2//National Key R&D Program of China/ ; Y819V11111//Youth Innovation Promotion Association of the Chinese Academy of Sciences/ ; G182402//Chenshan Special Fund for Shanghai Landscaping Administration Bureau Program/ ; 2019TQ0335//China Postdoctoral Science Foundation/ ; NA//Harold R. Burton Endowed Professorship/ ; NA//Postdoctoral fellowship from the University of Kentucky/ ; },
abstract = {Sweet potato (Ipomoea batatas) is one of the largest food crops in the world. Due to its abundance of starch, sweet potato is a valuable ingredient in food derivatives, dietary supplements, and industrial raw materials. In addition, due to its ability to adapt to a wide range of harsh climate and soil conditions, sweet potato is a crop that copes well with the environmental stresses caused by climate change. However, due to the complexity of the sweet potato genome and the long breeding cycle, our ability to modify sweet potato starch is limited. In this review, we cover the recent development in sweet potato breeding, understanding of starch properties, and the progress in sweet potato genomics. We describe the applicational values of sweet potato starch in food, industrial products, and biofuel, in addition to the effects of starch properties in different industrial applications. We also explore the possibility of manipulating starch properties through biotechnological means, such as the CRISPR/Cas-based genome editing. The ability to target the genome with precision provides new opportunities for reducing breeding time, increasing yield, and optimizing the starch properties of sweet potatoes.},
}

@article {pmid34502143,
year = {2021},
author = {Arango, D and Bittar, A and Esmeral, NP and Ocasión, C and Muñoz-Camargo, C and Cruz, JC and Reyes, LH and Bloch, NI},
title = {Understanding the Potential of Genome Editing in Parkinson's Disease.},
journal = {International journal of molecular sciences},
volume = {22},
number = {17},
pages = {},
doi = {10.3390/ijms22179241},
pmid = {34502143},
issn = {1422-0067},
support = {120484467666//Departamento Administrativo de Ciencia, Tecnología e Innovación (COLCIENCIAS)/ ; },
abstract = {CRISPR is a simple and cost-efficient gene-editing technique that has become increasingly popular over the last decades. Various CRISPR/Cas-based applications have been developed to introduce changes in the genome and alter gene expression in diverse systems and tissues. These novel gene-editing techniques are particularly promising for investigating and treating neurodegenerative diseases, including Parkinson's disease, for which we currently lack efficient disease-modifying treatment options. Gene therapy could thus provide treatment alternatives, revolutionizing our ability to treat this disease. Here, we review our current knowledge on the genetic basis of Parkinson's disease to highlight the main biological pathways that become disrupted in Parkinson's disease and their potential as gene therapy targets. Next, we perform a comprehensive review of novel delivery vehicles available for gene-editing applications, critical for their successful application in both innovative research and potential therapies. Finally, we review the latest developments in CRISPR-based applications and gene therapies to understand and treat Parkinson's disease. We carefully examine their advantages and shortcomings for diverse gene-editing applications in the brain, highlighting promising avenues for future research.},
}

@article {pmid34493846,
year = {2021},
author = {},
title = {License CRISPR patents for free to share gene editing globally.},
journal = {Nature},
volume = {597},
number = {7875},
pages = {152},
doi = {10.1038/d41586-021-02420-x},
pmid = {34493846},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; },
}

CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
*Gene Editing

@article {pmid34461727,
year = {2021},
author = {Shen, H and Qileng, A and Yang, H and Liang, H and Zhu, H and Liu, Y and Lei, H and Liu, W},
title = {"Dual-Signal-On" Integrated-Type Biosensor for Portable Detection of miRNA: Cas12a-Induced Photoelectrochemistry and Fluorescence Strategy.},
journal = {Analytical chemistry},
volume = {93},
number = {34},
pages = {11816-11825},
doi = {10.1021/acs.analchem.1c02395},
pmid = {34461727},
issn = {1520-6882},
mesh = {*Biosensing Techniques ; CRISPR-Cas Systems ; DNA ; Horseradish Peroxidase ; *MicroRNAs/genetics ; },
abstract = {The abnormal expression of microRNA (miRNA) can affect the RNA transcription and protein translation, leading to tumor progression and metastasis. Currently, the accurate detection of aberrant expression of miRNA, particularly using a portable detection system, remains a great challenge. Herein, a novel dual-mode biosensor with high sensitivity and robustness for miR-21 detection was developed based on the cis-cleavage and trans-cleavage activities of Cas12a. miRNA can be combined with hairpin DNA-horseradish peroxidase anchored on a CdS/g-C3N4/B-TiO2 photoelectrode, thus the nonenzymatic amplification was triggered to form numerous HRP-modified double-stranded DNA (HRP-dsDNA). Then, HRP-dsDNA can be specifically recognized and efficiently cis-cleaved by Cas12a nucleases to detach HRP from the substrate, while the remaining HRP on HRP-dsDNA can catalyze 4-chloro-1-naphthol (4-CN) to form biocatalytic precipitation (BCP) on the surface of the photoelectrode, and thus the photocurrent can be changed. Meanwhile, the trans-cleavage ability of Cas12a was activated, and nonspecifically degrade the FQ-reporter and a significant fluorescence signal can be generated. Such two different kinds of signals with independent transmission paths can mutually support to improve the performance of the detection platform. Besides, a portable device was constructed for the point-of-care (POC) detection of miR-21. Moreover, the dual-mode detection platform can be easily expanded for the specific detection of other types of biomarkers by changing the sequence of hairpin DNA, thereby promoting the establishment of POC detection for early cancer diagnosis.},
}

*Biosensing Techniques
CRISPR-Cas Systems
DNA
Horseradish Peroxidase
*MicroRNAs/genetics