@article {pmid31548319,
year = {2019},
author = {Velayutham, TS and Kumar, S and Zhang, X and Kose, N and Walker, DH and Winslow, G and Crowe, JE and McBride, JW},
title = {Ehrlichia chaffeensis Outer Membrane Protein 1-Specific Human Antibody-Mediated Immunity Is Defined by Intracellular TRIM21-Dependent Innate Immune Activation and Extracellular Neutralization.},
journal = {Infection and immunity},
volume = {87},
number = {12},
pages = {},
pmid = {31548319},
issn = {1098-5522},
mesh = {Adenine/analogs & derivatives/pharmacology ; Antibodies, Monoclonal/immunology ; Antibodies, Neutralizing/immunology ; Antigens, Bacterial/genetics/*immunology ; Autophagy/immunology ; Bacterial Adhesion/genetics ; Bacterial Outer Membrane Proteins/*genetics/*immunology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Ehrlichia chaffeensis/genetics/*immunology ; Gene Knockout Techniques ; Humans ; Immunity, Humoral/immunology ; NF-kappa B/genetics ; Ribonucleoproteins/*genetics ; THP-1 Cells ; },
abstract = {Antibodies are essential for immunity against Ehrlichia chaffeensis, and protective mechanisms involve blocking of ehrlichial attachment or complement and Fcγ-receptor-dependent destruction. In this study, we determined that major outer membrane protein 1 (OMP-19) hypervariable region 1 (HVR1)-specific human monoclonal antibodies (huMAbs) are protective through conventional extracellular neutralization and, more significantly, through a novel intracellular TRIM21-mediated mechanism. Addition of OMP-1-specific huMAb EHRL-15 (IgG1) prevented infection by blocking attachment/entry, a mechanism previously reported; conversely, OMP-1-specific huMAb EHRL-4 (IgG3) engaged intracellular TRIM21 and initiated an immediate innate immune response and rapid intracellular degradation of ehrlichiae. EHRL-4-TRIM21-mediated inhibition was significantly impaired in TRIM21 knockout THP-1 cells. EHRL-4 interacted with cytosolic Fc receptor TRIM21, observed by confocal microscopy and confirmed by co-immunoprecipitation. E. chaffeensis-EHRL-4-TRIM21 complexes caused significant upregulation of proinflammatory cytokine/chemokine transcripts and resulted in rapid (<30 min) nuclear accumulation of NF-κB and TRIM21 and ehrlichial destruction. We investigated the role of TRIM21 in the autophagic clearance of ehrlichiae in the presence of EHRL-4. Colocalization between EHRL-4-opsonized ehrlichiae, polyubiquitinated TRIM21, autophagy regulators (ULK1 and beclin 1) and effectors (LC3 and p62), and lysosome-associated membrane protein 2 (LAMP2) was observed. Moreover, autophagic flux defined by conversion of LC3I to LC3II and accumulation and degradation of p62 was detected, and EHRL-4-mediated degradation of E. chaffeensis was abrogated by the autophagy inhibitor 3-methyladenine. Our results demonstrate that huMAbs are capable of inhibiting E. chaffeensis infection by distinct effector mechanisms: extracellularly by neutralization and intracellularly by engaging TRIM21, which mediates a rapid innate immune response that mobilizes the core autophagy components, triggering localized selective autophagic degradation of ehrlichiae.},
}

Adenine/analogs & derivatives/pharmacology
Antibodies, Monoclonal/immunology
Antibodies, Neutralizing/immunology
Antigens, Bacterial/genetics/*immunology
Autophagy/immunology
Bacterial Adhesion/genetics
Bacterial Outer Membrane Proteins/*genetics/*immunology
CRISPR-Cas Systems
Cell Line, Tumor
Ehrlichia chaffeensis/genetics/*immunology
Gene Knockout Techniques
Humans
Immunity, Humoral/immunology
NF-kappa B/genetics
Ribonucleoproteins/*genetics
THP-1 Cells

@article {pmid31480315,
year = {2019},
author = {Zhang, S and Zhang, R and Gao, J and Gu, T and Song, G and Li, W and Li, D and Li, Y and Li, G},
title = {Highly Efficient and Heritable Targeted Mutagenesis in Wheat via the Agrobacteriumtumefaciens-Mediated CRISPR/Cas9 System.},
journal = {International journal of molecular sciences},
volume = {20},
number = {17},
pages = {},
doi = {10.3390/ijms20174257},
pmid = {31480315},
issn = {1422-0067},
support = {31601301, 31501312 and 31701428//National Natural Science Foundation of China/ ; LZ201905050290//Shandong Provincial Agricultural Variety Improvement Project/ ; 2017GNC10113//Key R&D Programme of Shandong Province/ ; 2018ZX08009-10B//the Ministry of Agriculture of China/ ; 2016YFD0100500//the Ministry of Science and Technology of China/ ; CXGC2016C09//the Agricultural Science and Technology Innovation Project of Shandong Academy of Agricultural Sciences/ ; without grant number//the Youth Talent Program of Shandong Academy of Agricultural Sciences/ ; },
mesh = {Agrobacterium/*metabolism ; Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Gene Editing ; Genes, Plant ; Genetic Vectors/metabolism ; Genotype ; Inheritance Patterns/*genetics ; Mutagenesis/*genetics ; Mutation Rate ; RNA, Guide/genetics ; Triticum/*genetics ; },
abstract = {The CRISPR/Cas9 system has been successfully used in hexaploid wheat. Although it has been reported that the induced mutations can be passed to the next generation, gene editing and transmission patterns in later generations still need to be studied. In this study, we demonstrated that the CRISPR/Cas9 system could achieve efficient mutagenesis in five wheat genes via Agrobacterium-mediated transformation of an sgRNA targeting the D genome, an sgRNA targeting both the A and B homologues and three tri-genome guides targeting the editing of all three homologues. High mutation rates and putative homozygous or biallelic mutations were observed in the T0 plants. The targeted mutations could be stably inherited by the next generation, and the editing efficiency of each mutant line increased significantly across generations. The editing types and inheritance of targeted mutagenesis were similar, which were not related to the targeted subgenome number. The presence of Cas9/sgRNA could cause new mutations in subsequent generations, while mutated lines without Cas9/sgRNA could retain the mutation type. Additionally, off-target mutations were not found in sequences that were highly homologous to the selected sgRNA sequences. Overall, the results suggested that CRISPR/Cas9-induced gene editing via Agrobacterium-mediated transformation plays important roles in wheat genome engineering.},
}

Agrobacterium/*metabolism
Base Sequence
CRISPR-Associated Protein 9/*metabolism
CRISPR-Cas Systems/*genetics
Gene Editing
Genes, Plant
Genetic Vectors/metabolism
Genotype
Inheritance Patterns/*genetics
Mutagenesis/*genetics
Mutation Rate
RNA, Guide/genetics
Triticum/*genetics

@article {pmid31446470,
year = {2019},
author = {Ren, C and Guo, Y and Gathunga, EK and Duan, W and Li, S and Liang, Z},
title = {Recovery of the non-functional EGFP-assisted identification of mutants generated by CRISPR/Cas9.},
journal = {Plant cell reports},
volume = {38},
number = {12},
pages = {1541-1549},
pmid = {31446470},
issn = {1432-203X},
support = {31772266//National Natural Science Foundation of China/ ; },
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/methods ; Genome, Plant/*genetics ; Green Fluorescent Proteins/genetics/metabolism ; Oxidoreductases/genetics/metabolism ; Plant Proteins/genetics/metabolism ; },
abstract = {KEY MESSAGE: The recovery of non-functional-enhanced green fluorescence protein can be used as indicator to facilitate the identification of mutants generated by CRISPR/Cas9. The CRISPR/Cas9 system is a powerful tool for genome editing and it has been employed to knock out genes of interest in multiple plant species. Identification of desired mutants from regenerated plants is necessary prior to functional study. Current screening methods work based on the purification of genomic DNA and it would be laborious and time consuming using these methods to screen mutants from a large population of seedlings. Here, we developed the non-functional enhanced green fluorescence protein (nEGFP) reporter gene by inserting a single guide RNA (sgRNA) and the protospacer adjacent motif in the 5' coding region of EGFP, and the activity of nEGFP could be recovered after successful targeted editing. Using the nEGFP as the reporter gene in Nicotiana tabacum, we found that over 94% of the plants exhibiting EGFP fluorescence were confirmed to be desired mutants. The use of this nEGFP reporter construct had limited negative effect on editing efficiency, and the expression of Cas9 and sgRNA was not affected. Moreover, this method was also applied in grape by targeting the phytoene desaturase gene (PDS), and the grape cells with EGFP signal were revealed to contain targeted mutations in VvPDS. Our results show that the nEGFP gene can be used as reporter to help screen mutants according to the recovered EGFP fluorescence during the application of CRISPR/Cas9 in plants.},
}

CRISPR-Cas Systems/*genetics
Gene Editing/methods
Genome, Plant/*genetics
Green Fluorescent Proteins/genetics/metabolism
Oxidoreductases/genetics/metabolism
Plant Proteins/genetics/metabolism

@article {pmid31352229,
year = {2019},
author = {Cai, C and Wang, X and Zhao, Y and Yi, C and Jin, Z and Zhang, A and Han, L},
title = {Construction of a mavs-inactivated MDCK cell line for facilitating the propagation of canine distemper virus (CDV).},
journal = {Molecular immunology},
volume = {114},
number = {},
pages = {133-138},
doi = {10.1016/j.molimm.2019.06.013},
pmid = {31352229},
issn = {1872-9142},
mesh = {Animals ; Antigens, CD/genetics ; CRISPR-Cas Systems/genetics ; Cell Line ; Chlorocebus aethiops ; Distemper/genetics ; Distemper Virus, Canine/*genetics ; Dogs ; Humans ; Madin Darby Canine Kidney Cells ; Vero Cells ; },
abstract = {Canine distemper is a highly contagious disease of wild and domestic carnivores. Obtaining of a suitable cell line for canine distemper virus (CDV) propagation is very important for field CDV isolation and vaccine antigen preparation. However, the cell line currently developed cell lines for CDV propagation are a marmoset lymphoid cell line (B95a), which could cause the virus to potentially infect human cells, and canine SLAM-expressing Vero cells, which may cause the virus to lose virulence. Therefore, a canine cell line constructed for efficient CDV propagation would be attractive. In the present study, a Madin-Darby Canine Kidney Epithelial (MDCK) cell line with mavs (mitochondrial antiviral signaling) inactivation was constructed by CRISPR/Cas9 technology. The interferon-I response induced by poly(I:C), an analogue of viral RNA, was significantly blocked in the constructed cell line, designated MDCK-KOmavs. Moreover, the propagation of a filed CDV strain was approximately 100 times higher in MDCK-KOmavs cells than in wild-type MDCK cells. Therefore, in the present study, a canine cell line facilitating CDV propagation was successfully constructed, and the results suggested that the constructed canine cell line was more efficient than the wild-type cell line for the isolation of field CDVs. In addition, the rapid propagation of CDVs to high titers in the constructed MDCK-KOmavs cell line indicated that this cell line could also be an alternative cell line for the preparation of vaccine antigens.},
}

Animals
Antigens, CD/genetics
CRISPR-Cas Systems/genetics
Cell Line
Chlorocebus aethiops
Distemper/genetics
Distemper Virus, Canine/*genetics
Dogs
Humans
Madin Darby Canine Kidney Cells
Vero Cells

@article {pmid31243056,
year = {2019},
author = {Ianiri, G and Dagotto, G and Sun, S and Heitman, J},
title = {Advancing Functional Genetics Through Agrobacterium-Mediated Insertional Mutagenesis and CRISPR/Cas9 in the Commensal and Pathogenic Yeast Malassezia.},
journal = {Genetics},
volume = {212},
number = {4},
pages = {1163-1179},
doi = {10.1534/genetics.119.302329},
pmid = {31243056},
issn = {1943-2631},
support = {R01 AI039115/AI/NIAID NIH HHS/United States ; R01 AI050113/AI/NIAID NIH HHS/United States ; R37 AI039115/AI/NIAID NIH HHS/United States ; },
mesh = {Agrobacterium/genetics ; *CRISPR-Cas Systems ; Drug Resistance, Fungal/genetics ; Gene Deletion ; Malassezia/*genetics ; *Mutagenesis ; Mutagenesis, Insertional ; Reverse Genetics/*methods ; Transformation, Genetic ; },
abstract = {Malassezia encompasses a monophyletic group of basidiomycetous yeasts naturally found on the skin of humans and other animals. Malassezia species have lost genes for lipid biosynthesis, and are therefore lipid-dependent and difficult to manipulate under laboratory conditions. In this study, we applied a recently-developed Agrobacterium tumefaciens-mediated transformation protocol to perform transfer (T)-DNA random insertional mutagenesis in Malassezia furfur A total of 767 transformants were screened for sensitivity to 10 different stresses, and 19 mutants that exhibited a phenotype different from the wild type were further characterized. The majority of these strains had single T-DNA insertions, which were identified within open reading frames of genes, untranslated regions, and intergenic regions. Some T-DNA insertions generated chromosomal rearrangements while others could not be characterized. To validate the findings of our forward genetic screen, a novel clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system was developed to generate targeted deletion mutants for two genes identified in the screen: CDC55 and PDR10 This system is based on cotransformation of M. furfur mediated by A. tumefaciens, to deliver both a CAS9-gRNA construct that induces double-strand DNA breaks and a gene replacement allele that serves as a homology-directed repair template. Targeted deletion mutants for both CDC55 and PDR10 were readily generated with this method. This study demonstrates the feasibility and reliability of A. tumefaciens-mediated transformation to aid in the identification of gene functions in M. furfur, through both insertional mutagenesis and CRISPR/Cas9-mediated targeted gene deletion.},
}

Agrobacterium/genetics
*CRISPR-Cas Systems
Drug Resistance, Fungal/genetics
Gene Deletion
Malassezia/*genetics
*Mutagenesis
Mutagenesis, Insertional
Reverse Genetics/*methods
Transformation, Genetic

@article {pmid30826063,
year = {2019},
author = {Chang, Y and Geng, F and Hu, Y and Ding, Y and Zhang, R},
title = {Zebrafish cysteine and glycine-rich protein 3 is essential for mechanical stability in skeletal muscles.},
journal = {Biochemical and biophysical research communications},
volume = {511},
number = {3},
pages = {604-611},
doi = {10.1016/j.bbrc.2019.02.115},
pmid = {30826063},
issn = {1090-2104},
mesh = {Animals ; Biomechanical Phenomena ; CRISPR-Cas Systems ; Gene Knockdown Techniques ; LIM Domain Proteins/genetics/*metabolism ; Muscle Proteins/genetics/*metabolism ; Muscle, Skeletal/growth & development/*metabolism/ultrastructure ; Mutation ; Stress, Mechanical ; Zebrafish/genetics/growth & development/*metabolism ; Zebrafish Proteins/genetics/*metabolism ; },
abstract = {Cysteine and glycine-rich protein 3 (CSRP3) is a striated muscle-specific cytoskeleton protein which participates in cardiac stretch sensing. Mutations in CSRP3 gene cause cardiomyopathies and deregulation of CSRP3 has been found in patients with heart failure and several skeletal muscle diseases. However, the mechanism underneath these disorders still remains poorly understood. Here we generated the first csrp3 knockout zebrafish. csrp3-/- embryos showed no gross morphological defects but csrp3 deficient skeletal muscle fibers were prone to lesions upon prolonged stretching force. Further studies revealed csrp3 cooperatively interacted with ilk to maintain skeletal muscle mechanical stability and regulated tcap activation. Thus, our work has established a zebrafish model to investigate the function of csrp3 gene, and provides novel insights towards how csrp3 defects may lead to skeletal myopathies by a mechanistic link between Csrp3 and force stimuli.},
}

Animals
Biomechanical Phenomena
CRISPR-Cas Systems
Gene Knockdown Techniques
LIM Domain Proteins/genetics/*metabolism
Muscle Proteins/genetics/*metabolism
Muscle, Skeletal/growth & development/*metabolism/ultrastructure
Mutation
Stress, Mechanical
Zebrafish/genetics/growth & development/*metabolism
Zebrafish Proteins/genetics/*metabolism

@article {pmid30745494,
year = {2019},
author = {Tobita, T and Kiyozumi, D and Muto, M and Noda, T and Ikawa, M},
title = {Lvrn expression is not critical for mouse placentation.},
journal = {The Journal of reproduction and development},
volume = {65},
number = {3},
pages = {239-244},
doi = {10.1262/jrd.2018-157},
pmid = {30745494},
issn = {1348-4400},
mesh = {Animals ; Blood Pressure ; CRISPR-Cas Systems ; Female ; Fetal Growth Retardation/metabolism ; Fetus/metabolism ; Gene Expression Profiling ; *Gene Expression Regulation, Developmental ; Lentivirus/metabolism ; Metalloproteases/genetics/*physiology ; Mice ; Mice, Knockout ; Placenta/*physiology ; *Placentation ; Pre-Eclampsia ; Pregnancy ; Pregnancy, Animal ; Trophoblasts/metabolism ; },
abstract = {Preeclampsia is a systemic disease caused by abnormal placentation that affects both mother and fetus. It was reported that Laeverin (LVRN, also known as Aminopeptidase Q) was up-regulated in the placenta of preeclamptic patients. However, physiological and pathological functions of LVRN remained to be unknown. Here we characterized Lvrn function during placentation in mice. RT-PCR showed that Lvrn is expressed in both fetus and placenta during embryogenesis, and several adult tissues. When we overexpressed Lvrn in a placenta-specific manner using lentiviral vectors, we did not see any defects in both placentae and fetuses. The mice carrying Lvrn overexpressing placentas did not show any preeclampsia-like symptoms such as maternal high blood pressure and fetal growth restriction. We next ablated Lvrn by CRISPR/Cas9-mediated genome editing to see physiological function. In Lvrn ablated mice, maternal blood pressure during pregnancy was not affected, and both placentas and fetuses grew normally. Collectively, these results suggest that, LVRN is irrelevant to preeclampsia and dispensable for normal placentation and embryonic development in mice.},
}

Animals
Blood Pressure
CRISPR-Cas Systems
Female
Fetal Growth Retardation/metabolism
Fetus/metabolism
Gene Expression Profiling
*Gene Expression Regulation, Developmental
Lentivirus/metabolism
Metalloproteases/genetics/*physiology
Mice
Mice, Knockout
Placenta/*physiology
*Placentation
Pre-Eclampsia
Pregnancy
Pregnancy, Animal
Trophoblasts/metabolism

@article {pmid30726783,
year = {2019},
author = {Tanihara, F and Hirata, M and Nguyen, NT and LE, QA and Hirano, T and Otoi, T},
title = {Effects of concentration of CRISPR/Cas9 components on genetic mosaicism in cytoplasmic microinjected porcine embryos.},
journal = {The Journal of reproduction and development},
volume = {65},
number = {3},
pages = {209-214},
doi = {10.1262/jrd.2018-116},
pmid = {30726783},
issn = {1348-4400},
mesh = {Alleles ; Animals ; Animals, Genetically Modified ; Blastocyst/cytology ; *CRISPR-Cas Systems ; Cytoplasm/*genetics ; Embryonic Development ; Female ; Fertilization in Vitro ; Gene Editing ; Male ; Microinjections ; *Mosaicism ; Mutation ; Nucleotides/genetics ; Oocytes/cytology ; Swine ; Zygote ; },
abstract = {Cytoplasmic microinjection (CI) of the CRISPR/Cas9 system enabled the induction of site-specific mutations in porcine zygotes and resulting pigs. However, mosaicism is a serious problem for genetically modified pigs. In the present study, we investigated suitable timing and concentration of CRISPR/Cas9 components for introduction into oocytes/zygotes by CI, to reduce mosaicism in the resulting blastocysts. First, we introduced 20 ng/μl of Cas9 protein and guide RNA (gRNA), targeting the α-1,3-galactosyltransferase (GalT) gene in oocytes before in vitro fertilization (IVF), in zygotes after IVF, or in oocytes/zygotes before and after IVF, twice. CI treatment had no detrimental effects on blastocyst formation rates. The highest value of the rate of mutant blastocysts was observed in zygotes injected after IVF. Next, we injected Cas9 protein and gRNA into zygotes after IVF at a concentration of 20 ng/μl each (20 ng/μl group) or 100 ng/μl each (100 ng/μl group). The ratio of the number of blastocysts that carried mutations to the total number of blastocysts examined in the 100 ng/μl group was significantly higher (P < 0.05) than that in the 20 ng/μl group. Although no blastocysts from the 20 ng/μl group carried a biallelic mutation, 16.7% of blastocysts from the 100 ng/μl group carried a biallelic mutation. In conclusion, increasing the concentration of Cas9 protein and gRNA is effective in generating biallelic mutant blastocysts. To reduce mosaicism, however, further optimization of the timing of CI, and the concentration of CRISPR/Cas9 components, is needed.},
}

Alleles
Animals
Animals, Genetically Modified
Blastocyst/cytology
*CRISPR-Cas Systems
Cytoplasm/*genetics
Embryonic Development
Female
Fertilization in Vitro
Gene Editing
Male
Microinjections
*Mosaicism
Mutation
Nucleotides/genetics
Oocytes/cytology
Swine
Zygote

@article {pmid30681833,
year = {2019},
author = {Tian, T and Kang, JW and Kang, A and Lee, TS},
title = {Redirecting Metabolic Flux via Combinatorial Multiplex CRISPRi-Mediated Repression for Isopentenol Production in Escherichia coli.},
journal = {ACS synthetic biology},
volume = {8},
number = {2},
pages = {391-402},
doi = {10.1021/acssynbio.8b00429},
pmid = {30681833},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Escherichia coli/genetics/*metabolism ; Gene Expression Regulation, Bacterial ; Metabolic Engineering/methods ; Pentanols/*metabolism ; Promoter Regions, Genetic/genetics ; },
abstract = {CRISPR interference (CRISPRi) via target guide RNA (gRNA) arrays and a deactivated Cas9 (dCas9) protein has been shown to simultaneously repress expression of multiple genomic DNA loci. By knocking down endogenous genes in competing pathways, CRISPRi technology can be utilized to redirect metabolic flux toward target metabolite. In this study, we constructed a CRISPRi-mediated multiplex repression system to silence transcription of several endogenous genes to increase precursor availability in a heterologous isopentenol biosynthesis pathway. To identify genomic knockdown targets in competing pathways, we first designed a single-gRNA library with 15 individual targets, where 3 gRNA cassettes targeting gene asnA, prpE, and gldA increased isopentenol titer by 18-24%. We then combined the 3 single-gRNA cassettes into a two- or three-gRNA array and observed up to 98% enhancement in production by fine-tuning the repression level through titrating dCas9 expression. Our strategy shows that multiplex combinatorial knockdown of competing genes using CRISPRi can increase production of the target metabolite, while the repression level needs to be adjusted to balance the metabolic network and achieve the maximum titer improvement.},
}

CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Escherichia coli/genetics/*metabolism
Gene Expression Regulation, Bacterial
Metabolic Engineering/methods
Pentanols/*metabolism
Promoter Regions, Genetic/genetics

@article {pmid30616338,
year = {2019},
author = {Shi, TQ and Gao, J and Wang, WJ and Wang, KF and Xu, GQ and Huang, H and Ji, XJ},
title = {CRISPR/Cas9-Based Genome Editing in the Filamentous Fungus Fusarium fujikuroi and Its Application in Strain Engineering for Gibberellic Acid Production.},
journal = {ACS synthetic biology},
volume = {8},
number = {2},
pages = {445-454},
doi = {10.1021/acssynbio.8b00478},
pmid = {30616338},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/*genetics ; Fungi/*genetics/*metabolism ; Fusarium/*genetics/*metabolism ; Gene Editing/methods ; Genome, Fungal/genetics ; Gibberellins/*metabolism ; },
abstract = {The filamentous fungus Fusarium fujikuroi is well-known for its production of natural plant growth hormones: a series of gibberellic acids (GAs). Some GAs, including GA1, GA3, GA4, and GA7, are biologically active and have been widely applied in agriculture. However, the low efficiency of traditional genetic tools limits the further research toward making this fungus more efficient and able to produce tailor-made GAs. Here, we established an efficient CRISPR/Cas9-based genome editing tool for F. fujikuroi. First, we compared three different nuclear localization signals (NLS) and selected an efficient NLS from histone H2B (HTBNLS) to enable the import of the Cas9 protein into the fungal nucleus. Then, different sgRNA expression strategies, both in vitro and different promoter-based in vivo strategies, were explored. The promoters of the U6 small nuclear RNA and 5S rRNA, which were identified in F. fujikuroi, had the highest editing efficiency. The 5S rRNA-promoter-driven genome editing efficiency reached up to 79.2%. What's more, multigene editing was also explored and showed good results. Finally, we used the developed genome editing tool to engineer the metabolic pathways responsible for the accumulation of a series GAs in the filamentous fungus F. fujikuroi, and successfully changed its GA product profile, from GA3 to tailor-made GA4 and GA7 mixtures. Since these mixtures are more efficient for agricultural use, especially for fruit growth, the developed strains will greatly improve industrial GA production.},
}

CRISPR-Cas Systems/*genetics
Fungi/*genetics/*metabolism
Fusarium/*genetics/*metabolism
Gene Editing/methods
Genome, Fungal/genetics
Gibberellins/*metabolism

@article {pmid31649350,
year = {2019},
author = {Opar, A},
title = {CRISPR-edited babies arrived, and regulators are still racing to catch up.},
journal = {Nature medicine},
volume = {25},
number = {11},
pages = {1634-1636},
doi = {10.1038/s41591-019-0641-x},
pmid = {31649350},
issn = {1546-170X},
mesh = {CRISPR-Cas Systems/*genetics ; Cloning, Organism/ethics ; Gene Editing/*ethics ; Humans ; Infant ; },
}

CRISPR-Cas Systems/*genetics
Cloning, Organism/ethics
Gene Editing/*ethics
Humans
Infant

@article {pmid31465436,
year = {2019},
author = {Bradford, J and Perrin, D},
title = {A benchmark of computational CRISPR-Cas9 guide design methods.},
journal = {PLoS computational biology},
volume = {15},
number = {8},
pages = {e1007274},
doi = {10.1371/journal.pcbi.1007274},
pmid = {31465436},
issn = {1553-7358},
mesh = {Animals ; Benchmarking/methods/statistics & numerical data ; *CRISPR-Cas Systems ; Computational Biology ; Databases, Nucleic Acid/statistics & numerical data ; Gene Editing/*methods/standards/statistics & numerical data ; Mice ; RNA, Guide/*genetics ; Software ; },
abstract = {The popularity of CRISPR-based gene editing has resulted in an abundance of tools to design CRISPR-Cas9 guides. This is also driven by the fact that designing highly specific and efficient guides is a crucial, but not trivial, task in using CRISPR for gene editing. Here, we thoroughly analyse the performance of 18 design tools. They are evaluated based on runtime performance, compute requirements, and guides generated. To achieve this, we implemented a method for auditing system resources while a given tool executes, and tested each tool on datasets of increasing size, derived from the mouse genome. We found that only five tools had a computational performance that would allow them to analyse an entire genome in a reasonable time, and without exhausting computing resources. There was wide variation in the guides identified, with some tools reporting every possible guide while others filtered for predicted efficiency. Some tools also failed to exclude guides that would target multiple positions in the genome. We also considered two collections with over a thousand guides each, for which experimental data is available. There is a lot of variation in performance between the datasets, but the relative order of the tools is partially conserved. Importantly, the most striking result is a lack of consensus between the tools. Our results show that CRISPR-Cas9 guide design tools need further work in order to achieve rapid whole-genome analysis and that improvements in guide design will likely require combining multiple approaches.},
}

Animals
Benchmarking/methods/statistics & numerical data
*CRISPR-Cas Systems
Computational Biology
Databases, Nucleic Acid/statistics & numerical data
Gene Editing/*methods/standards/statistics & numerical data
Mice
RNA, Guide/*genetics
Software

@article {pmid31449515,
year = {2019},
author = {Craig, M and Kaveh, K and Woosley, A and Brown, AS and Goldman, D and Eton, E and Mehta, RM and Dhawan, A and Arai, K and Rahman, MM and Chen, S and Nowak, MA and Goldman, A},
title = {Cooperative adaptation to therapy (CAT) confers resistance in heterogeneous non-small cell lung cancer.},
journal = {PLoS computational biology},
volume = {15},
number = {8},
pages = {e1007278},
doi = {10.1371/journal.pcbi.1007278},
pmid = {31449515},
issn = {1553-7358},
support = {DP5 OD019851/OD/NIH HHS/United States ; },
mesh = {Adaptation, Physiological/genetics ; CRISPR-Cas Systems ; Carcinoma, Non-Small-Cell Lung/*drug therapy/genetics/*physiopathology ; Cell Line, Tumor ; Cell Proliferation/drug effects/genetics/physiology ; Coculture Techniques ; Computational Biology ; Computer Simulation ; DEAD-box RNA Helicases/genetics ; Drug Resistance, Multiple/genetics ; Drug Resistance, Neoplasm/genetics ; Humans ; Lung Neoplasms/*drug therapy/genetics/*physiopathology ; Models, Biological ; Mutation ; Ribonuclease III/genetics ; },
abstract = {Understanding intrinsic and acquired resistance is crucial to overcoming cancer chemotherapy failure. While it is well-established that intratumor, subclonal genetic and phenotypic heterogeneity significantly contribute to resistance, it is not fully understood how tumor sub-clones interact with each other to withstand therapy pressure. Here, we report a previously unrecognized behavior in heterogeneous tumors: cooperative adaptation to therapy (CAT), in which cancer cells induce co-resistant phenotypes in neighboring cancer cells when exposed to cancer therapy. Using a CRISPR/Cas9 toolkit we engineered phenotypically diverse non-small cell lung cancer (NSCLC) cells by conferring mutations in Dicer1, a type III cytoplasmic endoribonuclease involved in small non-coding RNA genesis. We monitored three-dimensional growth dynamics of fluorescently-labeled mutant and/or wild-type cells individually or in co-culture using a substrate-free NanoCulture system under unstimulated or drug pressure conditions. By integrating mathematical modeling with flow cytometry, we characterized the growth patterns of mono- and co-cultures using a mathematical model of intra- and interspecies competition. Leveraging the flow cytometry data, we estimated the model's parameters to reveal that the combination of WT and mutants in co-cultures allowed for beneficial growth in previously drug sensitive cells despite drug pressure via induction of cell state transitions described by a cooperative game theoretic change in the fitness values. Finally, we used an ex vivo human tumor model that predicts clinical response through drug sensitivity analyses and determined that cellular and morphologic heterogeneity correlates to prognostic failure of multiple clinically-approved and off-label drugs in individual NSCLC patient samples. Together, these findings present a new paradox in drug resistance implicating non-genetic cooperation among tumor cells to thwart drug pressure, suggesting that profiling for druggable targets (i.e. mutations) alone may be insufficient to assign effective therapy.},
}

Adaptation, Physiological/genetics
CRISPR-Cas Systems
Carcinoma, Non-Small-Cell Lung/*drug therapy/genetics/*physiopathology
Cell Line, Tumor
Cell Proliferation/drug effects/genetics/physiology
Coculture Techniques
Computational Biology
Computer Simulation
DEAD-box RNA Helicases/genetics
Drug Resistance, Multiple/genetics
Drug Resistance, Neoplasm/genetics
Humans
Lung Neoplasms/*drug therapy/genetics/*physiopathology
Models, Biological
Mutation
Ribonuclease III/genetics

@article {pmid31131829,
year = {2019},
author = {Mangeot, PE},
title = {Nanoblades: Pseudoviral shuttles for CRISPR-CAS9 delivery.},
journal = {Virologie (Montrouge, France)},
volume = {23},
number = {1},
pages = {3-6},
doi = {10.1684/vir.2019.0759},
pmid = {31131829},
issn = {1267-8694},
mesh = {Biomedical Research/methods/trends ; CRISPR-Cas Systems/*genetics ; Drug Delivery Systems/methods/trends ; *Gene Editing/methods/trends ; *Gene Transfer Techniques ; Genetic Vectors/chemistry/*physiology ; Humans ; Nanotechnology/methods/trends ; Virion/*physiology ; Virus Assembly/physiology ; },
}

Biomedical Research/methods/trends
CRISPR-Cas Systems/*genetics
Drug Delivery Systems/methods/trends
*Gene Editing/methods/trends
*Gene Transfer Techniques
Genetic Vectors/chemistry/*physiology
Humans
Nanotechnology/methods/trends
Virion/*physiology
Virus Assembly/physiology

@article {pmid31094413,
year = {2019},
author = {Anower-E-Khuda, F and Singh, G and Deng, Y and Gordts, PLSM and Esko, JD},
title = {Triglyceride-rich lipoprotein binding and uptake by heparan sulfate proteoglycan receptors in a CRISPR/Cas9 library of Hep3B mutants.},
journal = {Glycobiology},
volume = {29},
number = {8},
pages = {582-592},
doi = {10.1093/glycob/cwz037},
pmid = {31094413},
issn = {1460-2423},
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Cells, Cultured ; Hepatocytes/metabolism ; Humans ; Lipoproteins/chemistry/*metabolism ; *Loss of Function Mutation ; N-Acetylglucosaminyltransferases/genetics/*metabolism ; Protein Binding ; Sulfotransferases/genetics/*metabolism ; Syndecan-1/genetics/metabolism ; Triglycerides/chemistry/metabolism ; },
abstract = {Binding and uptake of triglyceride-rich lipoproteins (TRLs) in mice depend on heparan sulfate and the hepatic proteoglycan, syndecan-1 (SDC1). Alteration of glucosamine N-sulfation by deletion of glucosamine N-deacetylase-N-sulfotransferase 1 (Ndst1) and 2-O-sulfation of uronic acids by deletion of uronyl 2-O-sulfotransferase (Hs2st) led to diminished lipoprotein metabolism, whereas inactivation of glucosaminyl 6-O-sulfotransferase 1 (Hs6st1), which encodes one of the three 6-O-sulfotransferases, had little effect on lipoprotein binding. However, other studies have suggested that 6-O-sulfation may be important for TRL binding and uptake. In order to explain these discrepant findings, we used CRISPR/Cas9 gene editing to create a library of mutants in the human hepatoma cell line, Hep3B. Inactivation of EXT1 encoding the heparan sulfate copolymerase, NDST1 and HS2ST dramatically reduced binding of TRLs. Inactivation of HS6ST1 had no effect, but deletion of HS6ST2 reduced TRL binding. Compounding mutations in HS6ST1 and HS6ST2 did not exacerbate this effect indicating that HS6ST2 is the dominant 6-O-sulfotransferase and that binding of TRLs indeed depends on 6-O-sulfation of glucosamine residues. Uptake studies showed that TRL internalization was also affected in 6-O-sulfation deficient cells. Interestingly, genetic deletion of SDC1 only marginally impacted binding of TRLs but reduced TRL uptake to the same extent as treating the cells with heparin lyases. These findings confirm that SDC1 is the dominant endocytic proteoglycan receptor for TRLs in human Hep3B cells and that binding and uptake of TRLs depend on SDC1 and N- and 2-O-sulfation as well as 6-O-sulfation of heparan sulfate chains catalyzed by HS6ST2.},
}

CRISPR-Cas Systems
Cell Line, Tumor
Cells, Cultured
Hepatocytes/metabolism
Humans
Lipoproteins/chemistry/*metabolism
*Loss of Function Mutation
N-Acetylglucosaminyltransferases/genetics/*metabolism
Protein Binding
Sulfotransferases/genetics/*metabolism
Syndecan-1/genetics/metabolism
Triglycerides/chemistry/metabolism

@article {pmid30375064,
year = {2019},
author = {Takeda, Y and Suzuki, S and Tobimatsu, Y and Osakabe, K and Osakabe, Y and Ragamustari, SK and Sakamoto, M and Umezawa, T},
title = {Lignin characterization of rice CONIFERALDEHYDE 5-HYDROXYLASE loss-of-function mutants generated with the CRISPR/Cas9 system.},
journal = {The Plant journal : for cell and molecular biology},
volume = {97},
number = {3},
pages = {543-554},
doi = {10.1111/tpj.14141},
pmid = {30375064},
issn = {1365-313X},
mesh = {Acrolein/*analogs & derivatives/metabolism ; Biomass ; CRISPR-Cas Systems ; Cell Wall/metabolism ; Lignin/*metabolism ; Loss of Function Mutation ; Mixed Function Oxygenases/genetics/*metabolism ; Oryza/enzymology/*genetics ; Plant Proteins/genetics/metabolism ; Propionates/metabolism ; },
abstract = {The aromatic composition of lignin is an important trait that greatly affects the usability of lignocellulosic biomass. We previously identified a rice (Oryza sativa) gene encoding coniferaldehyde 5-hydroxylase (OsCAld5H1), which was effective in modulating syringyl (S)/guaiacyl (G) lignin composition ratio in rice, a model grass species. Previously characterized OsCAld5H1-knockdown rice lines, which were produced via an RNA-interference approach, showed augmented G lignin units yet contained considerable amounts of residual S lignin units. In this study, to further investigate the effect of suppression of OsCAld5H1 on rice lignin structure, we generated loss-of-function mutants of OsCAld5H1 using the CRISPR/Cas9-mediated genome editing system. Homozygous OsCAld5H1-knockout lines harboring anticipated frame-shift mutations in OsCAld5H1 were successfully obtained. A series of wet-chemical and two-dimensional NMR analyses on cell walls demonstrated that although lignins in the mutant were predictably enriched in G units all the tested mutant lines produced considerable numbers of S units. Intriguingly, lignin γ-p-coumaroylation analysis by the derivatization followed by reductive cleavage method revealed that enrichment of G units in lignins of the mutants was limited to the non-γ-p-coumaroylated units, whereas grass-specific γ-p-coumaroylated lignin units were almost unaffected. Gene expression analysis indicated that no homologous genes of OsCAld5H1 were overexpressed in the mutants. These data suggested that CAld5H is mainly involved in the production of non-γ-p-coumaroylated S lignin units, common in both eudicots and grasses, but not in the production of grass-specific γ-p-coumaroylated S units in rice.},
}

Acrolein/*analogs & derivatives/metabolism
Biomass
CRISPR-Cas Systems
Cell Wall/metabolism
Lignin/*metabolism
Loss of Function Mutation
Mixed Function Oxygenases/genetics/*metabolism
Oryza/enzymology/*genetics
Plant Proteins/genetics/metabolism
Propionates/metabolism

@article {pmid31954772,
year = {2020},
author = {Beisel, CL},
title = {Methods for characterizing, applying, and teaching CRISPR-Cas systems.},
journal = {Methods (San Diego, Calif.)},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymeth.2020.01.004},
pmid = {31954772},
issn = {1095-9130},
}

@article {pmid31947593,
year = {2020},
author = {Jia, Y and Yang, B and Ross, P and Stanton, C and Zhang, H and Zhao, J and Chen, W},
title = {Comparative Genomics Analysis of Lactobacillus mucosae from Different Niches.},
journal = {Genes},
volume = {11},
number = {1},
pages = {},
doi = {10.3390/genes11010095},
pmid = {31947593},
issn = {2073-4425},
support = {Nos. 31771953, 31820103010//National Natural Science Foundation of China/ ; JUFSTR20180102//Food Science and Technology/ ; Not applicable//Collaborative innovation center of food safety and quality control in Jiangsu Province/ ; },
abstract = {The potential probiotic benefits of Lactobacillus mucosae have received increasing attention. To investigate the genetic diversity of L. mucosae, comparative genomic analyses of 93 strains isolated from different niches (human and animal gut, human vagina, etc.) and eight strains of published genomes were conducted. The results showed that the core genome of L. mucosae mainly encoded translation and transcription, amino acid biosynthesis, sugar metabolism, and defense function while the pan-genomic curve tended to be close. The genetic diversity of L. mucosae mainly reflected in carbohydrate metabolism and immune/competitive-related factors, such as exopolysaccharide (EPS), enterolysin A, and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas. It was worth noting that this research firstly predicted the complete EPS operon shared among L. mucosae. Additionally, the type IIIA CRISPR-Cas system was discovered in L. mucosae for the first time. This work provided new ideas for the study of this species.},
}

@article {pmid31028078,
year = {2019},
author = {Egorova, TV and Zotova, ED and Reshetov, DA and Polikarpova, AV and Vassilieva, SG and Vlodavets, DV and Gavrilov, AA and Ulianov, SV and Buchman, VL and Deykin, AV},
title = {CRISPR/Cas9-generated mouse model of Duchenne muscular dystrophy recapitulating a newly identified large 430 kb deletion in the human DMD gene.},
journal = {Disease models & mechanisms},
volume = {12},
number = {4},
pages = {},
doi = {10.1242/dmm.037655},
pmid = {31028078},
issn = {1754-8411},
mesh = {Animals ; Base Pairing/*genetics ; Biomechanical Phenomena ; CRISPR-Cas Systems/*genetics ; Cell Line ; Child ; Chromatin/metabolism ; Disease Models, Animal ; Dystrophin/*genetics ; Exons/genetics ; Female ; Humans ; Male ; Mice, Inbred C57BL ; Muscles/physiopathology ; Muscular Dystrophy, Duchenne/*genetics/physiopathology ; Phenotype ; RNA, Guide/metabolism ; *Sequence Deletion ; },
abstract = {Exon skipping is a promising strategy for Duchenne muscular dystrophy (DMD) disease-modifying therapy. To make this approach safe, ensuring that excluding one or more exons will restore the reading frame and that the resulting protein will retain critical functions of the full-length dystrophin protein is necessary. However, in vivo testing of the consequences of skipping exons that encode the N-terminal actin-binding domain (ABD) has been confounded by the absence of a relevant animal model. We created a mouse model of the disease recapitulating a novel human mutation, a large de novo deletion of exons 8-34 of the DMD gene, found in a Russian DMD patient. This mutation was achieved by deleting exons 8-34 of the X-linked mouse Dmd gene using CRISPR/Cas9 genome editing, which led to a reading frame shift and the absence of functional dystrophin production. Male mice carrying this deletion display several important signs of muscular dystrophy, including a gradual age-dependent decrease in muscle strength, increased creatine kinase, muscle fibrosis and central nucleation. The degrees of these changes are comparable to those observed in mdx mice, a standard laboratory model of DMD. This new model of DMD will be useful for validating therapies based on skipping exons that encode the N-terminal ABD and for improving our understanding of the role of the N-terminal domain and central rod domain in the biological function of dystrophin. Simultaneous skipping of exons 6 and 7 should restore the gene reading frame and lead to the production of a protein that might retain functionality despite the partial deletion of the ABD.},
}

Animals
Base Pairing/*genetics
Biomechanical Phenomena
CRISPR-Cas Systems/*genetics
Cell Line
Child
Chromatin/metabolism
Disease Models, Animal
Dystrophin/*genetics
Exons/genetics
Female
Humans
Male
Mice, Inbred C57BL
Muscles/physiopathology
Muscular Dystrophy, Duchenne/*genetics/physiopathology
Phenotype
RNA, Guide/metabolism
*Sequence Deletion

@article {pmid30849442,
year = {2019},
author = {Liu, B and Saber, A and Haisma, HJ},
title = {CRISPR/Cas9: a powerful tool for identification of new targets for cancer treatment.},
journal = {Drug discovery today},
volume = {24},
number = {4},
pages = {955-970},
doi = {10.1016/j.drudis.2019.02.011},
pmid = {30849442},
issn = {1878-5832},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing ; Humans ; Neoplasms/genetics/*therapy ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated nuclease 9 (Cas9), as a powerful genome-editing tool, has revolutionized genetic engineering. It is widely used to investigate the molecular basis of different cancer types. In this review, we present an overview of recent studies in which CRISPR/Cas9 has been used for the identification of potential molecular targets. Based on the collected data, we suggest here that CRISPR/Cas9 is an effective system to distinguish between mutant and wild-type alleles in cancer. We show that several new potential therapeutic targets, such as CD38, CXCR2, MASTL, and RBX2, as well as several noncoding (nc)RNAs have been identified using CRISPR/Cas9 technology. We also discuss the obstacles and challenges that we face for using CRISPR/Cas9 as a therapeutic.},
}

Animals
*CRISPR-Cas Systems
Gene Editing
Humans
Neoplasms/genetics/*therapy

@article {pmid31943634,
year = {2020},
author = {Palumbo, CM and Gutierrez-Bujari, JM and O'Geen, H and Segal, DJ and Beal, P},
title = {Versatile 3' Functionalization of CRISPR Single Guide RNA.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {},
number = {},
pages = {},
doi = {10.1002/cbic.201900736},
pmid = {31943634},
issn = {1439-7633},
abstract = {Specific applications of CRISPR/Cas genome editing systems benefit from chemical modifications of the sgRNA. Here we describe a versatile and efficient strategy for functionalization of the 3' end of a sgRNA. An exemplary collection of six chemically modified sgRNAs was prepared containing crosslinkers, a fluorophore and biotin. Modification of the sgRNA 3' end was broadly tolerated by S. pyogenes Cas9 in an in vitro DNA cleavage assay. The 3'-biotinylated sgRNA was used as an affinity reagent to identify IGF2BP1, YB1 and hnRNP K as sgRNA-binding proteins present in HEK293T cells. Overall, the modification strategy presented here has the potential to expand on current applications of CRISPR/Cas systems.},
}

@article {pmid31942685,
year = {2020},
author = {Hao, Y and Zong, W and Zeng, D and Han, J and Chen, S and Tang, J and Zhao, Z and Li, X and Ma, K and Xie, X and Zhu, Q and Chen, Y and Zhao, X and Guo, J and Liu, YG},
title = {Shortened snRNA promoters for efficient CRISPR/Cas-based multiplex genome editing in monocot plants.},
journal = {Science China. Life sciences},
volume = {},
number = {},
pages = {},
doi = {10.1007/s11427-019-1612-6},
pmid = {31942685},
issn = {1869-1889},
}

@article {pmid31942051,
year = {2020},
author = {Hampton, HG and Watson, BNJ and Fineran, PC},
title = {The arms race between bacteria and their phage foes.},
journal = {Nature},
volume = {577},
number = {7790},
pages = {327-336},
doi = {10.1038/s41586-019-1894-8},
pmid = {31942051},
issn = {1476-4687},
abstract = {Bacteria are under immense evolutionary pressure from their viral invaders-bacteriophages. Bacteria have evolved numerous immune mechanisms, both innate and adaptive, to cope with this pressure. The discovery and exploitation of CRISPR-Cas systems have stimulated a resurgence in the identification and characterization of anti-phage mechanisms. Bacteriophages use an extensive battery of counter-defence strategies to co-exist in the presence of these diverse phage defence mechanisms. Understanding the dynamics of the interactions between these microorganisms has implications for phage-based therapies, microbial ecology and evolution, and the development of new biotechnological tools. Here we review the spectrum of anti-phage systems and highlight their evasion by bacteriophages.},
}

@article {pmid31941083,
year = {2020},
author = {Yang, D and Wang, Z and Ma, J and Fu, Q and Wu, L and Wang, H and Wang, S and Yan, Y and Sun, J},
title = {Glycine Cleavage System and cAMP Receptor Protein Co-Regulate CRISPR/cas3 Expression to Resist Bacteriophage.},
journal = {Viruses},
volume = {12},
number = {1},
pages = {},
doi = {10.3390/v12010090},
pmid = {31941083},
issn = {1999-4915},
support = {31772744//National Natural Science Foundation of China/ ; 31571932//National Natural Science Foundation of China/ ; T20180205//Shanghai Agriculture Applied Technology Development Program/ ; 18391901900//Science and Technology Commission of Shanghai Municipality/ ; 16391903400//Science and Technology Commission of Shanghai Municipality/ ; 2018YFD0500100//National Key Research and Development Program of China/ ; Agri-X2017007//Shanghai Jiao Tong University Fund/ ; },
abstract = {The CRISPR/Cas system protects bacteria against bacteriophage and plasmids through a sophisticated mechanism where cas operon plays a crucial role consisting of cse1 and cas3. However, comprehensive studies on the regulation of cas3 operon of the Type I-E CRISPR/Cas system are scarce. Herein, we investigated the regulation of cas3 in Escherichia coli. The mutation in gcvP or crp reduced the CRISPR/Cas system interference ability and increased bacterial susceptibility to phage, when the casA operon of the CRISPR/Cas system was activated. The silence of the glycine cleavage system (GCS) encoded by gcvTHP operon reduced cas3 expression. Adding N5, N10-methylene tetrahydrofolate (N5, N10-mTHF), which is the product of GCS-catalyzed glycine, was able to activate cas3 expression. In addition, a cAMP receptor protein (CRP) encoded by crp activated cas3 expression via binding to the cas3 promoter in response to cAMP concentration. Since N5, N10-mTHF provides one-carbon unit for purine, we assumed GCS regulates cas3 through associating with CRP. It was evident that the mutation of gcvP failed to further reduce the cas3 expression with the crp deletion. These results illustrated a novel regulatory pathway which GCS and CRP co-regulate cas3 of the CRISPR/Cas system and contribute to the defence against invasive genetic elements, where CRP is indispensable for GCS regulation of cas3 expression.},
}

@article {pmid31937679,
year = {2020},
author = {Weissman, JL and Johnson, PLF},
title = {Network-Based Prediction of Novel CRISPR-Associated Genes in Metagenomes.},
journal = {mSystems},
volume = {5},
number = {1},
pages = {},
doi = {10.1128/mSystems.00752-19},
pmid = {31937679},
issn = {2379-5077},
abstract = {A diversity of clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems provide adaptive immunity to bacteria and archaea through recording "memories" of past viral infections. Recently, many novel CRISPR-associated proteins have been discovered via computational studies, but those studies relied on biased and incomplete databases of assembled genomes. We avoided these biases and applied a network theory approach to search for novel CRISPR-associated genes by leveraging subtle ecological cooccurrence patterns identified from environmental metagenomes. We validated our method using existing annotations and discovered 32 novel CRISPR-associated gene families. These genes span a range of putative functions, with many potentially regulating the response to infection.IMPORTANCE Every branch on the tree of life, including microbial life, faces the threat of viral pathogens. Over the course of billions of years of coevolution, prokaryotes have evolved a great diversity of strategies to defend against viral infections. One of these is the CRISPR adaptive immune system, which allows microbes to "remember" past infections in order to better fight them in the future. There has been much interest among molecular biologists in CRISPR immunity because this system can be repurposed as a tool for precise genome editing. Recently, a number of comparative genomics approaches have been used to detect novel CRISPR-associated genes in databases of genomes with great success, potentially leading to the development of new genome-editing tools. Here, we developed novel methods to search for these distinct classes of genes directly in environmental samples ("metagenomes"), thus capturing a more complete picture of the natural diversity of CRISPR-associated genes.},
}

@article {pmid31937548,
year = {2020},
author = {Deecker, SR and Ensminger, AW},
title = {Type I-F CRISPR-Cas Distribution and Array Dynamics in Legionella pneumophila.},
journal = {G3 (Bethesda, Md.)},
volume = {},
number = {},
pages = {},
doi = {10.1534/g3.119.400813},
pmid = {31937548},
issn = {2160-1836},
abstract = {In bacteria and archaea, several distinct types of CRISPR-Cas systems provide adaptive immunity through broadly similar mechanisms: short nucleic acid sequences derived from foreign DNA, known as spacers, engage in complementary base pairing with invasive genetic elements setting the stage for nucleases to degrade the target DNA. A hallmark of type I CRISPR-Cas systems is their ability to acquire spacers in response to both new and previously encountered invaders (naïve and primed acquisition, respectively). Our phylogenetic analyses of 43 L. pneumophila type I-F CRISPR-Cas systems and their resident genomes suggest that many of these systems have been horizontally acquired. These systems are frequently encoded on plasmids and can co-occur with nearly identical chromosomal loci. We show that two such co-occurring systems are highly protective and undergo efficient primed acquisition in the lab. Furthermore, we observe that targeting by one system's array can prime spacer acquisition in the other. Lastly, we provide experimental and genomic evidence for a model in which primed acquisition can efficiently replenish a depleted type I CRISPR array following a mass spacer deletion event.},
}

@article {pmid31936769,
year = {2020},
author = {Cui, L and Wang, X and Huang, D and Zhao, Y and Feng, J and Lu, Q and Pu, Q and Wang, Y and Cheng, G and Wu, M and Dai, M},
title = {CRISPR-cas3 of Salmonella Upregulates Bacterial Biofilm Formation and Virulence to Host Cells by Targeting Quorum-Sensing Systems.},
journal = {Pathogens (Basel, Switzerland)},
volume = {9},
number = {1},
pages = {},
doi = {10.3390/pathogens9010053},
pmid = {31936769},
issn = {2076-0817},
support = {2662017JC034//Fundamental Research Funds for the Central Universities/ ; 2017YFC1600100//National Basic Research Program of China (973 Program)/ ; 2017020201010228//Applied Basic Research Programs of Wuhan/ ; CARS-35//Earmarked Fund for China Agriculture Research System/ ; AI101973-01//Foundation for the National Institutes of Health/ ; AI109317-01A1//Foundation for the National Institutes of Health/ ; AI097532-01A1//Foundation for the National Institutes of Health/ ; P20GM103442//Foundation for the National Institutes of Health/ ; P20GM113123//Foundation for the National Institutes of Health/ ; },
abstract = {Salmonella is recognized as one of the most common microbial pathogens worldwide. The bacterium contains the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems, providing adaptive immunity against invading foreign nucleic acids. Previous studies suggested that certain bacteria employ the Cas proteins of CRISPR-Cas systems to target their own genes, which also alters the virulence during invasion of mammals. However, whether CRISPR-Cas systems in Salmonella have similar functions during bacterial invasion of host cells remains unknown. Here, we systematically analyzed the genes that are regulated by Cas3 in a type I-E CRISPR-Cas system and the virulence changes due to the deletion of cas3 in Salmonella enterica serovar Enteritidis. Compared to the cas3 gene wild-type (cas3 WT) Salmonella strain, cas3 deletion upregulated the lsrFGBE genes in lsr (luxS regulated) operon related to quorum sensing (QS) and downregulated biofilm-forming-related genes and Salmonella pathogenicity island 1 (SPI-1) genes related to the type three secretion system (T3SS). Consistently, the biofilm formation ability was downregulated in the cas3 deletion mutant (Δcas3). The bacterial invasive and intracellular capacity of Δcas3 to host cells was also reduced, thereby increasing the survival of infected host cells and live chickens. By the transcriptome-wide screen (RNA-Seq), we found that the cas3 gene impacts a series of genes related to QS, the flagellum, and SPI-1-T3SS system, thereby altering the virulence phenotypes. As QS SPI-1-T3SS and CRISPR-Cas systems are widely distributed in the bacteria kingdom, our findings extend our understanding of virulence regulation and pathogenicity in mammalian hosts for Salmonella and potentially other bacteria.},
}

@article {pmid31936280,
year = {2020},
author = {Wang, S and Yang, B and Ross, RP and Stanton, C and Zhao, J and Zhang, H and Chen, W},
title = {Comparative Genomics Analysis of Lactobacillus ruminis from Different Niches.},
journal = {Genes},
volume = {11},
number = {1},
pages = {},
doi = {10.3390/genes11010070},
pmid = {31936280},
issn = {2073-4425},
abstract = {Lactobacillus ruminis is a commensal motile lactic acid bacterium living in the intestinal tract of humans and animals. Although a few genomes of L. ruminis were published, most of them were animal derived. To explore the genetic diversity and potential niche-specific adaptation changes of L. ruminis, in the current work, draft genomes of 81 L. ruminis strains isolated from human, bovine, piglet, and other animals were sequenced, and comparative genomic analysis was performed. The genome size and GC content of L. ruminis on average were 2.16 Mb and 43.65%, respectively. Both the origin and the sampling distance of these strains had a great influence on the phylogenetic relationship. For carbohydrate utilization, the human-derived L. ruminis strains had a higher consistency in the utilization of carbon source compared to the animal-derived strains. L. ruminis mainly increased the competitiveness of niches by producing class II bacteriocins. The type of clustered regularly interspaced short palindromic repeats /CRISPR-associated (CRISPR/Cas) system presented in L. ruminis was mainly subtype IIA. The diversity of CRISPR/Cas locus depended on the high denaturation of spacer number and sequence, although cas1 protein was relatively conservative. The genetic differences in those newly sequenced L. ruminis strains highlighted the gene gains and losses attributed to niche adaptations.},
}

@article {pmid31934359,
year = {2020},
author = {Li, H and Yang, Y and Hong, W and Huang, M and Wu, M and Zhao, X},
title = {Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects.},
journal = {Signal transduction and targeted therapy},
volume = {5},
number = {},
pages = {1},
pmid = {31934359},
issn = {2059-3635},
abstract = {Based on engineered or bacterial nucleases, the development of genome editing technologies has opened up the possibility of directly targeting and modifying genomic sequences in almost all eukaryotic cells. Genome editing has extended our ability to elucidate the contribution of genetics to disease by promoting the creation of more accurate cellular and animal models of pathological processes and has begun to show extraordinary potential in a variety of fields, ranging from basic research to applied biotechnology and biomedical research. Recent progress in developing programmable nucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas-associated nucleases, has greatly expedited the progress of gene editing from concept to clinical practice. Here, we review recent advances of the three major genome editing technologies (ZFNs, TALENs, and CRISPR/Cas9) and discuss the applications of their derivative reagents as gene editing tools in various human diseases and potential future therapies, focusing on eukaryotic cells and animal models. Finally, we provide an overview of the clinical trials applying genome editing platforms for disease treatment and some of the challenges in the implementation of this technology.},
}

@article {pmid31932164,
year = {2020},
author = {Lau, RK and Ye, Q and Birkholz, EA and Berg, KR and Patel, L and Mathews, IT and Watrous, JD and Ego, K and Whiteley, AT and Lowey, B and Mekalanos, JJ and Kranzusch, PJ and Jain, M and Pogliano, J and Corbett, KD},
title = {Structure and Mechanism of a Cyclic Trinucleotide-Activated Bacterial Endonuclease Mediating Bacteriophage Immunity.},
journal = {Molecular cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molcel.2019.12.010},
pmid = {31932164},
issn = {1097-4164},
abstract = {Bacteria possess an array of defenses against foreign invaders, including a broadly distributed bacteriophage defense system termed CBASS (cyclic oligonucleotide-based anti-phage signaling system). In CBASS systems, a cGAS/DncV-like nucleotidyltransferase synthesizes cyclic di- or tri-nucleotide second messengers in response to infection, and these molecules activate diverse effectors to mediate bacteriophage immunity via abortive infection. Here, we show that the CBASS effector NucC is related to restriction enzymes but uniquely assembles into a homotrimer. Binding of NucC trimers to a cyclic tri-adenylate second messenger promotes assembly of a NucC homohexamer competent for non-specific double-strand DNA cleavage. In infected cells, NucC activation leads to complete destruction of the bacterial chromosome, causing cell death prior to completion of phage replication. In addition to CBASS systems, we identify NucC homologs in over 30 type III CRISPR/Cas systems, where they likely function as accessory nucleases activated by cyclic oligoadenylate second messengers synthesized by these systems' effector complexes.},
}

@article {pmid31927331,
year = {2019},
author = {Weng, Y and Huang, Q and Li, C and Yang, Y and Wang, X and Yu, J and Huang, Y and Liang, XJ},
title = {Improved Nucleic Acid Therapy with Advanced Nanoscale Biotechnology.},
journal = {Molecular therapy. Nucleic acids},
volume = {19},
number = {},
pages = {581-601},
doi = {10.1016/j.omtn.2019.12.004},
pmid = {31927331},
issn = {2162-2531},
abstract = {Due to a series of systemic and intracellular obstacles in nucleic acid (NA) therapy, including fast degradation in blood, renal clearance, poor cellular uptake, and inefficient endosomal escape, NAs may need delivery methods to transport to the cell nucleus or cytosol to be effective. Advanced nanoscale biotechnology-associated strategies, such as controlling the particle size, charge, drug loading, response to environmental signals, or other physical/chemical properties of delivery carriers, have provided great help for the in vivo and in vitro delivery of NA therapeutics. In this review, we introduce the characteristics of different NA modalities and illustrate how advanced nanoscale biotechnology assists NA therapy. The specific features and challenges of various nanocarriers in clinical and preclinical studies are summarized and discussed. With the help of advanced nanoscale biotechnology, some of the major barriers to the development of NA therapy will eventually be overcome in the near future.},
}

@article {pmid31926765,
year = {2020},
author = {Wang, R and Angenent, GC and Seymour, G and de Maagd, RA},
title = {Revisiting the Role of Master Regulators in Tomato Ripening.},
journal = {Trends in plant science},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tplants.2019.11.005},
pmid = {31926765},
issn = {1878-4372},
abstract = {The study of transcriptional regulation of tomato ripening has been led by spontaneous mutations in transcription factor (TF) genes that completely inhibit normal ripening, suggesting that they are 'master regulators'. Studies using CRISPR/Cas9 mutagenesis to produce knockouts of the underlying genes indicate a different picture, suggesting that the regulation is more robust than previously thought. This requires us to revisit our model of the regulation of ripening and replace it with one involving a network of partially redundant components. At the same time, the fast rise of CRISPR/Cas mutagenesis, resulting in unexpectedly weak phenotypes, compared with knockdown technology, suggests that compensatory mechanisms may obscure protein functions. This emphasises the need for assessment of these mechanisms in plants and for the careful design of mutagenesis experiments.},
}

@article {pmid31925391,
year = {2020},
author = {Jia, N and Xie, W and de la Cruz, MJ and Eng, ET and Patel, DJ},
title = {Structure-function insights into the initial step of DNA integration by a CRISPR-Cas-Transposon complex.},
journal = {Cell research},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41422-019-0272-2},
pmid = {31925391},
issn = {1748-7838},
}

@article {pmid31925273,
year = {2019},
author = {Sansbury, BM and Hewes, AM and Kmiec, EB},
title = {Understanding the diversity of genetic outcomes from CRISPR-Cas generated homology-directed repair.},
journal = {Communications biology},
volume = {2},
number = {1},
pages = {458},
doi = {10.1038/s42003-019-0705-y},
pmid = {31925273},
issn = {2399-3642},
support = {1558//BIRD Foundation (Israel-U.S. Binational Industrial Research and Development Foundation)/ ; },
abstract = {As CRISPR-Cas systems advance toward clinical application, it is essential to identify all the outcomes of gene-editing activity in human cells. Reports highlighting the remarkable success of homology-directed repair (HDR) in the treatment of inherited diseases may inadvertently underreport the collateral activity of this remarkable technology. We are utilizing an in vitro gene-editing system in which a CRISPR-Cas complex provides the double-stranded cleavage and a mammalian cell-free extract provides the enzymatic activity to promote non-homologous end joining, micro-homology mediated end joining, and homology-directed repair. Here, we detail the broad spectrum of gene-editing reaction outcomes utilizing Cas9 and Cas12a in combination with single-stranded donor templates of the sense and nonsense polarity. This system offers the opportunity to see the range of outcomes of gene-editing reactions in an unbiased fashion, detailing the distribution of DNA repair outcomes as a function of a set of genetic tools.},
}

@article {pmid31925225,
year = {2019},
author = {Young, JK and Gasior, SL and Jones, S and Wang, L and Navarro, P and Vickroy, B and Barrangou, R},
title = {The repurposing of type I-E CRISPR-Cascade for gene activation in plants.},
journal = {Communications biology},
volume = {2},
number = {1},
pages = {383},
doi = {10.1038/s42003-019-0637-6},
pmid = {31925225},
issn = {2399-3642},
abstract = {CRISPR-Cas systems are robust and facile tools for manipulating the genome, epigenome and transcriptome of eukaryotic organisms. Most groups use class 2 effectors, such as Cas9 and Cas12a, however, other CRISPR-Cas systems may provide unique opportunities for genome engineering. Indeed, the multi-subunit composition of class 1 systems offers to expand the number of domains and functionalities that may be recruited to a genomic target. Here we report DNA targeting in Zea mays using a class 1 type I-E CRISPR-Cas system from S. thermophilus. First, we engineer its Cascade complex to modulate gene expression by tethering a plant transcriptional activation domain to 3 different subunits. Next, using an immunofluorescent assay, we confirm Cascade cellular complex formation and observe enhanced gene activation when multiple subunits tagged with the transcriptional activator are combined. Finally, we examine Cascade mediated gene activation at chromosomal DNA targets by reprogramming Zea mays cells to change color.},
}

@article {pmid31924979,
year = {2019},
author = {Qiao, J and Li, W and Lin, S and Sun, W and Ma, L and Liu, Y},
title = {Co-expression of Cas9 and single-guided RNAs in Escherichia coli streamlines production of Cas9 ribonucleoproteins.},
journal = {Communications biology},
volume = {2},
number = {1},
pages = {161},
doi = {10.1038/s42003-019-0402-x},
pmid = {31924979},
issn = {2399-3642},
abstract = {CRISPR/Cas9 ribonucleoprotein (RNP) complexes are promising biological tools with diverse biomedical applications. However, to date there are no efficient methods that can produce these proteins at large scales and low cost. Here, we present a streamlined method for direct production of Cas9 RNPs from Escherichia coli by co-expression of Cas9 and the target-specific single-guided RNAs. Harnessing an ultrahigh-affinity CL7/Im7 purification system recently developed we achieve one-step purification of the self-assembling CRISPR/Cas RNPs, including the commonly used Cas9 and Cas12a, within half a day and with a ~fourfold higher yield than incumbent methods. The prepared Cas RNPs show remarkable stability in the absence of RNase inhibitors, as well as profound gene-editing efficiency in vitro and in vivo. Our method is convenient, cost-effective, and can be used to prepare other CRISPR/Cas RNPs.},
}

@article {pmid31922192,
year = {2020},
author = {Hidalgo-Cantabrana, C and Barrangou, R},
title = {Characterization and applications of Type I CRISPR-Cas systems.},
journal = {Biochemical Society transactions},
volume = {},
number = {},
pages = {},
doi = {10.1042/BST20190119},
pmid = {31922192},
issn = {1470-8752},
abstract = {CRISPR-Cas constitutes the adaptive immune system of bacteria and archaea. This RNA-mediated sequence-specific recognition and targeting machinery has been used broadly for diverse applications in a wide range of organisms across the tree of life. The compact class 2 systems, that hinge on a single Cas effector nuclease have been harnessed for genome editing, transcriptional regulation, detection, imaging and other applications, in different research areas. However, most of the CRISPR-Cas systems belong to class 1, and the molecular machinery of the most widespread and diverse Type I systems afford tremendous opportunities for a broad range of applications. These highly abundant systems rely on a multi-protein effector complex, the CRISPR associated complex for antiviral defense (Cascade), which drives DNA targeting and cleavage. The complexity of these systems has somewhat hindered their widespread usage, but the pool of thousands of diverse Type I CRISPR-Cas systems opens new avenues for CRISPR-based applications in bacteria, archaea and eukaryotes. Here, we describe the features and mechanism of action of Type I CRISPR-Cas systems, illustrate how endogenous systems can be reprogrammed to target the host genome and perform genome editing and transcriptional regulation by co-delivering a minimal CRISPR array together with a repair template. Moreover, we discuss how these systems can also be used in eukaryotes. This review provides a framework for expanding the CRISPR toolbox, and repurposing the most abundant CRISPR-Cas systems for a wide range of applications.},
}

@article {pmid31921246,
year = {2019},
author = {Wesseler, J and Politiek, H and Zilberman, D},
title = {The Economics of Regulating New Plant Breeding Technologies - Implications for the Bioeconomy Illustrated by a Survey Among Dutch Plant Breeders.},
journal = {Frontiers in plant science},
volume = {10},
number = {},
pages = {1597},
pmid = {31921246},
issn = {1664-462X},
abstract = {New plant breeding technologies (NPBTs) are increasingly used for developing new plants with novel traits. The science tells us that those plants in general are as safe as than those once developed using "conventional" plant breeding methods. The knowledge about the induced changes and properties of the new plants by using NPBTs is more precise. This should lead to the conclusion that plants developed using NPBTs should not be regulated differently than those developed using "conventional" plant breeding methods. This contribution discusses the economics of regulating new plant breeding technologies. We first develop the theoretical model and elaborate on the different regulatory approaches being used and compare their advantages and disadvantages. Then we provide a perspectives on EU regulation around mutagenesis-based New Plant Breeding Techniques (NPBT), formed by new insights from a survey among Dutch plant breeding companies. The survey measures the attitude of breeding companies towards the ruling of the EU Court of Justice that subjected the use of CRISPR-Cas in the development of new plant varieties under the general EU regulations around GMOs. The results show that plant breeders experience a financial barrier because of the ruling, with perceived negative impact on competitiveness and investments in CRISPR-Cas as a result. The degree of negative impact differs however significantly among seed-sectors and company sizes. One of the most striking results was the relative optimism of companies in the sector about more lenient legislation in the next five years, despite the stated negative effects.},
}

@article {pmid31921024,
year = {2019},
author = {Watanabe, S and Cui, B and Kiga, K and Aiba, Y and Tan, XE and Sato'o, Y and Kawauchi, M and Boonsiri, T and Thitiananpakorn, K and Taki, Y and Li, FY and Azam, AH and Nakada, Y and Sasahara, T and Cui, L},
title = {Composition and Diversity of CRISPR-Cas13a Systems in the Genus Leptotrichia.},
journal = {Frontiers in microbiology},
volume = {10},
number = {},
pages = {2838},
pmid = {31921024},
issn = {1664-302X},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a, previously known as CRISPR-C2c2, is the most recently identified RNA-guided RNA-targeting CRISPR-Cas system that has the unique characteristics of both targeted and collateral single-stranded RNA (ssRNA) cleavage activities. This system was first identified in Leptotrichia shahii. Here, the complete whole genome sequences of 11 Leptotrichia strains were determined and compared with 18 publicly available Leptotrichia genomes in regard to the composition, occurrence and diversity of the CRISPR-Cas13a, and other CRISPR-Cas systems. Various types of CRISPR-Cas systems were found to be unevenly distributed among the Leptotrichia genomes, including types I-B (10/29, 34.4%), II-C (1/29, 2.6%), III-A (6/29, 15.4%), III-D (6/29, 15.4%), III-like (3/29, 7.7%), and VI-A (11/29, 37.9%), while 8 strains (20.5%) had no CRISPR-Cas system at all. The Cas13a effectors were found to be highly divergent with amino acid sequence similarities ranging from 61% to 90% to that of L. shahii, but their collateral ssRNA cleavage activities leading to impediment of bacterial growth were conserved. CRISPR-Cas spacers represent a sequential achievement of former intruder encounters, and the retained spacers reflect the evolutionary phylogeny or relatedness of strains. Analysis of spacer contents and numbers among Leptotrichia species showed considerable diversity with only 4.4% of spacers (40/889) were shared by two strains. The organization and distribution of CRISPR-Cas systems (type I-VI) encoded by all registered Leptotrichia species revealed that effector or spacer sequences of the CRISPR-Cas systems were very divergent, and the prevalence of types I, III, and VI was almost equal. There was only one strain carrying type II, while none carried type IV or V. These results provide new insights into the characteristics and divergences of CRISPR-Cas systems among Leptotrichia species.},
}

@article {pmid31919672,
year = {2020},
author = {Zhao, Z and Zhang, RA and Fu, GY and Zhang, R and Nie, YF and Sun, C and Wu, M},
title = {The Complete Genome of Emcibacter congregatus ZYLT, a Marine Bacterium Encoding a CRISPR-Cas 9 Immune System.},
journal = {Current microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1007/s00284-019-01867-6},
pmid = {31919672},
issn = {1432-0991},
support = {2017FY100300//Science & Technology Basic Resources Investigation Program of China/ ; 2018M642382//China Postdoctoral Science Foundation/ ; LQ19C010005//Natural Science Foundation of Zhejiang Province/ ; },
abstract = {Emcibacter congregatus ZYLT was isolated from a sediment sample cultured in situ in a coast located in the East China Sea. The genome of E. congregatus ZYLT was sequenced and assembled into one single circular chromosome with the size of 4,189,011 bp and G+C content of 52.6%. Genomic annotation showed that E. congregatus ZYLT had an intact Type II-C CRISPR-Cas system consists of three cas genes (cas 9, cas 1, and cas 2), 34 direct repeat sequences with the length of 36 bp, and 33 spacers. The predicted Cas 9 protein was smaller than most of existing genome editing tools. This structure might have potential in developing new gene editing system and uncovering the regulatory mechanisms of CRISPR-Cas system. Besides, the comparison between E. congregatus ZYLT and its relative species living in neritic environments unraveled some common traits of the defective strategies of these bacteria to face inshore challenges including the motility, multidrug resistance, and universal efflux pumps.},
}

@article {pmid31919495,
year = {2020},
author = {Kukhtar, D and Rubio-Peña, K and Serrat, X and Cerón, J},
title = {Mimicking of splicing-related retinitis pigmentosa mutations in C. elegans allow drug screens and identification of disease modifiers.},
journal = {Human molecular genetics},
volume = {},
number = {},
pages = {},
doi = {10.1093/hmg/ddz315},
pmid = {31919495},
issn = {1460-2083},
abstract = {CRISPR/Cas and the high conservation of the spliceosome components facilitate the mimicking of human pathological mutations in splicing factors of model organisms. The degenerative retinal disease retinitis pigmentosa (RP) is caused by mutations in distinct types of genes, including missense mutations in splicing factors that provoke RP in an autosomal dominant form (s-adRP). Using CRISPR in Caenorhabditis elegans, we generated mutant strains to mimic s-adRP mutations reported in PRPF8 and SNRNP200. Whereas these inherited mutations are present in heterozygosis in patients, C. elegans allows the maintenance of these mutations as homozygotes, which is advantageous for genetic and drug screens. We found that snrp-200(cer23[V676 L]) and prp-8(cer14[H2302del]) display pleiotropic phenotypes, including reduced fertility. However, snrp-200(cer24[S1080 L]) and prp-8(cer22[R2303G]) are weak alleles suitable for RNAi screens for identifying genetic interactions, which could uncover potential disease modifiers. We screened a collection of RNAi clones for splicing-related genes and identified three splicing factors: isy-1/ISY1, cyn-15/PPWD1, and mog-2/SNRPA1 whose partial inactivation may modify the course of the disease. Interestingly, these three genes act as modifiers of prp-8(cer22) but not of snrp-200(cer24). Finally, a screen of the strong allele prp-8(cer14) with FDA-approved drugs did not identify molecules capable of alleviating the temperature-sensitive sterility. Instead, we detected drugs, such as dequalinium chloride, which exacerbated the phenotype, and therefore, are potentially harmful to s-adRP patients since they may accelerate the progression of the disease.},
}

@article {pmid31917244,
year = {2020},
author = {Gui, S and Taning, CNT and Wei, D and Smagghe, G},
title = {First report on CRISPR/Cas9-targeted mutagenesis in the Colorado potato beetle, Leptinotarsa decemlineata.},
journal = {Journal of insect physiology},
volume = {121},
number = {},
pages = {104013},
doi = {10.1016/j.jinsphys.2020.104013},
pmid = {31917244},
issn = {1879-1611},
abstract = {Leptinotarsa decemlineata (Say), commonly known as the Colorado potato beetle (CPB), is an agricultural important pest for potatoes and other solanaceous plants. The CRISPR/Cas system is an efficient genome editing technology, which could be exploited to study the biology of CPB and possibly also lead to the development of better environmentally friendly pest management strategies. However, the use of CRISPR/Cas9 has been limited to only a few model insects. Here, for the first time, a CRISPR/Cas9 protocol for mutagenesis studies in CPB was developed. A gene with a clear phenotype such as the vestigial gene (vest), known to be involved in wing development in other insect species, was selected as a good indicator for the knockout study. First, vest was functionally characterized in CPB by using RNAi technology for knockdown studies. Once the expected deformed wing phenotypes were observed, a CRISPR/Cas9 work flow was established for mutagenesis in CPB. By co-injecting the Cas9 protein and a vest-guide RNA into 539 CPB eggs of <1 h old, sixty-two successfully developed to adults, among which mutation in the vest loci was confirmed in 5 of the 18 wingless CPBs (29% phenotypic mutation efficiency). The mutation in vest resulted in a clear phenotype in the CPBs, which developed to adulthood with no hindwing and elytron formed. Altogether, this study provides for the first time a useful methodology involving the use of the CRISPR/Cas9 system for mutagenesis studies in one of the most important pest insects.},
}

@article {pmid31916673,
year = {2020},
author = {Wang, M and Xu, Z and Gosavi, G and Ren, B and Cao, Y and Kuang, Y and Zhou, C and Spetz, C and Yan, F and Zhou, X and Zhou, H},
title = {Targeted base editing in rice with CRISPR/ScCas9 system.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.13330},
pmid = {31916673},
issn = {1467-7652},
abstract = {The CRISPR/Cas system has rapidly become the preferred tool for genome engineering in various organisms due to high efficiency, specificity, simplicity and versatility. Currently, CRISPR/Cas-mediated base editing, a novel genome editing strategy that enables irreversible nucleotide changes at target loci without double-stranded DNA cleavage or any donor template, has been widely adopted for generating gain-of-function germplasms in functional genomics research and crop genetic improvement (Hua et al., 2019; Ren et al., 2018; Yan et al., 2018).},
}

@article {pmid31914418,
year = {2019},
author = {Ramachandran, A and Summerville, L and Learn, BA and DeBell, L and Bailey, S},
title = {Processing and integration of functionally oriented prespacers in the E. coli CRISPR system depends on bacterial host exonucleases.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1074/jbc.RA119.012196},
pmid = {31914418},
issn = {1083-351X},
abstract = {CRISPR/Cas systems provide bacteria with adaptive immunity against viruses. During spacer adaptation, the Cas1-Cas2 complex selects fragments of foreign DNA, called prespacers, and integrates them into CRISPR arrays in an orientation that provides functional immunity. Cas4 is involved in both the trimming of prespacers and the cleavage of protospacer adjacent motif (PAM) in several type I CRISPR-Cas systems, but how the prespacers are processed in systems lacking Cas4, such as the type I-E and I-F systems, is not understood. In Escherichia coli, which has a type I-E system, Cas1-Cas2 preferentially selects prespacers with 3' overhangs via specific recognition of a protospacer adjacent motif (PAM), but how these prespacers are integrated in a functional orientation in the absence of Cas4 is not known. Using a biochemical approach with purified proteins, as well as integration, prespacer protection, sequencing and quantitative PCR assays, we show here that the bacterial 3'-5' exonucleases DnaQ or ExoT can trim long 3' overhangs of prespacers and promote integration in the correct orientation. We found that trimming by these exonucleases results in an asymmetric intermediate, because Cas1-Cas2 protects the PAM sequence, which helps to define spacer orientation. Our findings implicate the E. coli host 3'-5' exonucleases DnaQ and ExoT in spacer adaptation and reveal a mechanism by which spacer orientation is defined in E. coli.},
}

@article {pmid31913359,
year = {2020},
author = {Wang, B and Xu, W and Yang, H},
title = {Structural basis of a Tn7-like transposase recruitment and DNA loading to CRISPR-Cas surveillance complex.},
journal = {Cell research},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41422-020-0274-0},
pmid = {31913359},
issn = {1748-7838},
support = {31971135//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
}

@article {pmid31913120,
year = {2020},
author = {Hickman, AB and Kailasan, S and Genzor, P and Haase, AD and Dyda, F},
title = {Casposase structure and the mechanistic link between DNA transposition and spacer acquisition by CRISPR-Cas.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
doi = {10.7554/eLife.50004},
pmid = {31913120},
issn = {2050-084X},
support = {Intramural Program/DK/NIDDK NIH HHS/United States ; },
abstract = {Key to CRISPR-Cas adaptive immunity is maintaining an ongoing record of invading nucleic acids, a process carried out by the Cas1-Cas2 complex that integrates short segments of foreign genetic material (spacers) into the CRISPR locus. It is hypothesized that Cas1 evolved from casposases, a novel class of transposases. We show here that the Methanosarcina mazei casposase can integrate varied forms of the casposon end in vitro, and recapitulates several properties of CRISPR-Cas integrases including site-specificity. The X-ray structure of the casposase bound to DNA representing the product of integration reveals a tetramer with target DNA bound snugly between two dimers in which single-stranded casposon end binding resembles that of spacer 3'-overhangs. The differences between transposase and CRISPR-Cas integrase are largely architectural, and it appears that evolutionary change involved changes in protein-protein interactions to favor Cas2 binding over tetramerization; this in turn led to preferred integration of single spacers over two transposon ends.},
}

@article {pmid31911695,
year = {2020},
author = {Ledford, H},
title = {Quest to use CRISPR against disease gains ground.},
journal = {Nature},
volume = {577},
number = {7789},
pages = {156},
doi = {10.1038/d41586-019-03919-0},
pmid = {31911695},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *HIV Infections ; Humans ; *Precursor Cell Lymphoblastic Leukemia-Lymphoma ; Stem Cells ; },
}

CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
*HIV Infections
Humans
*Precursor Cell Lymphoblastic Leukemia-Lymphoma
Stem Cells

@article {pmid31911609,
year = {2020},
author = {Munck, C and Sheth, RU and Freedberg, DE and Wang, HH},
title = {Recording mobile DNA in the gut microbiota using an Escherichia coli CRISPR-Cas spacer acquisition platform.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {95},
pmid = {31911609},
issn = {2041-1723},
support = {R01 AI132403/AI/NIAID NIH HHS/United States ; },
abstract = {The flow of genetic material between bacteria is central to the adaptation and evolution of bacterial genomes. However, our knowledge about DNA transfer within complex microbiomes is lacking, with most studies of horizontal gene transfer (HGT) relying on bioinformatic analyses of genetic elements maintained on evolutionary timescales or experimental measurements of phenotypically trackable markers. Here, we utilize the CRISPR-Cas spacer acquisition process to detect DNA acquisition events from complex microbiota in real-time and at nucleotide resolution. In this system, an E. coli recording strain is exposed to a microbial sample and spacers are acquired from transferred plasmids and permanently stored in genomic CRISPR arrays. Sequencing and analysis of acquired spacers enables identification of the transferred plasmids. This approach allowed us to identify individual mobile elements without relying on phenotypic markers or post-transfer replication. We found that HGT into the recording strain in human clinical fecal samples can be extensive and is driven by different plasmid types, with the IncX type being the most actively transferred.},
}

@article {pmid31911131,
year = {2019},
author = {Li, J and Hong, S and Chen, W and Zuo, E and Yang, H},
title = {Advances in detecting and reducing off-target effects generated by CRISPR-mediated genome editing.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jgg.2019.11.002},
pmid = {31911131},
issn = {1673-8527},
abstract = {CRISPR-mediated genome editing is a revolutionary technology for genome manipulation that uses the CRISPR-Cas systems and base editors. Currently, poor efficiency and off-target problems have impeded the application of CRISPR systems. The on-target efficiency has been improved in several advanced versions of CRISPR systems, whereas the off-target detection still remains a key challenge. Here, we outline the different versions of CRISPR systems and off-target detection strategies, discuss the merits and limitations of off-target detection methods, and provide potential implications for further gene editing research.},
}

@article {pmid31910089,
year = {2020},
author = {Tatineni, S and Stewart, LR and Sanfaçon, H and Wang, X and Navas-Castillo, J and Hajimorad, MR},
title = {Fundamental Aspects of Plant Viruses-An Overview on Focus Issue Articles.},
journal = {Phytopathology},
volume = {110},
number = {1},
pages = {6-9},
doi = {10.1094/PHYTO-10-19-0404-FI},
pmid = {31910089},
issn = {0031-949X},
abstract = {Given the importance of and rapid research progress in plant virology in recent years, this Focus Issue broadly emphasizes advances in fundamental aspects of virus infection cycles and epidemiology. This Focus Issue comprises three review articles and 18 research articles. The research articles cover broad research areas on the identification of novel viruses, the development of detection methods, reverse genetics systems and functional genomics for plant viruses, vector and seed transmission studies, viral population studies, virus-virus interactions and their effect on vector transmission, and management strategies of viral diseases. The three review articles discuss recent developments in application of prokaryotic clustered regularly interspaced short palindromic repeats/CRISPR-associated genes (CRISPR/Cas) technology for plant virus resistance, mixed viral infections and their role in disease synergism and cross-protection, and viral transmission by whiteflies. The following briefly summarizes the articles appearing in this Focus Issue.},
}

@article {pmid31907146,
year = {2019},
author = {Chen, G and Cheng, D and Chen, B},
title = {[Development of CRISPR technology and its application in bone and cartilage tissue engineering].},
journal = {Nan fang yi ke da xue xue bao = Journal of Southern Medical University},
volume = {39},
number = {12},
pages = {1515-1520},
doi = {10.12122/j.issn.1673-4254.2019.12.19},
pmid = {31907146},
issn = {1673-4254},
mesh = {CRISPR-Cas Systems ; Cartilage ; *Clustered Regularly Interspaced Short Palindromic Repeats ; RNA, Guide ; *Tissue Engineering ; },
abstract = {The CRISPR/Cas9 system, consisting of Cas9 nuclease and single guide RNA (sgRNA), is an emerging gene editing technology that can perform gene reprogramming operations such as deletion, insertion, and point mutation on DNA sequences targeted by sgRNA. In addition, CRISPR/dCas9 (a mutant that loses Cas9 nuclease activity) still retains the ability of sgRNA to target DNA. The fusion of dCas9 protein with transcriptional activator (CRISPRa) can activate the expression of the target gene, and fusion transcriptional repressors (CRISPRi) can also be used to suppress target gene expression. Efficient delivery of the CRISPR/Cas9 system is one of the main problems limiting its wide clinical application. Viral vectors are widely used to efficiently deliver CRISPR/Cas9 elements, but non-viral vector research is more attractive in terms of safety, simplicity, and flexibility. In this review, we summarize the principles and research advances of CRISPR technology, including CRISPR/ Cas9 delivery vectors, delivery methods, and obstacles to the delivery, and review the progress of CRISPR-based research in bone and cartilage tissue engineering. Finally, the challenges and future applications of CRISPR technology in bone and cartilage tissue engineering are discussed.},
}

CRISPR-Cas Systems
Cartilage
*Clustered Regularly Interspaced Short Palindromic Repeats
RNA, Guide
*Tissue Engineering

@article {pmid31906943,
year = {2020},
author = {Yeo, WL and Heng, E and Tan, LL and Lim, YW and Ching, KC and Tsai, DJ and Jhang, YW and Lauderdale, TL and Shia, KS and Zhao, H and Ang, EL and Zhang, MM and Lim, YH and Wong, FT},
title = {Biosynthetic engineering of the antifungal, anti-MRSA auroramycin.},
journal = {Microbial cell factories},
volume = {19},
number = {1},
pages = {3},
pmid = {31906943},
issn = {1475-2859},
support = {NRF2013-THE001-094//National Research Foundation Singapore/ ; NRF2013-THE001-094//National Research Foundation Singapore/ ; NRF2013-THE001-094//National Research Foundation Singapore/ ; Visiting Investigator Program//Agency for Science, Technology and Research, ASTAR, Singapore/ ; 1535j00137//ASTAR Visiting Investigator Program/ ; },
abstract = {Using an established CRISPR-Cas mediated genome editing technique for streptomycetes, we explored the combinatorial biosynthesis potential of the auroramycin biosynthetic gene cluster in Streptomyces roseosporous. Auroramycin is a potent anti-MRSA polyene macrolactam. In addition, auroramycin has antifungal activities, which is unique among structurally similar polyene macrolactams, such as incednine and silvalactam. In this work, we employed different engineering strategies to target glycosylation and acylation biosynthetic machineries within its recently elucidated biosynthetic pathway. Auroramycin analogs with variations in C-, N- methylation, hydroxylation and extender units incorporation were produced and characterized. By comparing the bioactivity profiles of five of these analogs, we determined that unique disaccharide motif of auroramycin is essential for its antimicrobial bioactivity. We further demonstrated that C-methylation of the 3, 5-epi-lemonose unit, which is unique among structurally similar polyene macrolactams, is key to its antifungal activity.},
}

@article {pmid31901636,
year = {2019},
author = {Broeders, M and Herrero-Hernandez, P and Ernst, MPT and van der Ploeg, AT and Pijnappel, WWMP},
title = {Sharpening the Molecular Scissors: Advances in Gene-Editing Technology.},
journal = {iScience},
volume = {23},
number = {1},
pages = {100789},
doi = {10.1016/j.isci.2019.100789},
pmid = {31901636},
issn = {2589-0042},
abstract = {The ability to precisely modify human genes has been made possible by the development of tools such as meganucleases, zinc finger nucleases, TALENs, and CRISPR/Cas. These now make it possible to generate targeted deletions, insertions, gene knock outs, and point variants; to modulate gene expression by targeting transcription factors or epigenetic machineries to DNA; or to target and modify RNA. Endogenous repair mechanisms are used to make the modifications required in DNA; they include non-homologous end joining, homology-directed repair, homology-independent targeted integration, microhomology-mediated end joining, base-excision repair, and mismatch repair. Off-target effects can be monitored using in silico prediction and sequencing and minimized using Cas proteins with higher accuracy, such as high-fidelity Cas9, enhanced-specificity Cas9, and hyperaccurate Cas9. Alternatives to Cas9 have been identified, including Cpf1, Cas12a, Cas12b, and smaller Cas9 orthologs such as CjCas9. Delivery of gene-editing components is performed ex vivo using standard techniques or in vivo using AAV, lipid nanoparticles, or cell-penetrating peptides. Clinical development of gene-editing technology is progressing in several fields, including immunotherapy in cancer treatment, antiviral therapy for HIV infection, and treatment of genetic disorders such as β-thalassemia, sickle cell disease, lysosomal storage disorders, and retinal dystrophy. Here we review these technological advances and the challenges to their clinical implementation.},
}

@article {pmid31901522,
year = {2019},
author = {Chevallereau, A and Meaden, S and Fradet, O and Landsberger, M and Maestri, A and Biswas, A and Gandon, S and van Houte, S and Westra, ER},
title = {Exploitation of the Cooperative Behaviors of Anti-CRISPR Phages.},
journal = {Cell host & microbe},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.chom.2019.12.004},
pmid = {31901522},
issn = {1934-6069},
abstract = {Bacteriophages encoding anti-CRISPR proteins (Acrs) must cooperate to overcome phage resistance mediated by the bacterial immune system CRISPR-Cas, where the first phage blocks CRISPR-Cas immunity in order to allow a second Acr phage to successfully replicate. However, in nature, bacteria are frequently not pre-immunized, and phage populations are often not clonal, exhibiting variations in Acr presence and strength. We explored how interactions between Acr phages and initially sensitive bacteria evolve, both in the presence and absence of competing phages lacking Acrs. We find that Acr phages benefit "Acr-negative" phages by limiting the evolution of CRISPR-based resistance and helping Acr-negative phages to replicate on resistant host sub-populations. These benefits depend on the strength of CRISPR-Cas inhibitors and result in strong Acrs providing smaller fitness advantages than weaker ones when Acr phages compete with Acr-negative phages. These results indicate that different Acr types shape the evolutionary dynamics and social interactions of phage populations in natural communities.},
}

@article {pmid31900288,
year = {2020},
author = {Agudelo, D and Carter, S and Velimirovic, M and Duringer, A and Rivest, JF and Levesque, S and Loehr, J and Mouchiroud, M and Cyr, D and Waters, PJ and Laplante, M and Moineau, S and Goulet, A and Doyon, Y},
title = {Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9.},
journal = {Genome research},
volume = {30},
number = {1},
pages = {107-117},
doi = {10.1101/gr.255414.119},
pmid = {31900288},
issn = {1549-5469},
abstract = {Targeting definite genomic locations using CRISPR-Cas systems requires a set of enzymes with unique protospacer adjacent motif (PAM) compatibilities. To expand this repertoire, we engineered nucleases, cytosine base editors, and adenine base editors from the archetypal Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9) system. We found that St1Cas9 strain variants enable targeting to five distinct A-rich PAMs and provide a structural basis for their specificities. The small size of this ortholog enables expression of the holoenzyme from a single adeno-associated viral vector for in vivo editing applications. Delivery of St1Cas9 to the neonatal liver efficiently rewired metabolic pathways, leading to phenotypic rescue in a mouse model of hereditary tyrosinemia. These robust enzymes expand and complement current editing platforms available for tailoring mammalian genomes.},
}

@article {pmid31896785,
year = {2020},
author = {Broniewski, JM and Meaden, S and Paterson, S and Buckling, A and Westra, ER},
title = {The effect of phage genetic diversity on bacterial resistance evolution.},
journal = {The ISME journal},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41396-019-0577-7},
pmid = {31896785},
issn = {1751-7370},
abstract = {CRISPR-Cas adaptive immune systems are found in bacteria and archaea and provide defence against phage by inserting phage-derived sequences into CRISPR loci on the host genome to provide sequence specific immunological memory against re-infection. Under laboratory conditions the bacterium Pseudomonas aeruginosa readily evolves the high levels of CRISPR-based immunity against clonal populations of its phage DMS3vir, which in turn causes rapid extinction of the phage. However, in nature phage populations are likely to be more genetically diverse, which could theoretically impact the frequency at which CRISPR-based immunity evolves which in turn can alter phage persistence over time. Here we experimentally test these ideas and found that a smaller proportion of infected bacterial populations evolved CRISPR-based immunity against more genetically diverse phage populations, with the majority of the population evolving a sm preventing phage adsorption and providing generalised defence against a broader range of phage genotypes. However, those cells that do evolve CRISPR-based immunity in response to infection with more genetically diverse phage acquire greater numbers of CRISPR memory sequences in order to resist a wider range of phage genotypes. Despite differences in bacterial resistance evolution, the rates of phage extinction were similar in the context of clonal and diverse phage infections suggesting selection for CRISPR-based immunity or sm-based resistance plays a relatively minor role in the ecological dynamics in this study. Collectively, these data help to understand the drivers of CRISPR-based immunity and their consequences for bacteria-phage coexistence, and, more broadly, when generalised defences will be favoured over more specific defences.},
}

@article {pmid31892993,
year = {2020},
author = {Yamaguchi, H and Suzuki, S and Osana, Y and Kawachi, M},
title = {Genomic Characteristics of the Toxic Bloom-Forming Cyanobacterium Microcystis aeruginosa NIES-102.},
journal = {Journal of genomics},
volume = {8},
number = {},
pages = {1-6},
pmid = {31892993},
issn = {1839-9940},
abstract = {Microcystis aeruginosa, a bloom-forming cyanobacterium distributed mainly in freshwater environments, can be divided into at least 12 groups (A-K and X) based on multi-locus phylogenetic analyses. In this study, we characterized the genome of microcystin-producing M. aeruginosa NIES-102, assigned to group A, isolated from Lake Kasumigaura, Japan. The complete genome sequence of M. aeruginosa NIES-102 comprised a 5.87-Mbp circular chromosome containing 5,330 coding sequences. The genome was the largest among all sequenced genomes for the species. In a comparison with the genome of M. aeruginosa NIES-843, which belongs to the same group, the microcystin biosynthetic gene cluster and CRISPR-Cas locus were highly similar. A synteny analysis revealed small-scale rearrangements between the two genomes. Genes encoding transposases were more abundant in these two genomes than in other Microcystis genomes. Our results improve our understanding of structural genomic changes and adaptation to a changing environment in the species.},
}

@article {pmid31890588,
year = {2020},
author = {Walker, JE and Lanahan, AA and Zheng, T and Toruno, C and Lynd, LR and Cameron, JC and Olson, DG and Eckert, CA},
title = {Development of both type I-B and type II CRISPR/Cas genome editing systems in the cellulolytic bacterium Clostridium thermocellum.},
journal = {Metabolic engineering communications},
volume = {10},
number = {},
pages = {e00116},
pmid = {31890588},
issn = {2214-0301},
abstract = {The robust lignocellulose-solubilizing activity of C. thermocellum makes it a top candidate for consolidated bioprocessing for biofuel production. Genetic techniques for C. thermocellum have lagged behind model organisms thus limiting attempts to improve biofuel production. To improve our ability to engineer C. thermocellum, we characterized a native Type I-B and heterologous Type II Clustered Regularly-Interspaced Short Palindromic Repeat (CRISPR)/cas (CRISPR associated) systems. We repurposed the native Type I-B system for genome editing. We tested three thermophilic Cas9 variants (Type II) and found that GeoCas9, isolated from Geobacillus stearothermophilus, is active in C. thermocellum. We employed CRISPR-mediated homology directed repair to introduce a nonsense mutation into pyrF. For both editing systems, homologous recombination between the repair template and the genome appeared to be the limiting step. To overcome this limitation, we tested three novel thermophilic recombinases and demonstrated that exo/beta homologs, isolated from Acidithiobacillus caldus, are functional in C. thermocellum. For the Type I-B system an engineered strain, termed LL1586, yielded 40% genome editing efficiency at the pyrF locus and when recombineering machinery was expressed this increased to 71%. For the Type II GeoCas9 system, 12.5% genome editing efficiency was observed and when recombineering machinery was expressed, this increased to 94%. By combining the thermophilic CRISPR system (either Type I-B or Type II) with the recombinases, we developed a new tool that allows for efficient CRISPR editing. We are now poised to enable CRISPR technologies to better engineer C. thermocellum for both increased lignocellulose degradation and biofuel production.},
}

@article {pmid31890142,
year = {2020},
author = {Liu, G and Zhang, Y and Zhang, T},
title = {Computational approaches for effective CRISPR guide RNA design and evaluation.},
journal = {Computational and structural biotechnology journal},
volume = {18},
number = {},
pages = {35-44},
pmid = {31890142},
issn = {2001-0370},
abstract = {The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/ CRISPR-associated (Cas) system has emerged as the main technology for gene editing. Successful editing by CRISPR requires an appropriate Cas protein and guide RNA. However, low cleavage efficiency and off-target effects hamper the development and application of CRISPR/Cas systems. To predict cleavage efficiency and specificity, numerous computational approaches have been developed for scoring guide RNAs. Most scores are empirical or trained by experimental datasets, and scores are implemented using various computational methods. Herein, we discuss these approaches, focusing mainly on the features or computational methods they utilise. Furthermore, we summarise these tools and give some suggestions for their usage. We also recommend three versatile web-based tools with user-friendly interfaces and preferable functions. The review provides a comprehensive and up-to-date overview of computational approaches for guide RNA design that could help users to select the optimal tools for their research.},
}

@article {pmid31890021,
year = {2019},
author = {Liu, Q and Zhang, Y and Li, F and Li, J and Sun, W and Tian, C},
title = {Upgrading of efficient and scalable CRISPR-Cas-mediated technology for genetic engineering in thermophilic fungus Myceliophthora thermophila.},
journal = {Biotechnology for biofuels},
volume = {12},
number = {},
pages = {293},
pmid = {31890021},
issn = {1754-6834},
abstract = {Background: Thermophilic filamentous fungus Myceliophthora thermophila has great capacity for biomass degradation and is an attractive system for direct production of enzymes and chemicals from plant biomass. Its industrial importance inspired us to develop genome editing tools to speed up the genetic engineering of this fungus. First-generation CRISPR-Cas9 technology was developed in 2017 and, since then, some progress has been made in thermophilic fungi genetic engineering, but a number of limitations remain. They include the need for complex independent expression cassettes for targeting multiplex genomic loci and the limited number of available selectable marker genes.

Results: In this study, we developed an Acidaminococcus sp. Cas12a-based CRISPR system for efficient multiplex genome editing, using a single-array approach in M. thermophila. These CRISPR-Cas12a cassettes worked well for simultaneous multiple gene deletions/insertions. We also developed a new simple approach for marker recycling that relied on the novel cleavage activity of the CRISPR-Cas12a system to make DNA breaks in selected markers. We demonstrated its performance by targeting nine genes involved in the cellulase production pathway in M. thermophila via three transformation rounds, using two selectable markers neo and bar. We obtained the nonuple mutant M9 in which protein productivity and lignocellulase activity were 9.0- and 18.5-fold higher than in the wild type. We conducted a parallel investigation using our transient CRISPR-Cas9 system and found the two technologies were complementary. Together we called them CRISPR-Cas-assisted marker recycling technology (Camr technology).

Conclusions: Our study described new approaches (Camr technology) that allow easy and efficient marker recycling and iterative stacking of traits in the same thermophilic fungus strain either, using the newly established CRISPR-Cas12a system or the established CRISPR-Cas9 system. This Camr technology will be a versatile and efficient tool for engineering, theoretically, an unlimited number of genes in fungi. We expect this advance to accelerate biotechnology-oriented engineering processes in fungi.},
}

@article {pmid31883145,
year = {2019},
author = {Xiong, Q and Xie, C and Zhang, Z and Liu, L and Powell, JT and Shen, Q and Lin, C},
title = {DNA origami post-processing by CRISPR-Cas12a.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {},
number = {},
pages = {},
doi = {10.1002/anie.201915555},
pmid = {31883145},
issn = {1521-3773},
support = {DP2 GM114830/GM/NIGMS NIH HHS/United States ; R01 GM132114/GM/NIGMS NIH HHS/United States ; },
abstract = {Customizable nanostructures built through the DNA-origami technique hold tremendous promise in nanomaterial fabrication and biotechnology. Despite the cutting-edge tools for DNA-origami design and preparation, it remains challenging to separate structural components of an architecture built from - thus held together by - a continuous scaffold strand, which in turn limits the modularity and function of the DNA-origami devices. To address this challenge, here we present an enzymatic method to clean up and reconfigure DNA-origami structures. We target single-stranded (ss) regions of DNA-origami structures and remove them with CRISPR-Cas12a, a hyper-active ssDNA endonuclease without sequence specificity. We demonstrate the utility of this facile, selective post-processing method on DNA structures with various geometrical and mechanical properties, realizing intricate structures and structural transformations that were previously difficult to engineer. Given the biocompatibility of Cas12a-like enzymes, this versatile tool may be programmed in the future to operate functional nanodevices in cells.},
}

@article {pmid31882576,
year = {2019},
author = {Heffel, MG and Finnigan, GC},
title = {Mathematical modeling of self-contained CRISPR gene drive reversal systems.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {20050},
pmid = {31882576},
issn = {2045-2322},
support = {Hatch Project 1013520//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; },
abstract = {There is a critical need for further research into methods to control biological populations. Numerous challenges to agriculture, ecological systems, and human health could be mitigated by the targeted reduction and management of key species (e.g. pests, parasites, and vectors for pathogens). The discovery and adaptation of the CRISPR/Cas editing platform co-opted from bacteria has provided a mechanism for a means to alter an entire population. A CRISPR-based gene drive system can allow for the forced propagation of a genetic element that bypasses Mendelian inheritance which can be used to bias sex determination, install exogenous information, or remove endogenous DNA within an entire species. Laboratory studies have demonstrated the potency by which gene drives can operate within insects and other organisms. However, continued research and eventual application face serious opposition regarding issues of policy, biosafety, effectiveness, and reversal. Previous mathematical work has suggested the use of modified gene drive designs that are limited in spread such as daisy chain or underdominance drives. However, no system has yet been proposed that allows for an inducible reversal mechanism without requiring the introduction of additional individuals. Here, we study gene drive effectiveness, fitness, and inducible drive systems that could respond to external stimuli expanding from a previous frequency-based population model. We find that programmed modification during gene drive propagation could serve as a potent safeguard to either slow or completely reverse drive systems and allow for a return to the original wild-type population.},
}

@article {pmid31880137,
year = {2019},
author = {Chen, X and Chen, L and Li, D},
title = {[Research progress of gene therapy in clinical application].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {35},
number = {12},
pages = {2295-2307},
doi = {10.13345/j.cjb.190363},
pmid = {31880137},
issn = {1000-3061},
mesh = {*CRISPR-Cas Systems ; Gene Editing ; *Genetic Therapy ; Humans ; },
abstract = {In the 1960s, scientists first raised the idea of curing genetic diseases using gene therapy. This new conceptual strategy aimed to achieve a much longer therapeutic effect by introducing exogenous genetic materials into the patients. After more than five decades of ups and downs, gene therapy has been brought into a new era by those milestone breakthroughs in the 21st century. Here we reviewed and summarized the history and breakthroughs of gene therapy, including some critical clinical trials, approved drugs, and emerging gene editing techniques. We believe that with their unique advantages over traditional therapies, more gene therapies will become practical approaches to genetic diseases and benefit the entire human race.},
}

*CRISPR-Cas Systems
Gene Editing
*Genetic Therapy
Humans

@article {pmid31879772,
year = {2019},
author = {Pinilla-Redondo, R and Mayo-Muñoz, D and Russel, J and Garrett, RA and Randau, L and Sørensen, SJ and Shah, SA},
title = {Type IV CRISPR-Cas systems are highly diverse and involved in competition between plasmids.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkz1197},
pmid = {31879772},
issn = {1362-4962},
abstract = {CRISPR-Cas systems provide prokaryotes with adaptive immune functions against viruses and other genetic parasites. In contrast to all other types of CRISPR-Cas systems, type IV has remained largely overlooked. Here, we describe a previously uncharted diversity of type IV gene cassettes, primarily encoded by plasmid-like elements from diverse prokaryotic taxa. Remarkably, via a comprehensive analysis of their CRISPR spacer content, these systems were found to exhibit a strong bias towards the targeting of other plasmids. Our data indicate that the functions of type IV systems have diverged from those of other host-related CRISPR-Cas immune systems to adopt a role in mediating conflicts between plasmids. Furthermore, we find evidence for cross-talk between certain type IV and type I CRISPR-Cas systems that co-exist intracellularly, thus providing a simple answer to the enigmatic absence of type IV adaptation modules. Collectively, our results lead to the expansion and reclassification of type IV systems and provide novel insights into the biological function and evolution of these elusive systems.},
}

@article {pmid31876473,
year = {2019},
author = {McConnell, SC},
title = {An Exclusive Interview With CRISPR.},
journal = {AMA journal of ethics},
volume = {21},
number = {12},
pages = {E1079-1088},
doi = {10.1001/amajethics.2019.1079},
pmid = {31876473},
issn = {2376-6980},
abstract = {This article chronicles a didactic encounter between an ethics-minded physician-scientist and a personified genome editing technology called clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins, commonly abbreviated as CRISPR/Cas, or simply CRISPR. The interview considers clinically and ethically relevant questions about this technology related to patient safety, therapeutic efficacy, equitable access, and global governance of humanity's genetic legacy.},
}

@article {pmid31876277,
year = {2019},
author = {Lomov, NA and Viushkov, VS and Petrenko, AP and Syrkina, MS and Rubtsov, MA},
title = {[Methods of Evaluating the Efficiency of CRISPR/Cas Genome Editing].},
journal = {Molekuliarnaia biologiia},
volume = {53},
number = {6},
pages = {982-997},
doi = {10.1134/S0026898419060119},
pmid = {31876277},
issn = {0026-8984},
abstract = {The CRISPR/Cas system is currently widely used for genome editing. The procedure of genome editing includes two necessary steps: (i) searching for the most effective guide RNA, and (ii) analyzing clones for presence of the desired mutation. This review presents the methods used to assess the efficiency of the CRISPR/Cas system and to confirm mutation in the target locus and discusses their advantages and disadvantages. It aims to provide information that could help researchers to choose a technique most appropriate for their specific tasks and available resources.},
}

@article {pmid31875674,
year = {2020},
author = {Wang, R and Zhao, X and Chen, X and Qiu, X and Qing, G and Zhang, H and Zhang, L and Hu, X and He, Z and Zhong, D and Wang, Y and Luo, Y},
title = {Rolling Circular Amplification (RCA)-Assisted CRISPR/Cas9 Cleavage (RACE) for Highly Specific Detection of Multiple Extracellular Vesicle MicroRNAs.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.9b04814},
pmid = {31875674},
issn = {1520-6882},
abstract = {Multiplexed detection of extracellular vesicle (EV)-derived microRNAs (miRNAs) plays a critical role in facilitating disease diagnosis and prognosis evaluation. Herein, we developed a highly specific nucleic acid detection platform for simultaneous quantification of several EV-derived miRNAs in constant temperature by integrating the advantages of a clustered regularly interspaced short palindromic repeats/CRISPR associated nucleases (CRISPR/Cas) system and rolling circular amplification (RCA) techniques. Particularly, the proposed approach demonstrated single-base resolution attributed to the dual-specific recognition from both padlock probe-mediated ligation and protospacer adjacent motif (PAM)-triggered cleavage. The high consistency between the proposed approach RCA-assisted CRISPR/Cas9 cleavage (RACE) and reverse transcription quantitative polymerase chain reaction (RT-qPCR) in detecting EV-derived miRNAs' abundance from both cultured cancer cells and clinical lung cancer patients validated its robustness, revealing its potentials in the screening, diagnosis, and prognosis of various diseases. In summary, RACE is a powerful tool for multiplexed, specific detection of nucleic acids in point-of-care diagnostics and field-deployable analysis.},
}

@article {pmid31875277,
year = {2019},
author = {Peng, Y and Yang, T and Tang, X and Chen, F and Wang, S},
title = {Construction of an Inducible CRISPR/Cas9 System for CXCR4 Gene and Demonstration of its Effects on MKN-45 Cells.},
journal = {Cell biochemistry and biophysics},
volume = {},
number = {},
pages = {},
doi = {10.1007/s12013-019-00898-x},
pmid = {31875277},
issn = {1559-0283},
support = {30700785//National Natural Science Foundation of China/ ; 2015MSXM040//the Chongqing Health and Family Planning Commission/ ; },
abstract = {The CRISPR/Cas9 system is an effective tool for gene editing. However, this conventional expression system cannot control the timing of gene editing and does not utilize resistance screening markers. Therefore, carrying out CRISPR/Cas9 experiments is extremely inconvenient. Our aim is to develop an inducible lentiviral vector-based gene-editing system for C-X-C chemokine receptor 4 (CXCR4) by CRISPR/Cas9, and to demonstrate its function in MKN-45 cell. The DNA fragments of Blasticidin and T2A-GFP were produced using the lenti-Cas9-BLAST and PX458 plasmids as templates. The PCR products were harvested and cloned into the lenti-guide-puro plasmid to yield the lenti-guide-BLAST-GFP plasmid. Three double-stranded guide RNA (gRNA) sequences targeting the exon 2 of CXCR4 gene were designed online (http://crispr.mit.edu), synthesized, and recombined into the lenti-guide-BLAST-GFP plasmid, to yield the lenti-guide-BLAST-GFP-gRNA plasmid. The pCW-Cas9 and lenti-guide-BLAST-GFP-gRNA plasmids were packaged with lentiviral vectors, which were then transfected into MKN-45 cells, to identify the CXCR4 gene-editing effects using the T7 endonuclease 1 (T7E1) and Western blot assays. The lenti-guide-BLAST-GFP and lenti-guide-BLAST-GFP-gRNA plasmids were successfully constructed and packaged, to yield lentiviral particles. Transfection of the pCW-Cas9 and lenti-guide-BLAST-GFP-gRNA viral vectors could decrease the expression of CXCR4 protein, and lead to gene editing in MKN-45 cells. The efficiencies of gRNA-1, gRNA-2, and gRNA-3 were 45.6%, 53.6%, and 56.7%, respectively. Furthermore, the chemotactic efficiency of the dual viral vector-infected MKN-45 cells was significantly decreased in response to SDF-1. The numbers of migratory cells in the lower chamber of the transwell system were 30.0 ± 0.23, 29.7 ± 1.55, 28.2 ± 1.11 and 36.1 ± 2.00 cells per field (400×) for gRNA-1, gRNA-2, gRNA-3 and the control, respectively (P < 0.05). We constructed an inducible CXCR4 gene-editing, dual-vector CRISPR/Cas9 system, which could induce CXCR4 gene editing in MKN-45 cells in a doxycycline-dependent manner and thus reduce the migration of MKN-45 cells.},
}

@article {pmid31872379,
year = {2019},
author = {Zhao, Y and Tian, J and Zheng, G and Chen, J and Sun, C and Yang, Z and Zimin, AA and Jiang, W and Deng, Z and Wang, Z and Lu, Y},
title = {Multiplex genome editing using a dCas9-cytidine deaminase fusion in Streptomyces.},
journal = {Science China. Life sciences},
volume = {},
number = {},
pages = {},
doi = {10.1007/s11427-019-1559-y},
pmid = {31872379},
issn = {1869-1889},
abstract = {CRISPR/Cas-mediated genome editing has greatly facilitated the study of gene function in Streptomyces. However, it could not be efficiently employed in streptomycetes with low homologous recombination (HR) ability. Here, a deaminase-assisted base editor dCas9-CDA-ULstr was developed in Streptomyces, which comprises the nuclease-deficient Cas9 (dCas9), the cytidine deaminase from Petromyzon marinus (PmCDA1), the uracil DNA glycosylase inhibitor (UGI) and the protein degradation tag (LVA tag). Using dCas9-CDA-ULstr, we achieved single-, double- and triple-point mutations (cytosine-to-thymine substitutions) at target sites in Streptomyces coelicolor with efficiency up to 100%, 60% and 20%, respectively. This base editor was also demonstrated to be highly efficient for base editing in the industrial strain, Streptomyces rapamycinicus, which produces the immunosuppressive agent rapamycin. Compared with base editors derived from the cytidine deaminase rAPOBEC1, the PmCDA1-assisted base editor dCas9-CDA-ULstr could edit cytosines preceded by guanosines with high efficiency, which is a great advantage for editing Streptomyces genomes (with high GC content). Collectively, the base editor dCas9-CDA-ULstr could be employed for efficient multiplex genome editing in Streptomyces. Since the dCas9-CDA-ULstr-based genome editing is independent of HR-mediated DNA repair, we believe this technology will greatly facilitate functional genome research and metabolic engineering in Streptomyces strains with weak HR ability.},
}

@article {pmid31872209,
year = {2019},
author = {Swartjes, T and Staals, RHJ and van der Oost, J},
title = {Editor's cut: DNA cleavage by CRISPR RNA-guided nucleases Cas9 and Cas12a.},
journal = {Biochemical Society transactions},
volume = {},
number = {},
pages = {},
doi = {10.1042/BST20190563},
pmid = {31872209},
issn = {1470-8752},
abstract = {Discovered as an adaptive immune system of prokaryotes, CRISPR-Cas provides many promising applications. DNA-cleaving Cas enzymes like Cas9 and Cas12a, are of great interest for genome editing. The specificity of these DNA nucleases is determined by RNA guides, providing great targeting adaptability. Besides this general method of programmable DNA cleavage, these nucleases have different biochemical characteristics, that can be exploited for different applications. Although Cas nucleases are highly promising, some room for improvement remains. New developments and discoveries like base editing, prime editing, and CRISPR-associated transposons might address some of these challenges.},
}

@article {pmid31862124,
year = {2020},
author = {Li, B and Niu, Y and Ji, W and Dong, Y},
title = {Strategies for the CRISPR-Based Therapeutics.},
journal = {Trends in pharmacological sciences},
volume = {41},
number = {1},
pages = {55-65},
doi = {10.1016/j.tips.2019.11.006},
pmid = {31862124},
issn = {1873-3735},
abstract = {The CRISPR (clustered regularly interspaced short palindromic repeats)-based genome editing technology is an emerging RNA-guided nuclease system initially identified from the microbial adaptive immune systems. In recent years, the CRISPR system has been reprogrammed to target specific regions of the eukaryotic genome and has become a powerful tool for genetic engineering. Researchers have explored many approaches to improve the genome editing activity of the CRISPR-Cas system and deliver its components both ex vivo and in vivo. Moreover, these strategies have been applied to genome editing in preclinical research and clinical trials. In this review, we focus on representative strategies for regulation and delivery of the CRISPR-Cas system, and outline current therapeutic applications in their clinical translation.},
}

@article {pmid31861790,
year = {2019},
author = {Hassa, J and Wibberg, D and Maus, I and Pühler, A and Schlüter, A},
title = {Genome Analyses and Genome-Centered Metatranscriptomics of Methanothermobacter wolfeii Strain SIV6, Isolated from a Thermophilic Production-Scale Biogas Fermenter.},
journal = {Microorganisms},
volume = {8},
number = {1},
pages = {},
doi = {10.3390/microorganisms8010013},
pmid = {31861790},
issn = {2076-2607},
support = {FKZ 22404015//Bundesministerium für Ernährung und Landwirtschaft/ ; },
abstract = {In the thermophilic biogas-producing microbial community, the genus Methanothermobacter was previously described to be frequently abundant. The aim of this study was to establish and analyze the genome sequence of the archaeal strain Methanothermobacter wolfeii SIV6 originating from a thermophilic industrial-scale biogas fermenter and compare it to related reference genomes. The circular chromosome has a size of 1,686,891 bases, featuring a GC content of 48.89%. Comparative analyses considering three completely sequenced Methanothermobacter strains revealed a core genome of 1494 coding sequences and 16 strain specific genes for M. wolfeii SIV6, which include glycosyltransferases and CRISPR/cas associated genes. Moreover, M. wolfeii SIV6 harbors all genes for the hydrogenotrophic methanogenesis pathway and genome-centered metatranscriptomics indicates the high metabolic activity of this strain, with 25.18% of all transcripts per million (TPM) belong to the hydrogenotrophic methanogenesis pathway and 18.02% of these TPM exclusively belonging to the mcr operon. This operon encodes the different subunits of the enzyme methyl-coenzyme M reductase (EC: 2.8.4.1), which catalyzes the final and rate-limiting step during methanogenesis. Finally, fragment recruitment of metagenomic reads from the thermophilic biogas fermenter on the SIV6 genome showed that the strain is abundant (1.2%) within the indigenous microbial community. Detailed analysis of the archaeal isolate M. wolfeii SIV6 indicates its role and function within the microbial community of the thermophilic biogas fermenter, towards a better understanding of the biogas production process and a microbial-based management of this complex process.},
}

@article {pmid31858277,
year = {2019},
author = {Van Vu, T and Sung, YW and Kim, J and Doan, DTH and Tran, MT and Kim, JY},
title = {Challenges and Perspectives in Homology-Directed Gene Targeting in Monocot Plants.},
journal = {Rice (New York, N.Y.)},
volume = {12},
number = {1},
pages = {95},
pmid = {31858277},
issn = {1939-8425},
support = {2017R1A4A1015515//National Research Foundation of Korea/ ; PJ01322601//the Next-Generation BioGreen 21 Program (SSAC, Grant PJ01322601/ ; },
abstract = {Continuing crop domestication/redomestication and modification is a key determinant of the adaptation and fulfillment of the food requirements of an exploding global population under increasingly challenging conditions such as climate change and the reduction in arable lands. Monocotyledonous crops are not only responsible for approximately 70% of total global crop production, indicating their important roles in human life, but also the first crops to be challenged with the abovementioned hurdles; hence, monocot crops should be the first to be engineered and/or de novo domesticated/redomesticated. A long time has passed since the first green revolution; the world is again facing the challenge of feeding a predicted 9.7 billion people in 2050, since the decline in world hunger was reversed in 2015. One of the major lessons learned from the first green revolution is the importance of novel and advanced trait-carrying crop varieties that are ideally adapted to new agricultural practices. New plant breeding techniques (NPBTs), such as genome editing, could help us succeed in this mission to create novel and advanced crops. Considering the importance of NPBTs in crop genetic improvement, we attempt to summarize and discuss the latest progress with major approaches, such as site-directed mutagenesis using molecular scissors, base editors and especially homology-directed gene targeting (HGT), a very challenging but potentially highly precise genome modification approach in plants. We therefore suggest potential approaches for the improvement of practical HGT, focusing on monocots, and discuss a potential approach for the regulation of genome-edited products.},
}

@article {pmid31857715,
year = {2019},
author = {Makarova, KS and Wolf, YI and Iranzo, J and Shmakov, SA and Alkhnbashi, OS and Brouns, SJJ and Charpentier, E and Cheng, D and Haft, DH and Horvath, P and Moineau, S and Mojica, FJM and Scott, D and Shah, SA and Siksnys, V and Terns, MP and Venclovas, Č and White, MF and Yakunin, AF and Yan, W and Zhang, F and Garrett, RA and Backofen, R and van der Oost, J and Barrangou, R and Koonin, EV},
title = {Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants.},
journal = {Nature reviews. Microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41579-019-0299-x},
pmid = {31857715},
issn = {1740-1534},
support = {R35 GM118160/GM/NIGMS NIH HHS/United States ; },
abstract = {The number and diversity of known CRISPR-Cas systems have substantially increased in recent years. Here, we provide an updated evolutionary classification of CRISPR-Cas systems and cas genes, with an emphasis on the major developments that have occurred since the publication of the latest classification, in 2015. The new classification includes 2 classes, 6 types and 33 subtypes, compared with 5 types and 16 subtypes in 2015. A key development is the ongoing discovery of multiple, novel class 2 CRISPR-Cas systems, which now include 3 types and 17 subtypes. A second major novelty is the discovery of numerous derived CRISPR-Cas variants, often associated with mobile genetic elements that lack the nucleases required for interference. Some of these variants are involved in RNA-guided transposition, whereas others are predicted to perform functions distinct from adaptive immunity that remain to be characterized experimentally. The third highlight is the discovery of numerous families of ancillary CRISPR-linked genes, often implicated in signal transduction. Together, these findings substantially clarify the functional diversity and evolutionary history of CRISPR-Cas.},
}

@article {pmid31857428,
year = {2019},
author = {Benati, D and Patrizi, C and Recchia, A},
title = {Gene editing prospects for treating inherited retinal diseases.},
journal = {Journal of medical genetics},
volume = {},
number = {},
pages = {},
doi = {10.1136/jmedgenet-2019-106473},
pmid = {31857428},
issn = {1468-6244},
abstract = {Retinal diseases (RD) include inherited retinal dystrophy (IRD), for example, retinitis pigmentosa and Leber's congenital amaurosis, or multifactorial forms, for example, age-related macular degeneration (AMD). IRDs are clinically and genetically heterogeneous in nature. To date, more than 200 genes are known to cause IRDs, which perturb the development, function and survival of rod and cone photoreceptors or retinal pigment epithelial cells. Conversely, AMD, the most common cause of blindness in the developed world, is an acquired disease of the macula characterised by progressive visual impairment. To date, available therapeutic approaches for RD include nutritional supplements, neurotrophic factors, antiangiogenic drugs for wet AMD and gene augmentation/interference strategy for IRDs. However, these therapies do not aim at correcting the genetic defect and result in inefficient and expensive treatments. The genome editing technology based on clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein (Cas) and an RNA that guides the Cas protein to a predetermined region of the genome, represents an attractive strategy to tackle IRDs without available cure. Indeed, CRISPR/Cas system can permanently and precisely replace or remove genetic mutations causative of a disease, representing a molecular tool to cure a genetic disorder. In this review, we will introduce the mechanism of CRISPR/Cas system, presenting an updated panel of Cas variants and delivery systems, then we will focus on applications of CRISPR/Cas genome editing in the retina, and, as emerging treatment options, in patient-derived induced pluripotent stem cells followed by transplantation of retinal progenitor cells into the eye.},
}

@article {pmid31853065,
year = {2020},
author = {Halpin-Healy, TS and Klompe, SE and Sternberg, SH and Fernández, IS},
title = {Structural basis of DNA targeting by a transposon-encoded CRISPR-Cas system.},
journal = {Nature},
volume = {577},
number = {7789},
pages = {271-274},
doi = {10.1038/s41586-019-1849-0},
pmid = {31853065},
issn = {1476-4687},
support = {GM103310/GM/NIGMS NIH HHS/United States ; },
abstract = {Bacteria use adaptive immune systems encoded by CRISPR and Cas genes to maintain genomic integrity when challenged by pathogens and mobile genetic elements1-3. Type I CRISPR-Cas systems typically target foreign DNA for degradation via joint action of the ribonucleoprotein complex Cascade and the helicase-nuclease Cas34,5, but nuclease-deficient type I systems lacking Cas3 have been repurposed for RNA-guided transposition by bacterial Tn7-like transposons6,7. How CRISPR- and transposon-associated machineries collaborate during DNA targeting and insertion remains unknown. Here we describe structures of a TniQ-Cascade complex encoded by the Vibrio cholerae Tn6677 transposon using cryo-electron microscopy, revealing the mechanistic basis of this functional coupling. The cryo-electron microscopy maps enabled de novo modelling and refinement of the transposition protein TniQ, which binds to the Cascade complex as a dimer in a head-to-tail configuration, at the interface formed by Cas6 and Cas7 near the 3' end of the CRISPR RNA (crRNA). The natural Cas8-Cas5 fusion protein binds the 5' crRNA handle and contacts the TniQ dimer via a flexible insertion domain. A target DNA-bound structure reveals critical interactions necessary for protospacer-adjacent motif recognition and R-loop formation. This work lays the foundation for a structural understanding of how DNA targeting by TniQ-Cascade leads to downstream recruitment of additional transposase proteins, and will guide protein engineering efforts to leverage this system for programmable DNA insertions in genome-engineering applications.},
}

@article {pmid31853027,
year = {2020},
author = {Neff, EP},
title = {CRISPR takes genetic screens forward.},
journal = {Lab animal},
volume = {49},
number = {1},
pages = {13-16},
doi = {10.1038/s41684-019-0445-0},
pmid = {31853027},
issn = {1548-4475},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Early Detection of Cancer ; Humans ; Immunotherapy ; *Neoplasms ; Protein Tyrosine Phosphatase, Non-Receptor Type 2 ; },
}

CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Early Detection of Cancer
Humans
Immunotherapy
*Neoplasms
Protein Tyrosine Phosphatase, Non-Receptor Type 2

@article {pmid31852806,
year = {2019},
author = {Nixon, SL and Daly, RA and Borton, MA and Solden, LM and Welch, SA and Cole, DR and Mouser, PJ and Wilkins, MJ and Wrighton, KC},
title = {Genome-Resolved Metagenomics Extends the Environmental Distribution of the Verrucomicrobia Phylum to the Deep Terrestrial Subsurface.},
journal = {mSphere},
volume = {4},
number = {6},
pages = {},
pmid = {31852806},
issn = {2379-5042},
abstract = {Bacteria of the phylum Verrucomicrobia are prevalent and are particularly common in soil and freshwater environments. Their cosmopolitan distribution and reported capacity for polysaccharide degradation suggests members of Verrucomicrobia are important contributors to carbon cycling across Earth's ecosystems. Despite their prevalence, the Verrucomicrobia are underrepresented in isolate collections and genome databases; consequently, their ecophysiological roles may not be fully realized. Here, we expand genomic sampling of the Verrucomicrobia phylum by describing a novel genus, "Candidatus Marcellius," belonging to the order Opitutales "Ca. Marcellius" was recovered from a shale-derived produced fluid metagenome collected 313 days after hydraulic fracturing, the deepest environment from which a member of the Verrucomicrobia has been recovered to date. We uncover genomic attributes that may explain the capacity of this organism to inhabit a shale gas well, including the potential for utilization of organic polymers common in hydraulic fracturing fluids, nitrogen fixation, adaptation to high salinities, and adaptive immunity via CRISPR-Cas. To illuminate the phylogenetic and environmental distribution of these metabolic and adaptive traits across the Verrucomicrobia phylum, we performed a comparative genomic analysis of 31 publicly available, nearly complete Verrucomicrobia genomes. Our genomic findings extend the environmental distribution of the Verrucomicrobia 2.3 kilometers into the terrestrial subsurface. Moreover, we reveal traits widely encoded across members of the Verrucomicrobia, including the capacity to degrade hemicellulose and to adapt to physical and biological environmental perturbations, thereby contributing to the expansive habitat range reported for this phylum.IMPORTANCE The Verrucomicrobia phylum of bacteria is widespread in many different ecosystems; however, its role in microbial communities remains poorly understood. Verrucomicrobia are often low-abundance community members, yet previous research suggests they play a major role in organic carbon degradation. While Verrucomicrobia remain poorly represented in culture collections, numerous genomes have been reconstructed from metagenomic data sets in recent years. The study of genomes from across the phylum allows for an extensive assessment of their potential ecosystem roles. The significance of this work is (i) the recovery of a novel genus of Verrucomicrobia from 2.3 km in the subsurface with the ability to withstand the extreme conditions that characterize this environment, and (ii) the most extensive assessment of ecophysiological traits encoded by Verrucomicrobia genomes to date. We show that members of this phylum are specialist organic polymer degraders that can withstand a wider range of environmental conditions than previously thought.},
}

@article {pmid31852637,
year = {2019},
author = {Lei, T and Xiao, B and He, Y and Qu, J and Sun, Z and Li, L},
title = {[Development and applications of CRISPR/Cas9 library screening technology in cancer research].},
journal = {Nan fang yi ke da xue xue bao = Journal of Southern Medical University},
volume = {39},
number = {11},
pages = {1381-1386},
doi = {10.12122/j.issn.1673-4254.2019.11.18},
pmid = {31852637},
issn = {1673-4254},
mesh = {Animals ; *CRISPR-Cas Systems ; Early Detection of Cancer ; Gene Knockout Techniques ; Gene Library ; Mice ; },
abstract = {The CRISPR/Cas9 technology has developed rapidly in recent years with fast, simple and accurate editing functions to allow gene knockout, knock in, activation and interference. It has become a powerful genetic screening tool and been widely used in various models including cell lines, mice and zebrafish. The application of CRISPR system in constructing genome library for high-throughput screening is the main strategy for target gene research of diseases, especially neoplasms. Here we summarize the rationales and recent development of CRISPR/Cas9 library screening technology, the strategies for improving the off-target effects, the basic workflow of library screening and the application of this technology in tumor research.},
}

Animals
*CRISPR-Cas Systems
Early Detection of Cancer
Gene Knockout Techniques
Gene Library
Mice

@article {pmid31850661,
year = {2019},
author = {Hussain, Q and Shi, J and Scheben, A and Zhan, J and Wang, X and Liu, G and Yan, G and King, GJ and Edwards, D and Wang, H},
title = {Genetic and signaling pathways of dry fruit size: targets for genome editing based crop improvement.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.13318},
pmid = {31850661},
issn = {1467-7652},
abstract = {Fruit are seed-bearing structures specific to angiosperm that form from the gynoecium after flowering. Fruit size is an important fitness character for plant evolution and an agronomical trait for crop domestication/improvement. Despite the functional and economic importance of fruit size, the underlying genes and mechanisms are poorly understood, especially for dry dehiscent fruit types. Improving our understanding of the genomic basis for fruit size opens the potential to apply gene-editing technology such as CRISPR/Cas to modulate fruit size in a range of species. This review examines the genes involved in the regulation of fruit size and identifies their genetic/signaling pathways, including the phytohormones, transcription and elongation factors, ubiquitin-proteasome, and microRNA pathways, G-protein and receptor kinases signaling, arabinogalactan, and RNA-binding proteins. Interestingly, different plant taxa have conserved functions for various fruit size regulators, suggesting that common genome edits across species may have similar outcomes. Many fruit size regulators identified to date are pleiotropic and affect other organs such as seeds, flowers, and leaves, indicating a coordinated regulation. The relationships between fruit size and fruit number/seed number per fruit/seed size, as well as future research questions, are also discussed.},
}

@article {pmid31848272,
year = {2019},
author = {Van Goethem, MW and Swenson, TL and Trubl, G and Roux, S and Northen, TR},
title = {Characteristics of Wetting-Induced Bacteriophage Blooms in Biological Soil Crust.},
journal = {mBio},
volume = {10},
number = {6},
pages = {},
pmid = {31848272},
issn = {2150-7511},
abstract = {Biological soil crusts (biocrusts) are photosynthetic "hot spots" in deserts and cover ∼12% of the Earth's terrestrial surface, and yet they face an uncertain future given expected shifts in rainfall events. Laboratory wetting of biocrust communities is known to cause a bloom of Firmicutes which rapidly become dominant community members within 2 days after emerging from a sporulated state. We hypothesized that their bacteriophages (phages) would respond to such a dramatic increase in their host's abundance. In our experiment, wetting caused Firmicutes to bloom and triggered a significant depletion of cyanobacterial diversity. We used genome-resolved metagenomics to link phage to their hosts and found that the bloom of the genus Bacillus correlated with a dramatic increase in the number of Caudovirales phages targeting these diverse spore-formers (r = 0.762). After 2 days, we observed dramatic reductions in the relative abundances of Bacillus, while the number of Bacillus phages continued to increase, suggestive of a predator-prey relationship. We found predicted auxiliary metabolic genes (AMGs) associated with sporulation in several Caudovirales genomes, suggesting that phages may influence and even benefit from sporulation dynamics in biocrusts. Prophage elements and CRISPR-Cas repeats in Firmicutes metagenome-assembled genomes (MAGs) provide evidence of recent infection events by phages, which were corroborated by mapping viral contigs to their host MAGs. Combined, these findings suggest that the blooming Firmicutes become primary targets for biocrust Caudovirales phages, consistent with the classical "kill-the-winner" hypothesis.IMPORTANCE This work forms part of an overarching research theme studying the effects of a changing climate on biological soil crust (biocrust) in the Southwestern United States. To our knowledge, this study was the first to characterize bacteriophages in biocrust and offers a view into the ecology of phages in response to a laboratory wetting experiment. The phages identified here represent lineages of Caudovirales, and we found that the dynamics of their interactions with their Firmicutes hosts explain the collapse of a bacterial bloom that was induced by wetting. Moreover, we show that phages carried host-altering metabolic genes and found evidence of proviral infection and CRISPR-Cas repeats within host genomes. Our results suggest that phages exert controls on population density by lysing dominant bacterial hosts and that they further impact biocrust by acquiring host genes for sporulation. Future research should explore how dominant these phages are in other biocrust communities and quantify how much the control and lysis of blooming populations contributes to nutrient cycling in biocrusts.},
}

@article {pmid31845410,
year = {2019},
author = {Tsoumani, KT and Meccariello, A and Mathiopoulos, KD and Papathanos, PA},
title = {Developing CRISPR-based sex-ratio distorters for the genetic control of fruit fly pests: A how to manual.},
journal = {Archives of insect biochemistry and physiology},
volume = {},
number = {},
pages = {e21652},
doi = {10.1002/arch.21652},
pmid = {31845410},
issn = {1520-6327},
support = {MIS-5001552//Hellenic State Scholarship Foundation (IKY)/ ; 79 0 4.02.2014//Italian Ministry Education, University and Research (MIUR)/ ; IS-5180-19//United States-Israel Binational Agricultural Research and Development Fund (BARD)/ ; 2388/19//Israel Science Foundation (ISF)/ ; },
abstract = {Agricultural pest control using genetic-based methods provides a species-specific and environmentally harmless way for population suppression of fruit flies. One way to improve the efficiency of such methods is through self-limiting, female-eliminating approaches that can alter an insect populations' sex ratio toward males. In this microreview, we summarize recent advances in synthetic sex ratio distorters based on X-chromosome shredding that can induce male-biased progeny. We outline the basic principles to guide the efficient design of an X-shredding system in an XY heterogametic fruit fly species of interest using CRISPR/Cas gene editing, newly developed computational tools, and insect genetic engineering. We also discuss technical aspects and challenges associated with the efficient transferability of this technology in fruit fly pest populations, toward the potential use of this new class of genetic control approaches for pest management purposes.},
}

@article {pmid31843968,
year = {2019},
author = {Takeishi, Y and Fujikane, R and Rikitake, M and Obayashi, Y and Sekiguchi, M and Hidaka, M},
title = {SMARCAD1-mediated recruitment of the DNA mismatch repair protein MutLα to MutSα on damaged chromatin induces apoptosis in human cells.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1074/jbc.RA119.008854},
pmid = {31843968},
issn = {1083-351X},
abstract = {The mismatch repair (MMR) complex is composed of MutSα (MSH2-MSH6) and MutLα (MLH1-PMS2) and specifically recognizes mismatched bases during DNA replication. O6-methylguanine is produced by treatment with alkylating agents such as N-methyl-N-nitrosourea (MNU) and during DNA replication forms a DNA mismatch, i.e. an O6-methylguanine/thymine pair, and induces a G/C to A/T transition mutation. To prevent this outcome, cells carrying this DNA mismatch are eliminated by MMR-dependent apoptosis, but the underlying molecular mechanism is unclear. In this study, we provide evidence that the chromatin-regulatory and ATP-dependent nucleosome-remodeling protein SMARCAD1 is involved in the induction of MMR-dependent apoptosis in human cells. Unlike control cells, SMARCAD1-knockout cells (∆SMARCAD1) were MNU resistant, and the appearances of a sub-G1 population and caspase-9 activation were significantly suppressed in the ∆SMARCAD1 cells. Furthermore, the MNU-induced mutation frequencies were increased in these cells. Immunoprecipitation analyses revealed that the recruitment of MutLα to chromatin-bound MutSα, observed in SMARCAD1-proficient cells, is suppressed in ∆SMARCAD1 cells. Of note, the effect of SMARCAD1 on the recruitment of MutLα exclusively depended on the ATPase activity of the protein. On the basis of these findings, we propose that SMARCAD1 induces apoptosis via its chromatin-remodeling activity, which helps recruit MutLα to MutSα on damaged chromatin.},
}

@article {pmid31841614,
year = {2019},
author = {Zhao, C and Devlin, AC and Chouhan, AK and Selvaraj, BT and Stavrou, M and Burr, K and Brivio, V and He, X and Mehta, AR and Story, D and Shaw, CE and Dando, O and Hardingham, GE and Miles, GB and Chandran, S},
title = {Mutant C9orf72 human iPSC-derived astrocytes cause non-cell autonomous motor neuron pathophysiology.},
journal = {Glia},
volume = {},
number = {},
pages = {},
doi = {10.1002/glia.23761},
pmid = {31841614},
issn = {1098-1136},
support = {//Euan MacDonald Centre for MND Research/ ; /MRC_/Medical Research Council/United Kingdom ; Miles/Oct 2014/878-792/MNDA_/Motor Neurone Disease Association/United Kingdom ; Miles/Oct12/862-792/MNDA_/Motor Neurone Disease Association/United Kingdom ; //MND Scotland/ ; //UK Dementia Research Institute/ ; //China Scholarship Council/ ; //MS Society/ ; },
abstract = {Mutations in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (ALS). Accumulating evidence implicates astrocytes as important non-cell autonomous contributors to ALS pathogenesis, although the potential deleterious effects of astrocytes on the function of motor neurons remains to be determined in a completely humanized model of C9orf72-mediated ALS. Here, we use a human iPSC-based model to study the cell autonomous and non-autonomous consequences of mutant C9orf72 expression by astrocytes. We show that mutant astrocytes both recapitulate key aspects of C9orf72-related ALS pathology and, upon co-culture, cause motor neurons to undergo a progressive loss of action potential output due to decreases in the magnitude of voltage-activated Na+ and K+ currents. Importantly, CRISPR/Cas-9 mediated excision of the C9orf72 repeat expansion reverses these phenotypes, confirming that the C9orf72 mutation is responsible for both cell-autonomous astrocyte pathology and non-cell autonomous motor neuron pathophysiology.},
}

@article {pmid31840103,
year = {2019},
author = {Sansbury, BM and Hewes, AM and Kmiec, EB},
title = {Understanding the diversity of genetic outcomes from CRISPR-Cas generated homology-directed repair.},
journal = {Communications biology},
volume = {2},
number = {},
pages = {458},
pmid = {31840103},
issn = {2399-3642},
abstract = {As CRISPR-Cas systems advance toward clinical application, it is essential to identify all the outcomes of gene-editing activity in human cells. Reports highlighting the remarkable success of homology-directed repair (HDR) in the treatment of inherited diseases may inadvertently underreport the collateral activity of this remarkable technology. We are utilizing an in vitro gene-editing system in which a CRISPR-Cas complex provides the double-stranded cleavage and a mammalian cell-free extract provides the enzymatic activity to promote non-homologous end joining, micro-homology mediated end joining, and homology-directed repair. Here, we detail the broad spectrum of gene-editing reaction outcomes utilizing Cas9 and Cas12a in combination with single-stranded donor templates of the sense and nonsense polarity. This system offers the opportunity to see the range of outcomes of gene-editing reactions in an unbiased fashion, detailing the distribution of DNA repair outcomes as a function of a set of genetic tools.},
}

@article {pmid31836139,
year = {2019},
author = {An, Y and Park, KH and Lee, M and Kim, TJ and Woo, EJ},
title = {Crystal structure of the Csm5 subunit of the type III-A CRISPR-Cas system.},
journal = {Biochemical and biophysical research communications},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.bbrc.2019.12.046},
pmid = {31836139},
issn = {1090-2104},
abstract = {The Csm complex eliminates foreign RNA and DNA in the microbial defense CRISPR-Cas system. Csm5, one of the five subunits in the complex, facilitates crRNA maturation and target RNA binding in the type III system. However, the exact functional mechanism of Csm5 has remained elusive. Here, we report the crystal structure of the apo form of the Csm5 subunit at a resolution of 2.6 Å. Structural comparison of amino acids in the complex bound to RNA exhibits notable conformational changes in the crRNA and the target RNA binding sites. Shifts in the β-hairpin motif (β5-β6), α13 helix (resides 352-383), and G-rich loop (residues 335-337) in the C-terminal domain indicate an induced movement by crRNA binding. The positively charged residues (Lys 92, Arg 95 and Lys 96) located in the β-α4 loop of the target RNA interface show high conformational flexibility, while three-helix bundles (α1-α3) of the N-domain involved in Csm2 binding exhibit a rotational shift. The altered architecture of the Csm5 subunit demonstrates remarkable versatility of the ferredoxin-like fold in the RNA binding protein and provides a structural basis for the mechanism for crRNA and target RNA binding in the type III-A Crispr-Cas system.},
}

@article {pmid31835565,
year = {2019},
author = {Dorn, A and Puchta, H},
title = {DNA Helicases as Safekeepers of Genome Stability in Plants.},
journal = {Genes},
volume = {10},
number = {12},
pages = {},
pmid = {31835565},
issn = {2073-4425},
abstract = {Genetic information of all organisms is coded in double-stranded DNA. DNA helicases are essential for unwinding this double strand when it comes to replication, repair or transcription of genetic information. In this review, we will focus on what is known about a variety of DNA helicases that are required to ensure genome stability in plants. Due to their sessile lifestyle, plants are especially exposed to harmful environmental factors. Moreover, many crop plants have large and highly repetitive genomes, making them absolutely dependent on the correct interplay of DNA helicases for safeguarding their stability. Although basic features of a number of these enzymes are conserved between plants and other eukaryotes, a more detailed analysis shows surprising peculiarities, partly also between different plant species. This is additionally of high relevance for plant breeding as a number of these helicases are also involved in crossover control during meiosis and influence the outcome of different approaches of CRISPR/Cas based plant genome engineering. Thus, gaining knowledge about plant helicases, their interplay, as well as the manipulation of their pathways, possesses the potential for improving agriculture. In the long run, this might even help us cope with the increasing obstacles of climate change threatening food security in completely new ways.},
}

@article {pmid31834846,
year = {2019},
author = {Liao, W and Liu, Y and Chen, C and Li, J and Du, F and Long, D and Zhang, W},
title = {Distribution of CRISPR-Cas Systems in Clinical Carbapenem-Resistant Klebsiella pneumoniae Strains in a Chinese Tertiary Hospital and Its Potential Relationship with Virulence.},
journal = {Microbial drug resistance (Larchmont, N.Y.)},
volume = {},
number = {},
pages = {},
doi = {10.1089/mdr.2019.0276},
pmid = {31834846},
issn = {1931-8448},
abstract = {Aim: In this study, we aimed to characterize the CRISPR-Cas systems in clinical carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates and to investigate the potential association of CRISPR-Cas systems with bacterial virulence. Methods: A total of 168 CRKP strains were collected from inpatients in a teaching hospital in Jiangxi Province. Five common carbapenemase genes, subtype genes of the CRISPR-Cas system, and 13 virulence genes were amplified by PCR using specific primers. The potential virulence of all the clinical CRKP strains was tested in a Galleria mellonella infection model. Results: PCR analysis of five common carbapenemase genes revealed the frequency of carbapenemase gene KPC-2 was the highest in the CRISPR-negative strains, compared to CRISPR type I-E* strains or CRISPR type I-E strains (p < 0.01). Isolates having the subtype I-E* CRISPR-Cas system tended to have more virulence genes such as magA, kfu, wcaG, and allS, compared to CRISPR-negative isolates and type I-E CRISPR-Cas isolates (p < 0.01). The average survival time of the larvae infected with the isolates having the subtype I-E* CRISPR-Cas system was significantly shorter than the other two group isolates (p < 0.05). Conclusion: The CRKP strains, which had the subtype I-E CRISPR-Cas system or the subtype I-E* CRISPR-Cas system, showed reduced acquisition of carbapenemase genes compared to CRISPR-negative isolates. Importantly, we first found that a small portion of "CR-hvKP" strains were selected from the CRKP clones, which had the type I-E* CRISPR-Cas systems.},
}

@article {pmid31830738,
year = {2019},
author = {Mc Carlie, S and Boucher, CE and Bragg, RR},
title = {Molecular basis of bacterial disinfectant resistance.},
journal = {Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy},
volume = {48},
number = {},
pages = {100672},
doi = {10.1016/j.drup.2019.100672},
pmid = {31830738},
issn = {1532-2084},
abstract = {Antibiotic resistance could accelerate humanity towards an already fast-approaching post-antibiotic era, where disinfectants and effective biosecurity measures will be critically important to control microbial diseases. Disinfectant resistance has the potential to change our way of life from compromising food security to threatening our medical health systems. Resistance to antimicrobial agents occurs through either intrinsic or acquired resistance mechanisms. Acquired resistance occurs through the efficient transfer of mobile genetic elements, which can carry single, or multiple resistance determinants. Drug resistance genes may form part of integrons, transposons and insertions sequences which are capable of intracellular transfer onto plasmids or gene cassettes. Thereafter, resistance plasmids and gene cassettes mobilize by self-transmission between bacteria, increasing the prevalence of drug resistance determinants in a bacterial population. An accumulation of drug resistance genes through these mechanisms gives rise to multidrug resistant (MDR) bacteria. The study of this mobility is integral to safeguard current antibiotics, disinfectants and other antimicrobials. Literature evidence, however, indicates that knowledge regarding disinfectant resistance is severly limited. Genome engineering such as the CRISPR-Cas system, has identified disinfectant resistance genes, and reversed resistance altogether in certain prokaryotes. Demonstrating that these techniques could prove invaluable in the combat against disinfectant resistance by uncovering the secrets of MDR bacteria.},
}

@article {pmid31824468,
year = {2019},
author = {Ipoutcha, T and Tsarmpopoulos, I and Talenton, V and Gaspin, C and Moisan, A and Walker, CA and Brownlie, J and Blanchard, A and Thebault, P and Sirand-Pugnet, P},
title = {Multiple Origins and Specific Evolution of CRISPR/Cas9 Systems in Minimal Bacteria (Mollicutes).},
journal = {Frontiers in microbiology},
volume = {10},
number = {},
pages = {2701},
pmid = {31824468},
issn = {1664-302X},
abstract = {CRISPR/Cas systems provide adaptive defense mechanisms against invading nucleic acids in prokaryotes. Because of its interest as a genetic tool, the Type II CRISPR/Cas9 system from Streptococcus pyogenes has been extensively studied. It includes the Cas9 endonuclease that is dependent on a dual-guide RNA made of a tracrRNA and a crRNA. Target recognition relies on crRNA annealing and the presence of a protospacer adjacent motif (PAM). Mollicutes are currently the bacteria with the smallest genome in which CRISPR/Cas systems have been reported. Many of them are pathogenic to humans and animals (mycoplasmas and ureaplasmas) or plants (phytoplasmas and some spiroplasmas). A global survey was conducted to identify and compare CRISPR/Cas systems found in the genome of these minimal bacteria. Complete or degraded systems classified as Type II-A and less frequently as Type II-C were found in the genome of 21 out of 52 representative mollicutes species. Phylogenetic reconstructions predicted a common origin of all CRISPR/Cas systems of mycoplasmas and at least two origins were suggested for spiroplasmas systems. Cas9 in mollicutes were structurally related to the S. aureus Cas9 except the PI domain involved in the interaction with the PAM, suggesting various PAM might be recognized by Cas9 of different mollicutes. Structure of the predicted crRNA/tracrRNA hybrids was conserved and showed typical stem-loop structures pairing the Direct Repeat part of crRNAs with the 5' region of tracrRNAs. Most mollicutes crRNA/tracrRNAs showed G + C% significantly higher than the genome, suggesting a selective pressure for maintaining stability of these secondary structures. Examples of CRISPR spacers matching with mollicutes phages were found, including the textbook case of Mycoplasma cynos strain C142 having no prophage sequence but a CRISPR/Cas system with spacers targeting prophage sequences that were found in the genome of another M. cynos strain that is devoid of a CRISPR system. Despite their small genome size, mollicutes have maintained protective means against invading DNAs, including restriction/modification and CRISPR/Cas systems. The apparent lack of CRISPR/Cas systems in several groups of species including main pathogens of humans, ruminants, and plants suggests different evolutionary routes or a lower risk of phage infection in specific ecological niches.},
}

@article {pmid31823826,
year = {2019},
author = {Vasquez-Rifo, A and Veksler-Lublinsky, I and Cheng, Z and Ausubel, FM and Ambros, V},
title = {The Pseudomonas aeruginosa accessory genome elements influence virulence towards Caenorhabditis elegans.},
journal = {Genome biology},
volume = {20},
number = {1},
pages = {270},
pmid = {31823826},
issn = {1474-760X},
support = {R01GM088365/NH/NIH HHS/United States ; R01GM034028/NH/NIH HHS/United States ; R01AI085581/NH/NIH HHS/United States ; P30DK040561/NH/NIH HHS/United States ; },
abstract = {BACKGROUND: Multicellular animals and bacteria frequently engage in predator-prey and host-pathogen interactions, such as the well-studied relationship between Pseudomonas aeruginosa and the nematode Caenorhabditis elegans. This study investigates the genomic and genetic basis of bacterial-driven variability in P. aeruginosa virulence towards C. elegans to provide evolutionary insights into host-pathogen relationships.

RESULTS: Natural isolates of P. aeruginosa that exhibit diverse genomes display a broad range of virulence towards C. elegans. Using gene association and genetic analysis, we identify accessory genome elements that correlate with virulence, including both known and novel virulence determinants. Among the novel genes, we find a viral-like mobile element, the teg block, that impairs virulence and whose acquisition is restricted by CRISPR-Cas systems. Further genetic and genomic evidence suggests that spacer-targeted elements preferentially associate with lower virulence while the presence of CRISPR-Cas associates with higher virulence.

CONCLUSIONS: Our analysis demonstrates substantial strain variation in P. aeruginosa virulence, mediated by specific accessory genome elements that promote increased or decreased virulence. We exemplify that viral-like accessory genome elements that decrease virulence can be restricted by bacterial CRISPR-Cas immune defense systems, and suggest a positive, albeit indirect, role for host CRISPR-Cas systems in virulence maintenance.},
}

@article {pmid31819262,
year = {2020},
author = {Mendoza, SD and Nieweglowska, ES and Govindarajan, S and Leon, LM and Berry, JD and Tiwari, A and Chaikeeratisak, V and Pogliano, J and Agard, DA and Bondy-Denomy, J},
title = {A bacteriophage nucleus-like compartment shields DNA from CRISPR nucleases.},
journal = {Nature},
volume = {577},
number = {7789},
pages = {244-248},
pmid = {31819262},
issn = {1476-4687},
support = {R01 GM127489/GM/NIGMS NIH HHS/United States ; DP5 OD021344/OD/NIH HHS/United States ; R01 GM104556/GM/NIGMS NIH HHS/United States ; R35 GM118099/GM/NIGMS NIH HHS/United States ; GM104556//Natiional Institutes of Health/International ; },
abstract = {All viruses require strategies to inhibit or evade the immune pathways of cells that they infect. The viruses that infect bacteria, bacteriophages (phages), must avoid immune pathways that target nucleic acids, such as CRISPR-Cas and restriction-modification systems, to replicate efficiently1. Here we show that jumbo phage ΦKZ segregates its DNA from immunity nucleases of its host, Pseudomonas aeruginosa, by constructing a proteinaceous nucleus-like compartment. ΦKZ is resistant to many immunity mechanisms that target DNA in vivo, including two subtypes of CRISPR-Cas3, Cas9, Cas12a and the restriction enzymes HsdRMS and EcoRI. Cas proteins and restriction enzymes are unable to access the phage DNA throughout the infection, but engineering the relocalization of EcoRI inside the compartment enables targeting of the phage and protection of host cells. Moreover, ΦKZ is sensitive to Cas13a-a CRISPR-Cas enzyme that targets RNA-probably owing to phage mRNA localizing to the cytoplasm. Collectively, we propose that Pseudomonas jumbo phages evade a broad spectrum of DNA-targeting nucleases through the assembly of a protein barrier around their genome.},
}

@article {pmid31819217,
year = {2020},
author = {Malone, LM and Warring, SL and Jackson, SA and Warnecke, C and Gardner, PP and Gumy, LF and Fineran, PC},
title = {A jumbo phage that forms a nucleus-like structure evades CRISPR-Cas DNA targeting but is vulnerable to type III RNA-based immunity.},
journal = {Nature microbiology},
volume = {5},
number = {1},
pages = {48-55},
doi = {10.1038/s41564-019-0612-5},
pmid = {31819217},
issn = {2058-5276},
abstract = {CRISPR-Cas systems provide bacteria with adaptive immunity against bacteriophages1. However, DNA modification2,3, the production of anti-CRISPR proteins4,5 and potentially other strategies enable phages to evade CRISPR-Cas. Here, we discovered a Serratia jumbo phage that evades type I CRISPR-Cas systems, but is sensitive to type III immunity. Jumbo phage infection resulted in a nucleus-like structure enclosed by a proteinaceous phage shell-a phenomenon only reported recently for distantly related Pseudomonas phages6,7. All three native CRISPR-Cas complexes in Serratia-type I-E, I-F and III-A-were spatially excluded from the phage nucleus and phage DNA was not targeted. However, the type III-A system still arrested jumbo phage infection by targeting phage RNA in the cytoplasm in a process requiring Cas7, Cas10 and an accessory nuclease. Type III, but not type I, systems frequently targeted nucleus-forming jumbo phages that were identified in global viral sequence datasets. The ability to recognize jumbo phage RNA and elicit immunity probably contributes to the presence of both RNA- and DNA-targeting CRISPR-Cas systems in many bacteria1,8. Together, our results support the model that jumbo phage nucleus-like compartments serve as a barrier to DNA-targeting, but not RNA-targeting, defences, and that this phenomenon is widespread among jumbo phages.},
}

@article {pmid31819086,
year = {2019},
author = {Goldstein, JM and Valido, A and Lewandowski, JP and Walker, RG and Mills, MJ and Messemer, KA and Besseling, P and Lee, KH and Wattrus, SJ and Cho, M and Lee, RT and Wagers, AJ},
title = {Variation in zygotic CRISPR/Cas9 gene editing outcomes generates novel reporter and deletion alleles at the Gdf11 locus.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {18613},
pmid = {31819086},
issn = {2045-2322},
abstract = {Recent advances in CRISPR/Cas gene editing technology have significantly expanded the possibilities and accelerated the pace of creating genetically engineered animal models. However, CRISPR/Cas-based strategies designed to precisely edit the genome can often yield unintended outcomes. Here, we report the use of zygotic CRISPR/Cas9 injections to generate a knock-in GFP reporter mouse at the Gdf11 locus. Phenotypic and genomic characterization of founder animals from these injections revealed a subset that contained the correct targeting event and exhibited GFP expression that, within the hematopoietic system, was restricted predominantly to lymphoid cells. Yet, in another subset of founder mice, we detected aberrant integration events at the target site that dramatically and inaccurately shifted hematopoietic GFP expression from the lymphoid to the myeloid lineage. Additionally, we recovered multiple Gdf11 deletion alleles that modified the C-terminus of the GDF11 protein. When bred to homozygosity, most of these alleles recapitulated skeletal phenotypes reported previously for Gdf11 knockout mice, suggesting that these represent null alleles. However, we also recovered one Gdf11 deletion allele that encodes a novel GDF11 variant protein ("GDF11-WE") predicted to contain two additional amino acids (tryptophan (W) and glutamic acid (E)) at the C-terminus of the mature ligand. Unlike the other Gdf11 deletion alleles recovered in this study, homozygosity for the Gdf11WE allele did not phenocopy Gdf11 knockout skeletal phenotypes. Further investigation using in vivo and in vitro approaches demonstrated that GDF11-WE retains substantial physiological function, indicating that GDF11 can tolerate at least some modifications of its C-terminus and providing unexpected insights into its biochemical activities. Altogether, our study confirms that one-step zygotic injections of CRISPR/Cas gene editing complexes provide a quick and powerful tool to generate gene-modified mouse models. Moreover, our findings underscore the critical importance of thorough characterization and validation of any modified alleles generated by CRISPR, as unintended on-target effects that fail to be detected by simple PCR screening can produce substantially altered phenotypic readouts.},
}

@article {pmid31817656,
year = {2019},
author = {Otterbein, H and Lehnert, H and Ungefroren, H},
title = {Negative Control of Cell Migration by Rac1b in Highly Metastatic Pancreatic Cancer Cells Is Mediated by Sequential Induction of Nonactivated Smad3 and Biglycan.},
journal = {Cancers},
volume = {11},
number = {12},
pages = {},
doi = {10.3390/cancers11121959},
pmid = {31817656},
issn = {2072-6694},
abstract = {Expression of the small GTPase, Ras-related C3 botulinum toxin substrate 1B (RAC1B), a RAC1-related member of the Rho GTPase family, in tumor tissues of pancreatic ductal adenocarcinoma (PDAC) has been shown previously to correlate positively with patient survival, but the underlying mechanism(s) and the target genes involved have remained elusive. Screening of a panel of established PDAC-derived cell lines by immunoblotting indicated that both RAC1B and Mothers against decapentaplegic homolog 3 (SMAD3) were more abundantly expressed in poorly metastatic and well-differentiated lines as opposed to highly metastatic, poorly differentiated ones. Both siRNA-mediated RAC1B knockdown in the transforming growth factor (TGF)-β-sensitive PDAC-derived cell lines, Panc1 and PaCa3, or CRISPR/Cas-mediated knockout of exon 3b of RAC1 in Panc1 cells resulted in a dramatic decrease in the expression of SMAD3. Unexpectedly, the knockdown of SMAD3 reproduced the promigratory activity of a RAC1B knockdown in Panc1 and PaCa3, but not in TGF-β-resistant BxPC3 and Capan1 cells, while forced expression of SMAD3 alone was able to mimic the antimigratory effect of ectopic RAC1B overexpression in Panc1 cells. Moreover, overexpression of SMAD3 was able to rescue Panc1 cells from the RAC1B knockdown-induced increase in cell migration, while knockdown of SMAD3 prevented the RAC1B overexpression-induced decrease in cell migration. Using pharmacological and dominant-negative inhibition of SMAD3 C-terminal phosphorylation, we further show that the migration-inhibiting effect of SMAD3 is independent of its activation by TGF-β. Finally, we provide evidence that the antimigratory program of RAC1B-SMAD3 in Panc1 cells is executed through upregulation of the migration and TGF-β inhibitor, biglycan (BGN). Together, our data suggest that a RAC1B-SMAD3-BGN axis negatively controls cell migration and that SMAD3 can induce antimigratory genes, i.e., BGN independent of its role as a signal transducer for TGF-β. Therefore, targeting this novel pathway for activation is a potential therapeutic strategy in highly metastatic PDAC to interfere with invasion and metastasis.},
}

@article {pmid31817229,
year = {2019},
author = {Zinn, R and Otterbein, H and Lehnert, H and Ungefroren, H},
title = {RAC1B: A Guardian of the Epithelial Phenotype and Protector Against Epithelial-Mesenchymal Transition.},
journal = {Cells},
volume = {8},
number = {12},
pages = {},
doi = {10.3390/cells8121569},
pmid = {31817229},
issn = {2073-4409},
abstract = {The small GTPase Ras-related C3 botulinum toxin substrate 1B (RAC1B) has been shown to potently inhibit transforming growth factor (TGF)-β1-induced cell migration and epithelial-mesenchymal transition (EMT) in pancreatic and breast epithelial cells, but the underlying mechanism has remained obscure. Using a panel of pancreatic ductal adenocarcinoma (PDAC)-derived cell lines of different differentiation stages, we show that RAC1B is more abundantly expressed in well differentiated as opposed to poorly differentiated cells. Interestingly, RNA interference-mediated knockdown of RAC1B decreased expression of the epithelial marker protein E-cadherin, encoded by CDH1, and enhanced its TGF-β1-induced downregulation, whereas ectopic overexpression of RAC1B upregulated CDH1 expression and largely prevented its TGF-β1-induced silencing of CDH1. Conversely, knockdown of RAC1B, or deletion of the RAC1B-specific exon 3b by CRISPR/Cas-mediated genomic editing, enhanced basal and TGF-β1-induced upregulation of mesenchymal markers like Vimentin, and EMT-associated transcription factors such as SNAIL and SLUG. Moreover, we demonstrate that knockout of RAC1B enhanced the cells' migratory activity and derepressed TGF-β1-induced activation of the mitogen-activated protein kinase ERK2. Pharmacological inhibition of ERK1/2 activation in RAC1B-depleted cells rescued cells from the RAC1B knockdown-induced enhancement of cell migration, TGF-β1-induced downregulation of CDH1, and upregulation of SNAI1. We conclude that RAC1B promotes epithelial gene expression and suppresses mesenchymal gene expression by interfering with TGF-β1-induced MEK-ERK signaling, thereby protecting cells from undergoing EMT and EMT-associated responses like acquisition of cell motility.},
}

@article {pmid31810603,
year = {2019},
author = {Xia, P and Liu, P and Fu, Q and Liu, C and Luo, Q and Zhang, X and Cheng, L and Qin, T and Zhang, H},
title = {Long noncoding RNA EPIC1 interacts with YAP1 to regulate the cell cycle and promote the growth of pancreatic cancer cells.},
journal = {Biochemical and biophysical research communications},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.bbrc.2019.11.167},
pmid = {31810603},
issn = {1090-2104},
abstract = {Pancreatic cancer (PC) is a fatal disease; most patients are asymptomatic before the disease enters the advanced stage, but molecular mechanisms of early PC that can be exploited for diagnosis are not clear. Long noncoding RNAs (lncRNAs) play key roles in the progression of PC. In this study, we found that the expression of the lncRNA EPIC1 (Lnc-EPIC1) is high in PC and closely related to tumor size, TNM staging and lymph node metastasis status. Silencing Lnc-EPIC1 by siRNA targeting could significantly inhibit the cell growth and colony formation ability of PC cells and induced G1/S cell cycle arrest and apoptosis in PC cells. Lnc-EPIC1-specific siRNAs could downregulate the expression of cyclins and CDKs, such as CDC20, CDK4 and Cyclin A1. Knocking out YAP1 with the CRISPR/Cas-9 gene editing method recapitulated the effects of the Lnc-EPIC1-specific siRNAs on cell growth, colony formation ability and apoptosis in PC cells. In addition, the Lnc-EPIC1-specific siRNAs did not further inhibit cell growth or promote apoptosis in YAP1-knockout (YAP1-KO) cells. RNA immunoprecipitation (RIP) results showed that there was a direct interaction between Lnc-EPIC1 and YAP1. An Lnc-EPIC1-overexpressing lentiviral vector promoted the growth of PC cells. The results show that Lnc-EPIC1 interacts with YAP1 to promote the progression of PC.},
}

@article {pmid31810530,
year = {2019},
author = {Wang, J and Bai, P and Li, Q and Lin, Y and Huo, D and Ke, F and Zhang, Q and Li, T and Zhao, J},
title = {Interaction between cyanophage MaMV-DC and eight Microcystis strains, revealed by genetic defense systems.},
journal = {Harmful algae},
volume = {85},
number = {},
pages = {101699},
doi = {10.1016/j.hal.2019.101699},
pmid = {31810530},
issn = {1878-1470},
abstract = {Cyanophage MaMV-DC is a member of Myoviridae that was reported to specifically infect and lyse Microcystis aeruginosa FACHB-524 among 21 selected cyanobacterial strains. We reidentified the infection specificity of MaMV-DC among seven other Microcystis strains of different species. In our experiments, MaMV-DC infected three Microcystis strains but did not form plaque in Microcystis lawns. This indicated that MaMV-DC is at least a genus- rather than strain-specific virus. Cyanophage MaMV-DC genes were transcribed in M. aeruginosa FACHB-524, M. flos-aquae TF09, M. aeruginosa TA09 and M. wesenbergii DW09, and the growth of these Microcystis strains was inhibited by the addition of MaMV-DC. The predicted defense of eight Microcystis strains by CRISPR-Cas systems has shown mixed consistency with the infection experiment results, suggesting other defense or anti-defense systems play roles during infection process. Restriction-modification (RM) system analysis revealed an abundance of four types of RM proteins that may play roles in defense against cyanophages.},
}

@article {pmid31809194,
year = {2019},
author = {Crowley, VM and Catching, A and Taylor, HN and Borges, AL and Metcalf, J and Bondy-Denomy, J and Jackson, RN},
title = {A Type IV-A CRISPR-Cas System in Pseudomonas aeruginosa Mediates RNA-Guided Plasmid Interference In Vivo.},
journal = {The CRISPR journal},
volume = {2},
number = {6},
pages = {434-440},
pmid = {31809194},
issn = {2573-1602},
abstract = {Bacteria and archaea use CRISPR-Cas adaptive immune systems to destroy complementary nucleic acids using RNAs derived from CRISPR loci. Here, we provide the first functional evidence for type IV CRISPR-Cas, demonstrating that the system from Pseudomonas aeruginosa strain PA83 mediates RNA-guided interference against a plasmid in vivo, both clearing the plasmid and inhibiting its uptake. This interference depends on the putative NTP-dependent helicase activity of Csf4/DinG.},
}

@article {pmid31805916,
year = {2019},
author = {Ahmed, HMM and Hildebrand, L and Wimmer, EA},
title = {Improvement and use of CRISPR/Cas9 to engineer a sperm-marking strain for the invasive fruit pest Drosophila suzukii.},
journal = {BMC biotechnology},
volume = {19},
number = {1},
pages = {85},
pmid = {31805916},
issn = {1472-6750},
abstract = {BACKGROUND: The invasive fruit pest Drosophila suzukii was reported for the first time in Europe and the USA in 2008 and has spread since then. The adoption of type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) as a tool for genome manipulation provides new ways to develop novel biotechnologically-based pest control approaches. Stage or tissue-specifically expressed genes are of particular importance in the field of insect biotechnology. The enhancer/promoter of the spermatogenesis-specific beta-2-tubulin (β2t) gene was used to drive the expression of fluorescent proteins or effector molecules in testes of agricultural pests and disease vectors for sexing, monitoring, and reproductive biology studies. Here, we demonstrate an improvement to CRISPR/Cas-based genome editing in D. suzukii and establish a sperm-marking system.

RESULTS: To improve genome editing, we isolated and tested the D. suzukii endogenous promoters of the small nuclear RNA gene U6 to drive the expression of a guide RNA and the Ds heat shock protein 70 promoter to express Cas9. For comparison, we used recombinant Cas9 protein and in vitro transcribed gRNA as a preformed ribonucleoprotein. We demonstrate the homology-dependent repair (HDR)-based genome editing efficiency by applying a previously established transgenic line that expresses DsRed ubiquitously as a target platform. In addition, we isolated the Ds_β2t gene and used its promoter to drive the expression of a red fluorescence protein in the sperm. A transgenic sperm-marking strain was then established by the improved HDR-based genome editing.

CONCLUSION: The deployment of the endogenous promoters of the D. suzukii U6 and hsp70 genes to drive the expression of gRNA and Cas9, respectively, enabled the effective application of helper plasmid co-injections instead of preformed ribonucleoproteins used in previous reports for HDR-based genome editing. The sperm-marking system should help to monitor the success of pest control campaigns in the context of the Sterile Insect Technique and provides a tool for basic research in reproductive biology of this invasive pest. Furthermore, the promoter of the β2t gene can be used in developing novel transgenic pest control approaches and the CRISPR/Cas9 system as an additional tool for the modification of previously established transgenes.},
}