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Bibliography on: CRISPR-Cas

The Electronic Scholarly Publishing Project: Providing world-wide, free access to classic scientific papers and other scholarly materials, since 1993.


ESP: PubMed Auto Bibliography 17 Oct 2019 at 01:35 Created: 


Clustered regularly interspaced short palindromic repeats (CRISPR, pronounced crisper) are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposures to foreign DNA (e.g a virus or plasmid). The CRISPR/Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present within plasmids and phages, and provides a form of acquired immunity. CRISPR associated proteins (Cas) use the CRISPR spacers to recognize and cut these exogenous genetic elements in a manner analogous to RNA interference in eukaryotic organisms. CRISPRs are found in approximately 40% of sequenced bacterial genomes and 90% of sequenced archaea. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added. The Cas9-gRNA complex corresponds with the CAS III crRNA complex in the above diagram. CRISPR/Cas genome editing techniques have many potential applications, including altering the germline of humans, animals, and food crops. The use of CRISPR Cas9-gRNA complex for genome editing was the AAAS's choice for breakthrough of the year in 2015.

Created with PubMed® Query: "CRISPR.CAS" OR "crispr/cas" NOT pmcbook NOT ispreviousversion

Citations The Papers (from PubMed®)

RevDate: 2019-10-16

Zhao Y, Yang X, Zhou G, et al (2019)

Engineering plants virus resistance: from RNA silencing to genome editing strategies.

Plant biotechnology journal [Epub ahead of print].

Viral diseases severely affect crop yield and quality, thereby threatening global food security. Genetic improvement of plant virus resistance is essential for sustainable agriculture. In the last decades, several modern technologies were applied in plant antiviral engineering. Here we summarized breakthroughs of the two major antiviral strategies, RNA silencing and genome editing. RNA silencing strategy has been used in antiviral breeding for more than thirty years, and many crops engineered to stably express small RNAs targeting various viruses have been approved for commercial release. Genome editing technology has emerged in the past decade, especially CRISPR/Cas, which provides new methods for genetic improvement of plant virus resistance and accelerates resistance breeding. Finally, we discuss the potential of these technologies for breeding crops, and the challenges and solutions they may face in the future.

RevDate: 2019-10-16

Yang X, Liu D, Tschaplinski TJ, et al (2019)

Comparative genomics can provide new insights into the evolutionary mechanisms and gene function in CAM plants.

Journal of experimental botany pii:5564849 [Epub ahead of print].

Crassulacean acid metabolism (CAM) photosynthesis is an important biological innovation enabling plant adaptation to hot and dry environments. CAM plants feature high water-use efficiency, with potential for sustainable crop production under water-limited conditions. A deep understanding of CAM-related gene function and molecular evolution of CAM plants is critical for exploiting the potential of engineering CAM into C3 crops to enhance crop production on semi-arid or marginal agricultural lands. With the newly emerging genomics resources for multiple CAM species, progress has been made in comparative genomics studies on the molecular basis and subsequently on the evolution of CAM. Here, recent advances in CAM comparative genomics research in constitutive and facultative CAM plants are reviewed, with a focus on the analyses of DNA/protein sequences and gene expression to provide new insights into the path and driving force of CAM evolution and to identify candidate genes involved in CAM-related biological processes. Potential applications of new computational and experimental technologies (e.g. CRISPR/Cas-mediated genome-editing technology) to the comparative and evolutionary genomics research on CAM plants are offered.

RevDate: 2019-10-16

Ren Q, Zhong Z, Wang Y, et al (2019)

Bidirectional Promoter-Based CRISPR-Cas9 Systems for Plant Genome Editing.

Frontiers in plant science, 10:1173.

CRISPR-Cas systems can be expressed in multiple ways, with different capabilities regarding tissue-specific expression, efficiency, and expression levels. Thus far, three expression strategies have been demonstrated in plants: mixed dual promoter systems, dual Pol II promoter systems, and single transcript unit (STU) systems. We explored a fourth strategy to express CRISPR-Cas9 in the model and crop plant, rice, where a bidirectional promoter (BiP) is used to express Cas9 and single guide RNA (sgRNA) in opposite directions. We first tested an engineered BiP system based on double-mini 35S promoter and an Arabidopsis enhancer, which resulted in 20.7% and 52.9% genome editing efficiencies at two target sites in T0 stable transgenic rice plants. We further improved the BiP system drastically by using a rice endogenous BiP, OsBiP1. The endogenous BiP expression system had higher expression strength and led to 75.9-93.3% genome editing efficiencies in rice T0 generation, when the sgRNAs were processed by either tRNA or Csy4. We provided a proof-of-concept study of applying BiP systems for expressing two-component CRISPR-Cas9 genome editing reagents in rice. Our work could promote future research and adoption of BiP systems for CRISPR-Cas-based genome engineering in plants.

RevDate: 2019-10-16
CmpDate: 2019-10-16

Liu Z, Chen M, Shan H, et al (2019)

Expanded targeting scope and enhanced base editing efficiency in rabbit using optimized xCas9(3.7).

Cellular and molecular life sciences : CMLS, 76(20):4155-4164.

Evolved xCas9(3.7) variant with broad PAM compatibility has been reported in cell lines, while its editing efficiency was site-specific. Here, we show that xCas9(3.7) can recognize a broad PAMs including NGG, NGA, and NGT, in both embryos and Founder (F0) rabbits. Furthermore, the codon-optimized xCas9-derived base editors, exBE4 and exABE, can dramatically improve the base editing efficiencies in rabbit embryos. Our results demonstrated that the optimized xCas9 with expanded PAM compatibility and enhanced base editing efficiency could be used for precise gene modifications in organisms.

RevDate: 2019-10-16
CmpDate: 2019-10-16

Zhang Z, Zhan X, Kim B, et al (2019)

A proteomic approach identifies SAFB-like transcription modulator (SLTM) as a bidirectional regulator of GLI family zinc finger transcription factors.

The Journal of biological chemistry, 294(14):5549-5561.

In Sonic hedgehog (SHH) signaling, GLI family zinc finger (GLI)-mediated diverse gene transcription outcomes are strictly regulated and are important for SHH function in both development and disease. However, how the GLI factors differentially regulate transcription in response to variable SHH activities is incompletely understood. Here, using a newly generated, tagged Gli3 knock-in mouse (Gli3TAP), we performed proteomic analyses and identified the chromatin-associated SAFB-like transcription modulator (SLTM) as a GLI-interacting protein that context-dependently regulates GLI activities. Using immunoprecipitation and immunoblotting, RT-quantitative PCR, and ChIP assays, we show that SLTM interacts with all three GLI proteins and that its cellular levels are regulated by SHH. We also found that SLTM enhances GLI3 binding to chromatin and increases GLI3 repressor (GLI3R) form protein levels. In a GLI3-dependent manner, SLTM promoted the formation of a repressive chromatin environment and functioned as a GLI3 co-repressor. In the absence of GLI3 or in the presence of low GLI3 levels, SLTM co-activated GLI activator (GLIA)-mediated target gene activation and cell differentiation. Moreover, in vivo Sltm deletion generated through CRISPR/Cas9-mediated gene editing caused perinatal lethality and SHH-related abnormal ventral neural tube phenotypes. We conclude that SLTM regulates GLI factor binding to chromatin and contributes to the transcriptional outcomes of SHH signaling via a novel molecular mechanism.

RevDate: 2019-10-16
CmpDate: 2019-10-16

Schröder M, Krötschel M, Conrad L, et al (2018)

Genetic screen in myeloid cells identifies TNF-α autocrine secretion as a factor increasing MDSC suppressive activity via Nos2 up-regulation.

Scientific reports, 8(1):13399.

The suppressive microenvironment of tumors remains one of the limiting factors for immunotherapies. In tumors, the function of effector T cells can be inhibited by cancer cells as well as myeloid cells including tumor associated macrophages and myeloid-derived suppressor cells (MDSC). A better understanding of how myeloid cells inhibit T cell function will guide the design of therapeutic strategies to increase anti-tumor responses. We have previously reported the in vitro differentiation of MDSC from immortalized mouse hematopoietic progenitors and characterized the impact of retinoic acid and 3-deazaneplanocin A on MDSC development and function. We describe here the effect of these compounds on MDSC transcriptome and identify genes and pathway affected by the treatment. In order to accelerate the investigation of gene function in MDSC suppressive activity, we developed protocols for CRISPR/Cas9-mediated gene editing in MDSC. Through screening of 217 genes, we found that autocrine secretion of TNF-α contributes to MDSC immunosuppressive activity through up-regulation of Nos2. The approach described here affords the investigation of gene function in myeloid cells such as MDSC with unprecedented ease and throughput.

RevDate: 2019-10-16
CmpDate: 2019-10-16

Lee W, Lee JH, Jun S, et al (2018)

Selective targeting of KRAS oncogenic alleles by CRISPR/Cas9 inhibits proliferation of cancer cells.

Scientific reports, 8(1):11879.

Mutations within the KRAS oncogene are associated with the proliferation of various cancers. Therapeutic approaches for treating cancers with such mutations have focused on targeting the downstream protein effectors of KRAS. However, to date, no approved treatment has targeted the mutated KRAS oncogene directly. Presently, we used the selectivity of the CRISPR/Cas9 system to directly target mutated KRAS alleles. We designed single-guide RNAs (sgRNAs) to target two specific single-nucleotide missense mutations on KRAS codon-12 located in the seed region adjacent to a protospacer adjacent motif (PAM). Lentiviral transduction of Cas9 and the sgRNAs into cancer cells with respective KRAS mutations resulted in high frequency of indels in the seed region. Indel-associated disruption of the mutant KRAS alleles correlated with reduced viability of the cancer cells. The results indicate that CRISPR-Cas9-mediated genome editing can potentially be used for the treatment of cancer patients, specifically those with oncogenic KRAS mutations.

RevDate: 2019-10-16
CmpDate: 2019-10-16

Xu X, Gao D, Wang P, et al (2018)

Efficient homology-directed gene editing by CRISPR/Cas9 in human stem and primary cells using tube electroporation.

Scientific reports, 8(1):11649.

CRISPR/Cas9 efficiently generates gene knock-out via nonhomologous end joining (NHEJ), but the efficiency of precise homology-directed repair (HDR) is substantially lower, especially in the hard-to-transfect human stem cells and primary cells. Herein we report a tube electroporation method that can effectively transfect human stem cells and primary cells with minimal cytotoxicity. When applied to genome editing using CRISPR/Cas9 along with single stranded DNA oligonucleotide (ssODN) template in human induced pluripotent stem cells (iPSCs), up to 42.1% HDR rate was achieved, drastically higher than many reported before. We demonstrated that the high HDR efficiency can be utilized to increase the gene ablation rate in cells relevant to clinical applications, by knocking-out β2-microglobulin (B2M) in primary human mesenchymal stem cells (MSCs, 37.3% to 80.2%), and programmed death-1 (PD-1) in primary human T cells (42.6% to 58.6%). Given the generality and efficiency, we expect that the method will have immediate impacts in cell research as well as immuno- and transplantation therapies.

RevDate: 2019-10-16
CmpDate: 2019-10-16

Hanada Y, Nakamura Y, Ozono Y, et al (2018)

Fibroblast growth factor 12 is expressed in spiral and vestibular ganglia and necessary for auditory and equilibrium function.

Scientific reports, 8(1):11491.

We investigated fibroblast growth factor 12 (FGF12) as a transcript enriched in the inner ear by searching published cDNA library databases. FGF12 is a fibroblast growth factor homologous factor, a subset of the FGF superfamily. To date, its localisation and function in the inner ear have not been determined. Here, we show that FGF12 mRNA is localised in spiral ganglion neurons (SGNs) and the vestibular ganglion. We also show that FGF12 protein is localised in SGNs, the vestibular ganglion, and nerve fibres extending beneath hair cells. Moreover, we investigated FGF12 function in auditory and vestibular systems using Fgf12-knockout (FGF12-KO) mice generated with CRISPR/Cas9 technology. Our results show that the inner ear morphology of FGF12-KO mice is not significantly different compared with wild-type mice. However, FGF12-KO mice exhibited an increased hearing threshold, as measured by the auditory brainstem response, as well as deficits in rotarod and balance beam performance tests. These results suggest that FGF12 is necessary for normal auditory and equilibrium function.

RevDate: 2019-10-15

Durut N, O Mittelsten Scheid (2019)

The Role of Noncoding RNAs in Double-Strand Break Repair.

Frontiers in plant science, 10:1155.

Genome stability is constantly threatened by DNA lesions generated by different environmental factors as well as endogenous processes. If not properly and timely repaired, damaged DNA can lead to mutations or chromosomal rearrangements, well-known reasons for genetic diseases or cancer in mammals, or growth abnormalities and/or sterility in plants. To prevent deleterious consequences of DNA damage, a sophisticated system termed DNA damage response (DDR) detects DNA lesions and initiates DNA repair processes. In addition to many well-studied canonical proteins involved in this process, noncoding RNA (ncRNA) molecules have recently been discovered as important regulators of the DDR pathway, extending the broad functional repertoire of ncRNAs to the maintenance of genome stability. These ncRNAs are mainly connected with double-strand breaks (DSBs), the most dangerous type of DNA lesions. The possibility to intentionally generate site-specific DSBs in the genome with endonucleases constitutes a powerful tool to study, in vivo, how DSBs are processed and how ncRNAs participate in this crucial event. In this review, we will summarize studies reporting the different roles of ncRNAs in DSB repair and discuss how genome editing approaches, especially CRISPR/Cas systems, can assist DNA repair studies. We will summarize knowledge concerning the functional significance of ncRNAs in DNA repair and their contribution to genome stability and integrity, with a focus on plants.

RevDate: 2019-10-15
CmpDate: 2019-10-15

Pignani S, Zappaterra F, Barbon E, et al (2019)

Tailoring the CRISPR system to transactivate coagulation gene promoters in normal and mutated contexts.

Biochimica et biophysica acta. Gene regulatory mechanisms, 1862(6):619-624.

Engineered transcription factors (TF) have expanded our ability to modulate gene expression and hold great promise as bio-therapeutics. The first-generation TF, based on Zinc Fingers or Transcription-Activator-like Effectors (TALE), required complex and time-consuming assembly protocols, and were indeed replaced in recent years by the CRISPR activation (CRISPRa) technology. Here, with coagulation F7/F8 gene promoters as models, we exploited a CRISPRa system based on deactivated (d)Cas9, fused with a transcriptional activator (VPR), which is driven to its target by a single guide (sg)RNA. Reporter gene assays in hepatoma cells identified a sgRNA (sgRNAF7.5) triggering a ~35-fold increase in the activity of F7 promoter, either wild-type, or defective due to the c.-61T>G mutation. The effect was higher (~15-fold) than that of an engineered TALE-TF (TF4) targeting the same promoter region. Noticeably, when challenged on the endogenous F7 gene, the dCas9-VPR/sgRNAF7.5 combination was more efficient (~6.5-fold) in promoting factor VII (FVII) protein secretion/activity than TF4 (~3.8-fold). The approach was translated to the promoter of F8, whose reduced expression causes hemophilia A. Reporter gene assays in hepatic and endothelial cells identified sgRNAs that, respectively, appreciably increased F8 promoter activity (sgRNAF8.1, ~8-fold and 3-fold; sgRNAF8.2, ~19-fold and 2-fold) with synergistic effects (~38-fold and 2.7-fold). Since modest increases in F7/F8 expression would ameliorate patients' phenotype, the CRISPRa-mediated transactivation extent might approach the low therapeutic threshold. Through this pioneer study we demonstrated that the CRISPRa system is easily tailorable to increase expression, or rescue disease-causing mutations, of different promoters, with potential intriguing implications for human disease models.

RevDate: 2019-10-15
CmpDate: 2019-10-15

Laughery MF, Mayes HC, Pedroza IK, et al (2019)

R-loop formation by dCas9 is mutagenic in Saccharomyces cerevisiae.

Nucleic acids research, 47(5):2389-2401.

Cas9 binds and cleaves specific DNA sequences by inducing the formation of an R-loop between the guide RNA and its genomic target. While targeting of active Cas9 to a genomic locus is highly mutagenic because Cas9 creates DNA double strand breaks, targeting of dead Cas9 (dCas9) is presumed not to be mutagenic, as dCas9 lacks DNA endonuclease activity. Here, we show that dCas9 targeting induces mutations in yeast, particularly when targeted to the non-transcribed strand of a gene. dCas9-induced mutations cluster near the guide RNA target region and are comprised of single nucleotide substitutions, small insertions and deletions, and even complex mutations, depending upon the particular guide RNA target. We show that many of these mutations are a consequence of cytosine deamination events occurring on the non-target strand of the dCas9-induced R-loop, while others are associated with homopolymer instability or translesion DNA synthesis. Targeting of dCas9 by a mismatch-containing guide RNA also increases CAN1 mutation frequency, particularly in an ung1Δ mutant strain, suggesting that dCas9 induces mutations through similar mechanisms at off-target sites. These findings indicate that DNA binding by dCas9 is mutagenic in yeast, likely because dCas9 induces the formation of an R-loop at its target site.

RevDate: 2019-10-15
CmpDate: 2019-10-15

Abbasi F, Miyata H, Shimada K, et al (2018)

RSPH6A is required for sperm flagellum formation and male fertility in mice.

Journal of cell science, 131(19):.

The flagellum is an evolutionarily conserved appendage used for sensing and locomotion. Its backbone is the axoneme and a component of the axoneme is the radial spoke (RS), a protein complex implicated in flagellar motility regulation. Numerous diseases occur if the axoneme is improperly formed, such as primary ciliary dyskinesia (PCD) and infertility. Radial spoke head 6 homolog A (RSPH6A) is an ortholog of Chlamydomonas RSP6 in the RS head and is evolutionarily conserved. While some RS head proteins have been linked to PCD, little is known about RSPH6A. Here, we show that mouse RSPH6A is testis-enriched and localized in the flagellum. Rsph6a knockout (KO) male mice are infertile as a result of their short immotile spermatozoa. Observation of the KO testis indicates that the axoneme can elongate but is disrupted before accessory structures are formed. Manchette removal is also impaired in the KO testis. Further, RSPH9, another radial spoke protein, disappeared in the Rsph6a KO flagella. These data indicate that RSPH6A is essential for sperm flagellar assembly and male fertility in mice.This article has an associated First Person interview with the first author of the paper.

RevDate: 2019-10-15
CmpDate: 2019-10-15

Li S, Li J, Zhang J, et al (2018)

Synthesis-dependent repair of Cpf1-induced double strand DNA breaks enables targeted gene replacement in rice.

Journal of experimental botany, 69(20):4715-4721.

The recently developed CRISPR (clustered regularly interspaced short palindromic repeats)/Cpf1 system expands the range of genome editing and is emerging as an alternative powerful tool for both plant functional genomics and crop improvement. Cpf1-CRISPR RNA (crRNA) produces double strand DNA breaks (DSBs) with long 5'-protruding ends, which may facilitate the pairing and insertion of repair templates through homology-directed repair (HDR) for targeted gene replacement and introduction of the desired DNA elements at specific gene loci for crop improvement. However, the potential mechanism underlying HDR of DSBs generated by Cpf1-crRNA remains to be investigated, and the inherent low efficiency of HDR and poor availability of exogenous donor DNA as repair templates strongly impede the use of HDR for precise genome editing in crop plants. Here, we provide evidence of synthesis-dependent repair of Cpf1-induced DSBs, which enables us precisely to replace the wild-type ALS gene with the intended mutant version that carries two discrete point mutations conferring herbicide resistance to rice plants. Our observation that the donor repair template (DRT) with only the left homologous arm is sufficient for precise targeted allele replacement offers a better understanding of the mechanism underlying HDR in plants, and greatly simplifies the design of DRTs for precision genome editing in crop improvement.

RevDate: 2019-10-15
CmpDate: 2019-10-15

Ljepoja B, García-Roman J, Sommer AK, et al (2018)

A proteomic analysis of an in vitro knock-out of miR-200c.

Scientific reports, 8(1):6927.

Loss of miR-200c is correlated to advanced cancer-subtypes due to increased EMT and decreased treatment efficacy by chemotherapeutics. As miRNAs regulate a multitude of targets, the analysis of differentially expressed proteins upon a genomic knock-out (KO) is of interest. In this study, we generated a TALENs KO of miR-200c in MCF7 breast cancer cells, excluded its compensation by family-members and evaluated the impact on the proteome by analyzing three individual KO-clones. We identified 26 key proteins and a variety of enrichments in metabolic and cytoskeletal pathways. In six of these targets (AGR2, FLNA/B, ALDH7A1, SCIN, GSTM3) the differential expression was additionally detected at mRNA level. Together, these alterations in protein abundance accounted for the observed biological phenotypes, i.e. increased migration and chemoresistance and altered metabolism, found in the miR-200c-KO clones. These findings provide novel insights into miR-200c and pave the way for further studies.

RevDate: 2019-10-15
CmpDate: 2019-10-15

Brown D, Samsa LA, Ito C, et al (2018)

Neuregulin-1 is essential for nerve plexus formation during cardiac maturation.

Journal of cellular and molecular medicine, 22(3):2007-2017.

The Neuregulin-1 (Nrg1)/ErbB pathway plays multiple, critical roles in early cardiac and nervous system development and has been implicated in both heart and nerve repair processes. However, the early embryonic lethality of mouse Nrg1 mutants precludes an analysis of Nrg1's function in later cardiac development and homeostasis. In this study, we generated a novel nrg1 null allele targeting all known isoforms of nrg1 in zebrafish and examined cardiac structural and functional parameters throughout development. We found that zebrafish nrg1 mutants instead survived until young adult stages when they exhibited reduced survivorship. This coincided with structural and functional defects in the developing juvenile and young adult hearts, as demonstrated by reduced intracardiac myocardial density, cardiomyocyte cell number, swimming performance and dysregulated heartbeat. Interestingly, nrg1 mutant hearts were missing long axons on the ventricle surface by standard length (SL) 5 mm, which preceded juvenile and adult cardiac defects. Given that the autonomic nervous system normally exerts fine control of cardiac output through this nerve plexus, these data suggest that Nrg1 may play a critical role in establishing the cardiac nerve plexus such that inadequate innervation leads to deficits in cardiac maturation, function and survival.

RevDate: 2019-10-13

Girardin L, Calvez V, F Débarre (2019)

Catch Me If You Can: A Spatial Model for a Brake-Driven Gene Drive Reversal.

Bulletin of mathematical biology pii:10.1007/s11538-019-00668-z [Epub ahead of print].

Population management using artificial gene drives (alleles biasing inheritance, increasing their own transmission to offspring) is becoming a realistic possibility with the development of CRISPR-Cas genetic engineering. A gene drive may, however, have to be stopped. "Antidotes" (brakes) have been suggested, but have been so far only studied in well-mixed populations. Here, we consider a reaction-diffusion system modeling the release of a gene drive (of fitness [Formula: see text]) and a brake (fitness [Formula: see text], [Formula: see text]) in a wild-type population (fitness 1). We prove that whenever the drive fitness is at most 1/2 while the brake fitness is close to 1, coextinction of the brake and the drive occurs in the long run. On the contrary, if the drive fitness is greater than 1/2, then coextinction is impossible: the drive and the brake keep spreading spatially, leaving in the invasion wake a complicated spatiotemporally heterogeneous genetic pattern. Based on numerical experiments, we argue in favor of a global coextinction conjecture provided the drive fitness is at most 1/2, irrespective of the brake fitness. The proof relies upon the study of a related predator-prey system with strong Allee effect on the prey. Our results indicate that some drives may be unstoppable and that if gene drives are ever deployed in nature, threshold drives, that only spread if introduced in high enough frequencies, should be preferred.

RevDate: 2019-10-14
CmpDate: 2019-10-14

Reardon S (2019)

World Health Organization panel weighs in on CRISPR-babies debate.

Nature, 567(7749):444-445.

RevDate: 2019-10-14
CmpDate: 2019-10-14

O'Connor CM, Hoffa MT, Taylor SE, et al (2019)

Protein phosphatase 2A Aα regulates Aβ protein expression and stability.

The Journal of biological chemistry, 294(15):5923-5934.

Protein phosphatase 2A (PP2A) represses many oncogenic signaling pathways and is an important tumor suppressor. PP2A comprises three distinct subunits and forms through a highly regulated biogenesis process, with the scaffolding A subunit existing as two highly related isoforms, Aα and Aβ. PP2A's tumor-suppressive functions have been intensely studied, and PP2A inactivation has been shown to be a prerequisite for tumor formation. Interestingly, although partial loss of the Aα isoform is growth promoting, complete Aα loss has no transformative properties. Additionally, in cancer patients, Aα is found to be inactivated in a haploinsufficient manner. Using both cellular and in vivo systems, colorectal and endometrial cancer cell lines, and biochemical and cellular assays, here we examined why the complete loss of Aα does not promote tumorigenesis. CRISPR/Cas9-mediated homozygous Aα deletion resulted in decreased colony formation and tumor growth across multiple cell lines. Protein expression analysis of PP2A family members revealed that the Aα deletion markedly up-regulates Aβ protein expression by increasing Aβ protein stability. Aβ knockdown in control and Aα knockout cell lines indicated that Aβ is necessary for cell survival in the Aα knockout cells. In the setting of Aα deficiency, co-immunoprecipitation analysis revealed increased binding of specific PP2A regulatory subunits to Aβ, and knockdown of these regulatory subunits restored colony-forming ability. Taken together, our results uncover a mechanism by which PP2A Aα regulates Aβ protein stability and activity and suggests why homozygous loss of Aα is rarely seen in cancer patients.

RevDate: 2019-10-14
CmpDate: 2019-10-14

Komatsubara AT, Goto Y, Kondo Y, et al (2019)

Single-cell quantification of the concentrations and dissociation constants of endogenous proteins.

The Journal of biological chemistry, 294(15):6062-6072.

Kinetic simulation is a useful approach for elucidating complex cell-signaling systems. The numerical simulations required for kinetic modeling in live cells critically require parameters such as protein concentrations and dissociation constants (Kd). However, only a limited number of parameters have been measured experimentally in living cells. Here we describe an approach for quantifying the concentration and Kd of endogenous proteins at the single-cell level with CRISPR/Cas9-mediated knock-in and fluorescence cross-correlation spectroscopy. First, the mEGFP gene was knocked in at the end of the mitogen-activated protein kinase 1 (MAPK1) gene, encoding extracellular signal-regulated kinase 2 (ERK2), through homology-directed repair or microhomology-mediated end joining. Next, the HaloTag gene was knocked in at the end of the ribosomal S6 kinase 2 (RSK2) gene. We then used fluorescence correlation spectroscopy to measure the protein concentrations of endogenous ERK2-mEGFP and RSK2-HaloTag fusion constructs in living cells, revealing substantial heterogeneities. Moreover, fluorescence cross-correlation spectroscopy analyses revealed temporal changes in the apparent Kd values of the binding between ERK2-mEGFP and RSK2-HaloTag in response to epidermal growth factor stimulation. Our approach presented here provides a robust and efficient method for quantifying endogenous protein concentrations and dissociation constants in living cells.

RevDate: 2019-10-14
CmpDate: 2019-10-14

Mann BJ, P Wadsworth (2018)

Distribution of Eg5 and TPX2 in mitosis: Insight from CRISPR tagged cells.

Cytoskeleton (Hoboken, N.J.), 75(12):508-521.

The mitotic spindle is a dynamic bipolar structure that mediates chromosome segregation in mitosis. In most organisms, spindle formation requires the action of kinesin-5 motor proteins that generate outward force on antiparallel microtubules to establish spindle bipolarity. Previous work has shown that Eg5 and TPX2, a spindle microtubule-associated protein that suppresses Eg5 motor activity, are enriched on parallel microtubules near spindle poles. This distribution is inconsistent with the requirement for Eg5-dependent force production during mitosis. To investigate this, we used CRISPR/Cas9 gene editing to tag Eg5 and TPX2 with EGFP and quantify protein distribution throughout mitosis. The results show that at metaphase both Eg5-EGFP and TPX2-EGFP are enriched toward spindle poles, but only TPX2-EGFP is enriched relative to microtubules. Eg5-EGFP and TPX2-EGFP show distinct localization patterns in anaphase, with Eg5-EGFP relocalizing to the midzone earlier than TPX2-EGFP. Analysis of spindles oriented at 90° to the coverslip confirmed that Eg5-EGFP was present on bridge microtubules in metaphase and anaphase; in contrast, TPX2 was not enriched, or enriched at later times, on these microtubules. Overall, TPX2 was present at 3.6X the level of Eg5 on the spindle and Eg5 was locally enriched at the prophase centrosome (~7×) compared to the whole cell. Our results show that using cells with fluorescent tags at the endogenous locus can provide novel insight into protein distribution during mitosis.

RevDate: 2019-10-14
CmpDate: 2019-10-14

Jeffries MA (2018)

Epigenetic editing: How cutting-edge targeted epigenetic modification might provide novel avenues for autoimmune disease therapy.

Clinical immunology (Orlando, Fla.), 196:49-58.

Autoimmune diseases are enigmatic and complex, and most been associated with epigenetic changes. Epigenetics describes changes in gene expression related to environmental influences mediated by a variety of effectors that alter the three-dimensional structure of chromatin and facilitate transcription factor or repressor binding. Recent years have witnessed a dramatic change and acceleration in epigenetic editing approaches, spurred on by the discovery and later development of the CRISPR/Cas9 system as a highly modular and efficient site-specific DNA binding domain. The purpose of this article is to offer a review of epigenetic editing approaches to date, with a focus on alterations of DNA methylation, and to describe a few prominent published examples of epigenetic editing. We will also offer as an example work done by our laboratory demonstrating epigenetic editing of the FOXP3 gene in human T cells. Finally, we discuss briefly the future of epigenetic editing in autoimmune disease.

RevDate: 2019-10-12

Fuchsbauer O, Swuec P, Zimberger C, et al (2019)

Cas9 Allosteric Inhibition by the Anti-CRISPR Protein AcrIIA6.

Molecular cell pii:S1097-2765(19)30697-5 [Epub ahead of print].

In the arms race against bacteria, bacteriophages have evolved diverse anti-CRISPR proteins (Acrs) that block CRISPR-Cas immunity. Acrs play key roles in the molecular coevolution of bacteria with their predators, use a variety of mechanisms of action, and provide tools to regulate Cas-based genome manipulation. Here, we present structural and functional analyses of AcrIIA6, an Acr from virulent phages, exploring its unique anti-CRISPR action. Our cryo-EM structures and functional data of AcrIIA6 binding to Streptococcus thermophilus Cas9 (St1Cas9) show that AcrIIA6 acts as an allosteric inhibitor and induces St1Cas9 dimerization. AcrIIA6 reduces St1Cas9 binding affinity for DNA and prevents DNA binding within cells. The PAM and AcrIIA6 recognition sites are structurally close and allosterically linked. Mechanistically, AcrIIA6 affects the St1Cas9 conformational dynamics associated with PAM binding. Finally, we identify a natural St1Cas9 variant resistant to AcrIIA6 illustrating Acr-driven mutational escape and molecular diversification of Cas9 proteins.

RevDate: 2019-10-11

Chen W, Zhang H, Zhang Y, et al (2019)

Molecular basis for the PAM expansion and fidelity enhancement of an evolved Cas9 nuclease.

PLoS biology, 17(10):e3000496 pii:PBIOLOGY-D-19-02313 [Epub ahead of print].

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have been harnessed as powerful genome editing tools in diverse organisms. However, the off-target effects and the protospacer adjacent motif (PAM) compatibility restrict the therapeutic applications of these systems. Recently, a Streptococcus pyogenes Cas9 (SpCas9) variant, xCas9, was evolved to possess both broad PAM compatibility and high DNA fidelity. Through determination of multiple xCas9 structures, which are all in complex with single-guide RNA (sgRNA) and double-stranded DNA containing different PAM sequences (TGG, CGG, TGA, and TGC), we decipher the molecular mechanisms of the PAM expansion and fidelity enhancement of xCas9. xCas9 follows a unique two-mode PAM recognition mechanism. For non-NGG PAM recognition, xCas9 triggers a notable structural rearrangement in the DNA recognition domains and a rotation in the key PAM-interacting residue R1335; such mechanism has not been observed in the wild-type (WT) SpCas9. For NGG PAM recognition, xCas9 applies a strategy similar to WT SpCas9. Moreover, biochemical and cell-based genome editing experiments pinpointed the critical roles of the E1219V mutation for PAM expansion and the R324L, S409I, and M694I mutations for fidelity enhancement. The molecular-level characterizations of the xCas9 nuclease provide critical insights into the mechanisms of the PAM expansion and fidelity enhancement of xCas9 and could further facilitate the engineering of SpCas9 and other Cas9 orthologs.

RevDate: 2019-10-11

Walter JM, Schubert MG, Kung SH, et al (2019)

Method for Multiplexed Integration of Synergistic Alleles and Metabolic Pathways in Yeasts via CRISPR-Cas9.

Methods in molecular biology (Clifton, N.J.), 2049:39-72.

CRISPR-Cas has proven to be a powerful tool for precision genetic engineering in a variety of difficult genetic systems. In the highly tractable yeast S. cerevisiae, CRISPR-Cas can be used to conduct multiple engineering steps in parallel, allowing for engineering of complex metabolic pathways at multiple genomic loci in as little as 1 week. In addition, CRISPR-Cas can be used to consolidate multiple causal alleles into a single strain, bypassing the laborious traditional methods using marked constructs, or mating. These tools compress the engineering timeline sixfold or more, greatly increasing the productivity of the strain engineer.

RevDate: 2019-10-11

Shiriaeva AA, Savitskaya E, Datsenko KA, et al (2019)

Detection of spacer precursors formed in vivo during primed CRISPR adaptation.

Nature communications, 10(1):4603 pii:10.1038/s41467-019-12417-w.

Type I CRISPR-Cas loci provide prokaryotes with a nucleic-acid-based adaptive immunity against foreign DNA. Immunity involves adaptation, the integration of ~30-bp DNA fragments, termed prespacers, into the CRISPR array as spacers, and interference, the targeted degradation of DNA containing a protospacer. Interference-driven DNA degradation can be coupled with primed adaptation, in which spacers are acquired from DNA surrounding the targeted protospacer. Here we develop a method for strand-specific, high-throughput sequencing of DNA fragments, FragSeq, and apply this method to identify DNA fragments accumulated in Escherichia coli cells undergoing robust primed adaptation by a type I-E or type I-F CRISPR-Cas system. The detected fragments have sequences matching spacers acquired during primed adaptation and function as spacer precursors when introduced exogenously into cells by transformation. The identified prespacers contain a characteristic asymmetrical structure that we propose is a key determinant of integration into the CRISPR array in an orientation that confers immunity.

RevDate: 2019-10-11
CmpDate: 2019-10-11

Leynaud-Kieffer LMC, Curran SC, Kim I, et al (2019)

A new approach to Cas9-based genome editing in Aspergillus niger that is precise, efficient and selectable.

PloS one, 14(1):e0210243 pii:PONE-D-18-32577.

Aspergillus niger and other filamentous fungi are widely used in industry, but efficient genetic engineering of these hosts remains nascent. For example, while molecular genetic tools have been developed, including CRISPR/Cas9, facile genome engineering of A. niger remains challenging. To address these challenges, we have developed a simple Cas9-based gene targeting method that provides selectable, iterative, and ultimately marker-free generation of genomic deletions and insertions. This method leverages locus-specific "pop-out" recombination to suppress off-target integrations. We demonstrated the effectiveness of this method by targeting the phenotypic marker albA and validated it by targeting the glaA and mstC loci. After two selection steps, we observed 100% gene editing efficiency across all three loci. This method greatly reduces the effort required to engineer the A. niger genome and overcomes low Cas9 transformations efficiency by eliminating the need for extensive screening. This method represents a significant addition to the A. niger genome engineering toolbox and could be adapted for use in other organisms. It is expected that this method will impact several areas of industrial biotechnology, such as the development of new strains for the secretion of heterologous enzymes and the discovery and optimization of metabolic pathways.

RevDate: 2019-10-11
CmpDate: 2019-10-11

Sanjana NE (2018)

A genome-wide net to catch and understand cancer.

Science translational medicine, 10(453):.

Genome-scale forward genetic screens elucidate the genetic basis of therapeutic resistance, tumor evolution, and metastasis in diverse human cancers.

RevDate: 2019-10-11
CmpDate: 2019-10-11

Ma L, Wang Y, Wang H, et al (2018)

Screen and Verification for Transgene Integration Sites in Pigs.

Scientific reports, 8(1):7433.

Efficient transgene expression in recipient cells constitutes the primary step in gene therapy. However, random integration in host genome comprises too many uncertainties. Our study presents a strategy combining bioinformatics and functional verification to find transgene integration sites in pig genome. Using an in silico approach, we screen out two candidate sites, namely, Pifs302 and Pifs501, located in actively transcribed intergenic regions with low nucleosome formation potential and without potential non-coding RNAs. After CRISPR/Cas9-mediated site-specific integration on Pifs501, we detected high EGFP expression in different pig cell types and ubiquitous EGFP expression in diverse tissues of transgenic pigs without adversely affecting 600 kb neighboring gene expression. Promoters integrated on Pifs501 exhibit hypomethylated modification, which suggest a permissive epigenetic status of this locus. We establish a versatile master cell line on Pifs501, which allows us to achieve site-specific exchange of EGFP to Follistatin with Cre/loxP system conveniently. Through in vitro and in vivo functional assays, we demonstrate the effectiveness of this screening method, and take Pifs501 as a potential site for transgene insertion in pigs. We anticipate that Pifs501 will have useful applications in pig genome engineering, though the identification of genomic safe harbor should over long-term various functional studies.

RevDate: 2019-10-11
CmpDate: 2019-10-11

Lancrey A, Joubert A, JB Boulé (2018)

Locus specific engineering of tandem DNA repeats in the genome of Saccharomyces cerevisiae using CRISPR/Cas9 and overlapping oligonucleotides.

Scientific reports, 8(1):7127.

DNA repeats constitute a large part of genomes of multicellular eucaryotes. For a longtime considered as junk DNA, their role in genome organization and tuning of gene expression is being increasingly documented. Synthetic biology has so far largely ignored DNA repeats as regulatory elements to manipulate functions in engineered genomes. The yeast Saccharomyces cerevisiae has been a workhorse of synthetic biology, owing to its genetic tractability. Here we demonstrate the ability to synthetize, in a simple manner, tandem DNA repeats of various size by Cas9-assisted oligonucleotide in vivo assembly in this organism. We show that long tandem DNA repeats of several kilobases can be assembled in one step for different monomer size and G/C content. The combinatorial nature of the approach allows exploring a wide variety of design for building synthetic tandem repeated DNA directly at a given locus in the Saccharomyces cerevisiae genome. This approach provides a simple way to incorporate tandem DNA repeat in synthetic genome designs to implement regulatory functions.

RevDate: 2019-10-10

Chen S, Soehnlen M, Blom J, et al (2019)

Comparative genomic analyses reveal diverse virulence factors and antimicrobial resistance mechanisms in clinical Elizabethkingia meningoseptica strains.

PloS one, 14(10):e0222648 pii:PONE-D-19-17060.

Three human clinical isolates of bacteria (designated strains Em1, Em2 and Em3) had high average nucleotide identity (ANI) to Elizabethkingia meningoseptica. Their genome sizes (3.89, 4.04 and 4.04 Mb) were comparable to those of other Elizabethkingia species and strains, and exhibited open pan-genome characteristics, with two strains being nearly identical and the third divergent. These strains were susceptible only to trimethoprim/sulfamethoxazole and ciprofloxacin amongst 16 antibiotics in minimum inhibitory tests. The resistome exhibited a high diversity of resistance genes, including 5 different lactamase- and 18 efflux protein- encoding genes. Forty-four genes encoding virulence factors were conserved among the strains. Sialic acid transporters and curli synthesis genes were well conserved in E. meningoseptica but absent in E. anophelis and E. miricola. E. meningoseptica carried several genes contributing to biofilm formation. 58 glycoside hydrolases (GH) and 25 putative polysaccharide utilization loci (PULs) were found. The strains carried numerous genes encoding two-component system proteins (56), transcription factor proteins (187~191), and DNA-binding proteins (6~7). Several prophages and CRISPR/Cas elements were uniquely present in the genomes.

RevDate: 2019-10-10
CmpDate: 2019-10-09

Lewis TW, Barthelemy JR, Virts EL, et al (2019)

Deficiency of the Fanconi anemia E2 ubiqitin conjugase UBE2T only partially abrogates Alu-mediated recombination in a new model of homology dependent recombination.

Nucleic acids research, 47(7):3503-3520.

The primary function of the UBE2T ubiquitin conjugase is in the monoubiquitination of the FANCI-FANCD2 heterodimer, a central step in the Fanconi anemia (FA) pathway. Genetic inactivation of UBE2T is responsible for the phenotypes of FANCT patients; however, a FANCT patient carrying a maternal duplication and a paternal deletion in the UBE2T loci displayed normal peripheral blood counts and UBE2T protein levels in B-lymphoblast cell lines. To test whether reversion by recombination between UBE2T AluYa5 elements could have occurred in the patient's hematopoietic stem cells despite the defects in homologous recombination (HR) in FA cells, we constructed HeLa cell lines containing the UBE2T AluYa5 elements and neighboring intervening sequences flanked by fluorescent reporter genes. Introduction of a DNA double strand break in the model UBE2T locus in vivo promoted single strand annealing (SSA) between proximal Alu elements and deletion of the intervening color marker gene, recapitulating the reversion of the UBE2T duplication in the FA patient. To test whether UBE2T null cells retain HR activity, the UBE2T genes were knocked out in HeLa cells and U2OS cells. CRISPR/Cas9-mediated genetic knockout of UBE2T only partially reduced HR, demonstrating that UBE2T-independent pathways can compensate for the recombination defect in UBE2T/FANCT null cells.

RevDate: 2019-10-10
CmpDate: 2019-10-10

Zhen S, Lu J, Chen W, et al (2018)

Synergistic Antitumor Effect on Bladder Cancer by Rational Combination of Programmed Cell Death 1 Blockade and CRISPR-Cas9-Mediated Long Non-Coding RNA Urothelial Carcinoma Associated 1 Knockout.

Human gene therapy, 29(12):1352-1363.

Targeted therapy produces objective responses in bladder cancer patients, although the responses can be short. Meanwhile, response rates to immune therapy are lower, but the effects are more durable. Based on these findings, it was hypothesized that urothelial carcinoma associated 1 (UCA1)-targeted therapy could synergize with programmed cell death 1 (PD-1) blockade to enhance antitumor activity. To test this hypothesis, the effects of CRISPR-Cas9 targeting of UCA1 and PD-1 were assessed in vitro and in vivo. It was found that gRNA/cas9-targeted UCA1 induced apoptosis of 5637 bladder cancer cells, whereas PD-1 gene knockout could be achieved by electroporation of gRNA/cas9 targeting PD-1, as detected by polymerase chain reaction. In 5637 cell-xenografted humanized SCID mice, stimulation with CRISPR-Cas9 systems, immune phenotypes, and cytokine expression of human dendritic cells (DCs) was detected by flow cytometry, and polymerase chain reaction, respectively. The results of these assays suggested that the gRNA/cas9 treatment upregulated expression of CD80, CD83, and CD86 and significantly increased interleukin (IL)-6, IL-12, and IL-23 and tumor necrosis factor alpha mRNA levels. Co-administration of anti-PD-1 and anti-UCA1 treatment suppressed tumor growth and markedly improved survival of 5637 xenografted mice. Additionally, the combination treatment increased interferon gamma production by T cells that subsequently enhanced the expression of Th1-associated immune-stimulating genes to reduce transcription of regulatory/suppressive immune genes and reshape the tumor microenvironment from an immunosuppressive to a stimulatory state. Finally, anti-UCA1 treatment was shown to induce interferon gamma-dependent programmed cell death ligand 1 expression within 5637 xenograft tumors in vivo. Together, these results demonstrate potent synergistic effects of a combination therapy using LncRNA UCA1-targeted therapy and immune checkpoint blockade of PD-1, thus supporting the translational potential of this combination strategy for clinical treatment of bladder cancer.

RevDate: 2019-10-10
CmpDate: 2019-10-09

Brajic A, Franckaert D, Burton O, et al (2018)

The Long Non-coding RNA Flatr Anticipates Foxp3 Expression in Regulatory T Cells.

Frontiers in immunology, 9:1989.

Mammalian genomes encode a plethora of long non-coding RNA (lncRNA). These transcripts are thought to regulate gene expression, influencing biological processes from development to pathology. Results from the few lncRNA that have been studied in the context of the immune system have highlighted potentially critical functions as network regulators. Here we explored the nature of the lncRNA transcriptome in regulatory T cells (Tregs), a subset of CD4+ T cells required to establish and maintain immunological self-tolerance. The identified Treg lncRNA transcriptome showed distinct differences from that of non-regulatory CD4+ T cells, with evidence of direct shaping of the lncRNA transcriptome by Foxp3, the master transcription factor driving the distinct mRNA profile of Tregs. Treg lncRNA changes were disproportionally reversed in the absence of Foxp3, with an enrichment for colocalisation with Foxp3 DNA binding sites, indicating a direct coordination of transcription by Foxp3 independent of the mRNA coordination function. We further identified a novel lncRNA Flatr, as a member of the core Treg lncRNA transcriptome. Flatr expression anticipates Foxp3 expression during in vitro Treg conversion, and Flatr-deficient mice show a mild delay in in vitro and peripheral Treg induction. These results implicate Flatr as part of the upstream cascade leading to Treg conversion, and may provide clues as to the nature of this process.

RevDate: 2019-10-10
CmpDate: 2019-10-10

Zhong G, Li H, Bai J, et al (2018)

Advancing the predictivity of skin sensitization by applying a novel HMOX1 reporter system.

Archives of toxicology, 92(10):3103-3115.

Reporter cell lines are a particularly useful tool to screen for the skin sensitization potential of chemicals. Current cell models based on Keap1-Nrf2 mimic induction by conducting antioxidant response element-luciferase plasmids. However, plasmid-based reporters may ignore comprehensive aspects of induction, thus affecting the accuracy of hazard identification. Herein, we developed a novel HaCaT-based reporter system, EndoSens, whereby luciferase was specifically inserted into the cassette for heme oxygenase (decycling) 1 (HMOX1, the most consistent marker induced by skin sensitizers) by CRISPR/Cas9. Testing data from 20 coded substances showed an accuracy of 90%, sensitivity of 91.7%, and specificity of 87.5%, which exceeded the OECD requirement. Among the 35 chemicals examined, predictivity was better than reported for the validated KeratinoSens™. These results indicate that the EndoSens assay could advance the predictivity of skin sensitization, thus making it a promising tool for in vitro skin sensitization testing.

RevDate: 2019-10-10
CmpDate: 2019-10-10

Shariati L, Rohani F, Heidari Hafshejani N, et al (2018)

Disruption of SOX6 gene using CRISPR/Cas9 technology for gamma-globin reactivation: An approach towards gene therapy of β-thalassemia.

Journal of cellular biochemistry, 119(11):9357-9363.

Elevation of Hemoglobin F ameliorates symptoms of β-thalassemia, a common autosomal recessive disorder. The transcription factor SOX6 plays a key role in the γ to β-globin gene switching. In the current investigation, a mutation was induced using the CRISPR/Cas9 technology in the binding domain region of SOX6 to reactivate γ-globin expression. Three CRISPR/Cas9 cassettes were provided, whose single-guide RNAs targeted different regions in the SOX6 gene-binding domain. After transfection of K562 cells with CRISPR a, b and c, and subsequent erythroid differentiation, the indel percentage of the cells was about 30%, 25%, and 24%, respectively. Relative quantification showed that the γ-globin mRNA level increased to 1.3-, 2.1-, and 1.1-fold in the cells treated with CRISPR/Cas9 a, b, and c, respectively, compared with untreated cells. Our results show that mutation induction in the binding site of the SOX6 gene leads to γ-globin reactivation. These findings support the idea that CRISPR interrupts the SOX6 binding site, and, as a result, SOX6 is incapable of binding the γ-globin promoter. In conclusion, SOX6 disruption could be considered as a therapeutic approach for β-thalassemia treatment. CRISPR/Cas9 was selected for this purpose as it is the most rapidly evolving technology.

RevDate: 2019-10-10
CmpDate: 2019-10-09

Vo BT, Kwon JA, Li C, et al (2018)

Mouse medulloblastoma driven by CRISPR activation of cellular Myc.

Scientific reports, 8(1):8733.

MYC-driven Group 3 (G3) medulloblastoma (MB) is the most aggressive of four molecular subgroups classified by transcriptome, genomic landscape and clinical outcomes. Mouse models that recapitulate human G3 MB all rely on retroviral vector-induced Myc expression driven by viral regulatory elements (Retro-Myc tumors). We used nuclease-deficient CRISPR/dCas9-based gene activation with combinatorial single guide RNAs (sgRNAs) to enforce transcription of endogenous Myc in Trp53-null neurospheres that were orthotopically transplanted into the brains of naïve animals. Three combined sgRNAs linked to dCas9-VP160 induced cellular Myc expression and large cell anaplastic MBs (CRISPR-Myc tumors) which recapitulated the molecular characteristics of mouse and human G3 MBs. The BET inhibitor JQ1 suppressed MYC expression in a human G3 MB cell line (HD-MB03) and CRISPR-Myc, but not in Retro-Myc MBs. This G3 MB mouse model in which Myc expression is regulated by its own promoter will facilitate pre-clinical studies with drugs that regulate Myc transcription.

RevDate: 2019-10-10
CmpDate: 2019-10-09

Sakaguchi Y, Nishikawa K, Seno S, et al (2018)

Roles of Enhancer RNAs in RANKL-induced Osteoclast Differentiation Identified by Genome-wide Cap-analysis of Gene Expression using CRISPR/Cas9.

Scientific reports, 8(1):7504.

Bidirectional transcription has been proposed to play a role associated with enhancer activity. Transcripts called enhancer RNAs (eRNAs) play important roles in gene regulation; however, their roles in osteoclasts are unknown. To analyse eRNAs in osteoclasts comprehensively, we used cap-analysis of gene expression (CAGE) to detect adjacent transcription start sites (TSSs) that were distant from promoters for protein-coding gene expression. When comparing bidirectional TSSs between osteoclast precursors and osteoclasts, we found that bidirectional TSSs were located in the 5'-flanking regions of the Nrp2 and Dcstamp genes. We also detected bidirectional TSSs in the intron region of the Nfatc1 gene. To investigate the role of bidirectional transcription in osteoclasts, we performed loss of function analyses using the CRISPR/Cas9 system. Targeted deletion of the DNA regions between the bidirectional TSSs led to decreased expression of the bidirectional transcripts, as well as the protein-coding RNAs of Nrp2, Dcstamp, and Nfatc1, suggesting that these transcripts act as eRNAs. Furthermore, osteoclast differentiation was impaired by targeted deletion of bidirectional eRNA regions. The combined results show that eRNAs play important roles in osteoclastogenic gene regulation, and may therefore provide novel insights to elucidate the transcriptional mechanisms that control osteoclast differentiation.

RevDate: 2019-10-10
CmpDate: 2019-10-09

Lee MH, Lin JJ, Lin YJ, et al (2018)

Genome-wide prediction of CRISPR/Cas9 targets in Kluyveromyces marxianus and its application to obtain a stable haploid strain.

Scientific reports, 8(1):7305.

Kluyveromyces marxianus, a probiotic yeast, is important in industrial applications because it has a broad substrate spectrum, a rapid growth rate and high thermotolerance. To date, however, there has been little effort in its genetic engineering by the CRISPR/Cas9 system. Therefore, we aimed at establishing the CRISPR/Cas9 system in K. marxianus and creating stable haploid strains, which will make genome engineering simpler. First, we predicted the genome-wide target sites of CRISPR/Cas9 that have been conserved among the eight sequenced genomes of K. marxianus strains. Second, we established the CRISPR/Cas9 system in the K. marxianus 4G5 strain, which was selected for its high thermotolerance, rapid growth, a pH range of pH3-9, utilization of xylose, cellobiose and glycerol, and toxin tolerance, and we knocked out its MATα3 to prevent mating-type switching. Finally, we used K. marxianus MATα3 knockout diploid strains to obtain stable haploid strains with a growth rate comparable to that of the diploid 4G5 strain. In summary, we present the workflow from identifying conserved CRISPR/Cas9 targets in the genome to knock out the MATα3 genes in K. marxianus to obtain a stable haploid strain, which can facilitate genome engineering applications.

RevDate: 2019-10-10
CmpDate: 2019-10-10

Yin L, Hu S, Mei S, et al (2018)

CRISPR/Cas9 Inhibits Multiple Steps of HIV-1 Infection.

Human gene therapy, 29(11):1264-1276.

CRISPR/Cas9 is an adaptive immune system where bacteria and archaea have evolved to resist the invading viruses and plasmid DNA by creating site-specific double-strand breaks in DNA. This study tested this gene editing system in inhibiting human immunodeficiency virus type 1 (HIV-1) infection by targeting the viral long terminal repeat and the gene coding sequences. Strong inhibition of HIV-1 infection by Cas9/gRNA was observed, which resulted not only from insertions and deletions (indels) that were introduced into viral DNA due to Cas9 cleavage, but also from the marked decrease in the levels of the late viral DNA products and the integrated viral DNA. This latter defect might have reflected the degradation of viral DNA that has not been immediately repaired after Cas9 cleavage. It was further observed that Cas9, when solely located in the cytoplasm, inhibits HIV-1 as strongly as the nuclear Cas9, except that the cytoplasmic Cas9 does not act on the integrated HIV-1 DNA and thus cannot be used to excise the latent provirus. Together, the results suggest that Cas9/gRNA is able to target and edit HIV-1 DNA both in the cytoplasm and in the nucleus. The inhibitory effect of Cas9 on HIV-1 is attributed to both the indels in viral DNA and the reduction in the levels of viral DNA.

RevDate: 2019-10-10
CmpDate: 2019-10-10

Erlich-Hadad T, Hadad R, Feldman A, et al (2018)

TAT-MTS-MCM fusion proteins reduce MMA levels and improve mitochondrial activity and liver function in MCM-deficient cells.

Journal of cellular and molecular medicine, 22(3):1601-1613.

Methylmalonic aciduria (MMA) is a disorder of organic acid metabolism resulting from a functional defect of the mitochondrial enzyme, methylmalonyl-CoA mutase (MCM). The main treatments for MMA patients are dietary restriction of propiogenic amino acids and carnitine supplementation. Liver or combined liver/kidney transplantation has been used to treat those with the most severe clinical manifestations. Thus, therapies are necessary to help improve quality of life and prevent liver, renal and neurological complications. Previously, we successfully used the TAT-MTS-Protein approach for replacing a number of mitochondrial-mutated proteins. In this targeted system, TAT, an 11 a.a peptide, which rapidly and efficiently can cross biological membranes, is fused to a mitochondrial targeting sequence (MTS), followed by the mitochondrial mature protein which sends the protein into the mitochondria. In the mitochondria, the TAT-MTS is cleaved off and the native protein integrates into its natural complexes and is fully functional. In this study, we used heterologous MTSs of human, nuclear-encoded mitochondrial proteins, to target the human MCM protein into the mitochondria. All fusion proteins reached the mitochondria and successfully underwent processing. Treatment of MMA patient fibroblasts with these fusion proteins restored mitochondrial activity such as ATP production, mitochondrial membrane potential and oxygen consumption, indicating the importance of mitochondrial function in this disease. Treatment with the fusion proteins enhanced cell viability and most importantly reduced MMA levels. Treatment also enhanced albumin and urea secretion in a CRISPR/Cas9-engineered HepG2 MUT (-/-) liver cell line. Therefore, we suggest using this TAT-MTS-Protein approach for the treatment of MMA.

RevDate: 2019-10-08
CmpDate: 2019-10-08

Shen W, Zhang J, Geng B, et al (2019)

Establishment and application of a CRISPR-Cas12a assisted genome-editing system in Zymomonas mobilis.

Microbial cell factories, 18(1):162 pii:10.1186/s12934-019-1219-5.

BACKGROUND: Efficient and convenient genome-editing toolkits can expedite genomic research and strain improvement for desirable phenotypes. Zymomonas mobilis is a highly efficient ethanol-producing bacterium with a small genome size and desirable industrial characteristics, which makes it a promising chassis for biorefinery and synthetic biology studies. While classical techniques for genetic manipulation are available for Z. mobilis, efficient genetic engineering toolkits enabling rapidly systematic and high-throughput genome editing in Z. mobilis are still lacking.

RESULTS: Using Cas12a (Cpf1) from Francisella novicida, a recombinant strain with inducible cas12a expression for genome editing was constructed in Z. mobilis ZM4, which can be used to mediate RNA-guided DNA cleavage at targeted genomic loci. gRNAs were then designed targeting the replicons of native plasmids of ZM4 with about 100% curing efficiency for three native plasmids. In addition, CRISPR-Cas12a recombineering was used to promote gene deletion and insertion in one step efficiently and precisely with efficiency up to 90%. Combined with single-stranded DNA (ssDNA), CRISPR-Cas12a system was also applied to introduce minor nucleotide modification precisely into the genome with high fidelity. Furthermore, the CRISPR-Cas12a system was employed to introduce a heterologous lactate dehydrogenase into Z. mobilis with a recombinant lactate-producing strain constructed.

CONCLUSIONS: This study applied CRISPR-Cas12a in Z. mobilis and established a genome editing tool for efficient and convenient genome engineering in Z. mobilis including plasmid curing, gene deletion and insertion, as well as nucleotide substitution, which can also be employed for metabolic engineering to help divert the carbon flux from ethanol production to other products such as lactate demonstrated in this work. The CRISPR-Cas12a system established in this study thus provides a versatile and powerful genome-editing tool in Z. mobilis for functional genomic research, strain improvement, as well as synthetic microbial chassis development for economic biochemical production.

RevDate: 2019-10-08
CmpDate: 2019-10-08

Han S, Miyoshi K, Shikada S, et al (2019)

TULP3 is required for localization of membrane-associated proteins ARL13B and INPP5E to primary cilia.

Biochemical and biophysical research communications, 509(1):227-234.

The primary cilia are known as biosensors that transduce signals through the ciliary membrane proteins in vertebrate cells. The ciliary membrane contains transmembrane proteins and membrane-associated proteins. Tubby-like protein 3 (TULP3), a member of the tubby family, has been shown to interact with the intraflagellar transport-A complex (IFT-A) and to be involved in the ciliary localization of transmembrane proteins, although its role in the ciliary entry of membrane-associated proteins has remained unclear. Here, to determine whether TULP3 is required for the localization of ciliary membrane-associated proteins, we generated and analyzed TULP3-knockout (KO) hTERT RPE-1 (RPE1) cells. Immunofluorescence analysis demonstrated that ciliary formation was downregulated in TULP3-KO cells and that membrane-associated proteins, ADP-ribosylation factor-like 13B (ARL13B) and inositol polyphosphate-5-phosphatase E (INPP5E), failed to localize to primary cilia in TULP3-KO cells. These defects in the localization of ARL13B and INPP5E in TULP3-KO cells were rescued by the exogenous expression of wild-type TULP3, but not that of mutant TULP3 lacking the ability to bind IFT-A. In addition, the expression of TUB protein, another member of the tubby family whose endogenous expression is absent in RPE1 cells, also rescued the defective ciliary localization of ARL13B and INPP5E in TULP3-KO cells, suggesting that there is functional redundancy between TULP3 and TUB. Our findings indicate that TULP3 participates in ciliogenesis, and targets membrane-associated proteins to primary cilia via binding to IFT-A.

RevDate: 2019-10-08
CmpDate: 2019-10-08

Sekine R, Kawata T, T Muramoto (2018)

CRISPR/Cas9 mediated targeting of multiple genes in Dictyostelium.

Scientific reports, 8(1):8471.

CRISPR/Cas9 has emerged in various organisms as a powerful technology for targeted gene knockout; however, no reports of editing the Dictyostelium genome efficiently using this system are available. We describe here the application of CRISPR/Cas9-mediated gene modification in Dictyostelium. The endogenous tRNA-processing system for expressing sgRNA was approximately 10 times more effective than the commonly used U6 promoter. The resulting sgRNA affected the sub-nuclear localisation of Cas9, indicating that the expression level of sgRNA was sufficiently high to form Cas9 and sgRNA complexes within the nucleus. The all-in-one vector containing Cas9 and sgRNA was transiently expressed to generate mutants in five PI3K genes. Mutation detective PCR revealed the mutagenesis frequency of the individual genes to be between 72.9% and 100%. We confirmed that all five targeting loci in the four independent clones had insertion/deletion mutations in their target sites. Thus, we show that the CRISPR/Cas9 system can be used in Dictyostelium cells to enable efficient genome editing of multiple genes. Since this system utilises transient expression of the all-in-one vector, it has the advantage that the drug resistance cassette is not integrated into the genome and simple vector construction, involving annealing two oligo-DNAs.

RevDate: 2019-10-08
CmpDate: 2019-10-08

Khan M, Jabeen N, Khan T, et al (2018)

The evolutionarily conserved genes: Tex37, Ccdc73, Prss55 and Nxt2 are dispensable for fertility in mice.

Scientific reports, 8(1):4975.

There are more than 2300 genes that are predominantly expressed in mouse testes. The role of hundreds of these genes has been studied in mouse spermatogenesis but still there are many genes whose function is unknown. Gene knockout (KO) strategy in mice is widely used for in vivo study of gene function. The present study was designed to explore the function of the four genes: Tex37, Ccdc73, Prss55 and Nxt2, which were evolutionarily conserved in eutherians. We found that these genes had a testis-enriched expression pattern in mice except Nxt2. We knocked out these genes by CRISPR/Cas9 individually and found that all the KO mice had normal fertility with no detectable difference in testis/body weight ratios, epididymal sperm counts, as well as testicular and epididymal histology from wild type mice. Although these genes are evolutionarily conserved in eutherians including human and mouse, they are not individually essential for spermatogenesis, testis development and male fertility in mice in laboratory conditions. Our report of these fertile KO data could avoid the repetition and duplication of efforts which will help in prioritizing efforts to focus on genes that are indispensable for male reproduction.

RevDate: 2019-10-07

Jin Y, Liu M, Sa R, et al (2019)

Mouse Models of Thyroid Cancer: Bridging Pathogenesis and Novel Therapeutics.

Cancer letters pii:S0304-3835(19)30489-6 [Epub ahead of print].

Due to a global increase in the incidence of thyroid cancer, numerous novel mouse models were established to reveal thyroid cancer pathogenesis and test promising therapeutic strategies, necessitating a comprehensive review of translational medicine that covers (i) the role of mouse models in the research of thyroid cancer pathogenesis, and (ii) preclinical testing of potential anti-thyroid cancer therapeutics. The present review article aims to: (i) describe the current approaches for mouse modeling of thyroid cancer, (ii) provide insight into the biology and genetics of thyroid cancers, and (iii) offer guidance on the use of mouse models for testing potential therapeutics in preclinical settings. Based on research with mouse models of thyroid cancer pathogenesis involving the RTK, RAS/RAF/MEK/ERK, PI3K/AKT/mTOR, SRC, and JAK-STAT signaling pathways, inhibitors of VEGFR, MEK, mTOR, SRC, and STAT3 have been developed as anti-thyroid cancer drugs for "bench-to-bedside" translation. In the future, mouse models of thyroid cancer will be designed to be ''humanized" and "patient-like," offering opportunities to: (i) investigate the pathogenesis of thyroid cancer through target screening based on the CRISPR/Cas system, (ii) test drugs based on new mouse models, and (iii) explore the underlying mechanisms based on multi-omics.

RevDate: 2019-10-07

Greisch JF, Tamara S, Scheltema RA, et al (2019)

Expanding the mass range for UVPD-based native top-down mass spectrometry.

Chemical science, 10(30):7163-7171 pii:c9sc01857c.

Native top-down mass spectrometry is emerging as a methodology that can be used to structurally investigate protein assemblies. To extend the possibilities of native top-down mass spectrometry to larger and more heterogeneous biomolecular assemblies, advances in both the mass analyzer and applied fragmentation techniques are still essential. Here, we explore ultraviolet photodissociation (UVPD) of protein assemblies on an Orbitrap with extended mass range, expanding its usage to large and heterogeneous macromolecular complexes, reaching masses above 1 million Da. We demonstrate that UVPD can lead not only to the ejection of intact subunits directly from such large intact complexes, but also to backbone fragmentation of these subunits, providing enough sequence information for subunit identification. The Orbitrap mass analyzer enables simultaneous monitoring of the precursor, the subunits, and the subunit fragments formed upon UVPD activation. While only partial sequence coverage of the subunits is observed, the UVPD data yields information about the localization of chromophores covalently attached to the subunits of the light harvesting complex B-phycoerythrin, extensive backbone fragmentation in a subunit of a CRISPR-Cas Csy (type I-F Cascade) complex, and sequence modifications in a virus-like proteinaceous nano-container. Through these multiple applications we demonstrate for the first time that UVPD based native top-down mass spectrometry is feasible for large and heterogeneous particles, including ribonucleoprotein complexes and MDa virus-like particles.

RevDate: 2019-10-07
CmpDate: 2019-10-04

Zheng R, Fang X, He L, et al (2018)

Generation of a Primary Hyperoxaluria Type 1 Disease Model Via CRISPR/Cas9 System in Rats.

Current molecular medicine, 18(7):436-447.

BACKGROUND: Primary hyperoxaluria type 1 (PH1) is an inherited disease caused by mutations in alanine-glyoxylate aminotransferase (AGXT). It is characterized by abnormal metabolism of glyoxylic acid in the liver leading to endogenous oxalate overproduction and deposition of oxalate in multiple organs, mainly the kidney. Patients of PH1 often suffer from recurrent urinary tract stones, and finally renal failure. There is no effective treatment other than combined liver-kidney transplantation.

METHODS: Microinjection was administered to PH1 rats. Urine samples were collected for urine analysis. Kidney tissues were for Western blotting, quantitative PCR, AGT assays and histological evaluation.

RESULTS: In this study, we generated a novel PH1 disease model through CRISPR/Cas9 mediated disruption of mitochondrial localized Agxt gene isoform in rats. Agxt-deficient rats excreted more oxalate in the urine than WT animals. Meanwhile, mutant rats exhibited crystalluria and showed a slight dilatation of renal tubules with mild fibrosis in the kidney. When supplied with 0.4% ethylene glycol (EG) in drinking water, mutant rats excreted greater abundance of oxalate and developed severe nephrocalcinosis in contrast to WT animals. Significantly elevated expression of inflammation- and fibrosisrelated genes was also detected in mutants.

CONCLUSION: These data suggest that Agxt-deficiency in mitochondria impairs glyoxylic acid metabolism and leads to PH1 in rats. This rat strain would not only be a useful model for the study of the pathogenesis and pathology of PH1 but also a valuable tool for the development and evaluation of innovative drugs and therapeutics.

RevDate: 2019-10-07
CmpDate: 2019-10-07

Schaks M, Singh SP, Kage F, et al (2018)

Distinct Interaction Sites of Rac GTPase with WAVE Regulatory Complex Have Non-redundant Functions in Vivo.

Current biology : CB, 28(22):3674-3684.e6.

Cell migration often involves the formation of sheet-like lamellipodia generated by branched actin filaments. The branches are initiated when Arp2/3 complex [1] is activated by WAVE regulatory complex (WRC) downstream of small GTPases of the Rac family [2]. Recent structural studies defined two independent Rac binding sites on WRC within the Sra-1/PIR121 subunit of the pentameric WRC [3, 4], but the functions of these sites in vivo have remained unknown. Here we dissect the mechanism of WRC activation and the in vivo relevance of distinct Rac binding sites on Sra-1, using CRISPR/Cas9-mediated gene disruption of Sra-1 and its paralog PIR121 in murine B16-F1 cells combined with Sra-1 mutant rescue. We show that the A site, positioned adjacent to the binding region of WAVE-WCA mediating actin and Arp2/3 complex binding, is the main site for allosteric activation of WRC. In contrast, the D site toward the C terminus is dispensable for WRC activation but required for optimal lamellipodium morphology and function. These results were confirmed in evolutionarily distant Dictyostelium cells. Moreover, the phenotype seen in D site mutants was recapitulated in Rac1 E31 and F37 mutants; we conclude these residues are important for Rac-D site interaction. Finally, constitutively activated WRC was able to induce lamellipodia even after both Rac interaction sites were lost, showing that Rac interaction is not essential for membrane recruitment. Our data establish that physical interaction with Rac is required for WRC activation, in particular through the A site, but is not mandatory for WRC accumulation in the lamellipodium.

RevDate: 2019-10-07
CmpDate: 2019-10-03

Bourgeois L, Pyne ME, VJJ Martin (2018)

A Highly Characterized Synthetic Landing Pad System for Precise Multicopy Gene Integration in Yeast.

ACS synthetic biology, 7(11):2675-2685.

A fundamental undertaking of metabolic engineering involves identifying and troubleshooting metabolic bottlenecks that arise from imbalances in pathway flux. To expedite the systematic screening of enzyme orthologs in conjunction with DNA copy number tuning, here we develop a simple and highly characterized CRISPR-Cas9 integration system in Saccharomyces cerevisiae. Our engineering strategy introduces a series of synthetic DNA landing pads (LP) into the S. cerevisiae genome to act as sites for high-level gene integration. LPs facilitate multicopy gene integration of one, two, three, or four DNA copies in a single transformation, thus providing precise control of DNA copy number. We applied our LP system to norcoclaurine synthase (NCS), an enzyme with poor kinetic properties involved in the first committed step of the production of high-value benzylisoquinoline alkaloids. The platform enabled rapid construction of a 40-strain NCS library by integrating ten NCS orthologs in four gene copies each. Six active NCS variants were identified, whereby production of (S)-norcoclaurine could be further enhanced by increasing NCS copy number. We anticipate the LP system will aid in metabolic engineering efforts by providing strict control of gene copy number and expediting strain and pathway engineering campaigns.

RevDate: 2019-10-07
CmpDate: 2019-10-04

Sinha R, P Shukla (2019)

Antimicrobial Peptides: Recent Insights on Biotechnological Interventions and Future Perspectives.

Protein and peptide letters, 26(2):79-87.

With the unprecedented rise of drug-resistant pathogens, particularly antibiotic-resistant bacteria, and no new antibiotics in the pipeline over the last three decades, the issue of antimicrobial resistance has emerged as a critical public health threat. Antimicrobial Peptides (AMP) have garnered interest as a viable solution to this grave issue and are being explored for their potential antimicrobial applications. Given their low bioavailability in nature, tailoring new AMPs or strategizing approaches for increasing the yield of AMPs, therefore, becomes pertinent. The present review focuses on biotechnological interventions directed towards enhanced AMP synthesis and revisits existing genetic engineering and synthetic biology strategies for production of AMPs. This review further underscores the importance and potential applications of advanced gene editing technologies for the synthesis of novel AMPs in future.

RevDate: 2019-10-07
CmpDate: 2019-10-03

Löbs AK, Schwartz C, Thorwall S, et al (2018)

Highly Multiplexed CRISPRi Repression of Respiratory Functions Enhances Mitochondrial Localized Ethyl Acetate Biosynthesis in Kluyveromyces marxianus.

ACS synthetic biology, 7(11):2647-2655.

The emergence of CRISPR-Cas9 for targeted genome editing and regulation has enabled the manipulation of desired traits and enhanced strain development of nonmodel microorganisms. The natural capacity of the yeast Kluyveromyces marxianus to produce volatile esters at high rate and at elevated temperatures make it a potentially valuable production platform for industrial biotechnology. Here, we identify the native localization of ethyl acetate biosynthesis in K. marxianus and use this information to develop a multiplexed CRISPRi system for redirecting carbon flux along central metabolic pathways, increasing ethyl acetate productivity. First, we identified the primary pathways of precursor and acetate ester biosynthesis. A genetic knockout screen revealed that the alcohol acetyltransferase Eat1 is the critical enzyme for ethyl, isoamyl, and phenylethyl acetate production. Truncation studies revealed that high ester biosynthesis is contingent on Eat1 mitochondrial localization. As ethyl acetate is formed from the condensation of ethanol and acetyl-CoA, we modulated expression of the TCA cycle and electron transport chain genes using a highly multiplexed CRISPRi approach. The simultaneous knockdown of ACO2b, SDH2, RIP1, and MSS51 resulted in a 3.8-fold increase in ethyl acetate productivity over the already high natural capacity. This work demonstrates that multiplexed CRISPRi regulation of central carbon flux, supported by a fundamental understanding of pathway biochemistry, is a potent strategy for metabolic engineering in nonconventional microorganisms.

RevDate: 2019-10-07
CmpDate: 2019-10-03

Shao Y, Lu N, Qin Z, et al (2018)

CRISPR-Cas9 Facilitated Multiple-Chromosome Fusion in Saccharomyces cerevisiae.

ACS synthetic biology, 7(11):2706-2708.

Eukaryotic cells usually contain multiple linear chromosomes. Recently, we artificially created a functional single-chromosome yeast via sequential two-chromosome fusion utilizing the high performance of the CRISPR-Cas9 system and homologous recombination in Saccharomyces cerevisiae. In this paper, we adapted this method for the simultaneous fusion of multiple chromosomes. We demonstrated the fusion of two, two-chromosome sets with a 75% positive rate and three-chromosome fusions with a 50% positive rate. We also found that by using an additional selection marker, the positive rate of two-chromosome fusions reached 100%. Due to the simplicity, efficiency, and portability of this method, we expect that it can be easily adapted for multiple-chromosome fusions in other organisms.

RevDate: 2019-10-07
CmpDate: 2019-10-07

Liu Y, Yu C, Daley TP, et al (2018)

CRISPR Activation Screens Systematically Identify Factors that Drive Neuronal Fate and Reprogramming.

Cell stem cell, 23(5):758-771.e8.

Comprehensive identification of factors that can specify neuronal fate could provide valuable insights into lineage specification and reprogramming, but systematic interrogation of transcription factors, and their interactions with each other, has proven technically challenging. We developed a CRISPR activation (CRISPRa) approach to systematically identify regulators of neuronal-fate specification. We activated expression of all endogenous transcription factors and other regulators via a pooled CRISPRa screen in embryonic stem cells, revealing genes including epigenetic regulators such as Ezh2 that can induce neuronal fate. Systematic CRISPR-based activation of factor pairs allowed us to generate a genetic interaction map for neuronal differentiation, with confirmation of top individual and combinatorial hits as bona fide inducers of neuronal fate. Several factor pairs could directly reprogram fibroblasts into neurons, which shared similar transcriptional programs with endogenous neurons. This study provides an unbiased discovery approach for systematic identification of genes that drive cell-fate acquisition.

RevDate: 2019-10-07
CmpDate: 2019-10-07

Ali R, Al-Kawaz A, Toss MS, et al (2018)

Targeting PARP1 in XRCC1-Deficient Sporadic Invasive Breast Cancer or Preinvasive Ductal Carcinoma In Situ Induces Synthetic Lethality and Chemoprevention.

Cancer research, 78(24):6818-6827.

: Targeting PARP1 for synthetic lethality is a new strategy for breast cancers harboring germline mutations in BRCA. However, these mutations are rare, and reactivation of BRCA-mediated pathways may result in eventual resistance to PARP1 inhibitor therapy. Alternative synthetic lethality approaches targeting more common sporadic breast cancers and preinvasive ductal carcinoma in situ (DCIS) are desirable. Here we show that downregulation of XRCC1, which interacts with PARP1 and coordinates base excision repair, is an early event in human breast cancer pathogenesis. XRCC1-deficient DCIS were aggressive and associated with increased risk of local recurrence. Human invasive breast cancers deficient in XRCC1 and expressing high PARP1 levels also manifested aggressive features and poor outcome. The PARP1 inhibitor olaparib was synthetically lethal in XRCC1-deficient DCIS and invasive breast cancer cells. We conclude that targeting PARP1 is an attractive strategy for synthetic lethality and chemoprevention in XRCC1-deficient breast cancers, including preinvasive DCIS. SIGNIFICANCE: These findings show that loss of XRCC1, which is associated with more malignant DCIS, can be exploited by PARP inhibition, suggesting its application as a promising therapeutic and chemoprevention strategy in XRCC1-deficient tumor cells.

RevDate: 2019-10-07
CmpDate: 2019-10-03

Wu Z, Zhang L, Qiao D, et al (2018)

Functional Analyses of Cassette Chromosome Recombinase C2 (CcrC2) and Its Use in Eliminating Methicillin Resistance by Combining CRISPR-Cas9.

ACS synthetic biology, 7(11):2590-2599.

Worldwide occurrence of methicillin-resistant Staphylococcus aureus (MRSA) poses enormous challenges for both communities and health care settings. Cassette chromosome recombinases (Ccr) specifically perform excision and acquisition of a staphylococcal cassette chromosome mec (SCC mec) in staphylococci and are responsible for the spread of methicillin resistance. This study explored the roles of CcrC2, a recently discovered Ccr, in the horizontal transfer of SCC mec and developed a potential means to control the spread of methicillin resistance. Knockout of CcrC2 completely aborted the excision of SCC mec, while overexpression of CcrC2 partially removed the SCC mec from the genome and transformed methicillin-resistant Staphylococcus aureus (MRSA) into methicillin-susceptible Staphylococcus aureus (MSSA). Moreover, two nucleotide residues (G5C6) in the direct repeat sequence within an att site were found to be critical for excision and acquisition efficiencies. To block the horizontal transfer of methicillin resistance, a SCC mec killer system was developed by combining the CcrC2-mediated SCC mec excision and the mecA-targeting CRISPR-Cas9 machinery. The SCC mec killer transformed MRSA to MSSA and disrupted the mecA-carrying SCC mec intermediate, thereby eliminating methicillin resistance determinant mecA gene inside a MRSA cell and blocking the horizontal transfer of SCC mec. The SCC mec killer was versatile for efficiently removing multiple types of SCC mec elements. It is envisioned that this approach could offer a new means to control the spread of methicillin resistance.

RevDate: 2019-10-07
CmpDate: 2019-10-03

Huang JF, Shen ZY, Mao QL, et al (2018)

Systematic Analysis of Bottlenecks in a Multibranched and Multilevel Regulated Pathway: The Molecular Fundamentals of l-Methionine Biosynthesis in Escherichia coli.

ACS synthetic biology, 7(11):2577-2589.

To produce chemicals and fuels from renewable resources, various strategies and genetic tools have been developed to redesign pathways and optimize the metabolic flux in microorganisms. However, in most successful cases, the target chemicals are synthesized through a linear pathway, and regular methodologies for the identification of bottlenecks and metabolic flux optimization in multibranched and multilevel regulated pathways, such as the l-methionine biosynthetic pathway, have rarely been reported. In the present study, a systematic analysis strategy was employed to gradually reveal and remove the potential bottlenecks limiting the l-methionine biosynthesis in E. coli. 80 genes in central metabolism and selected amino acids biosynthetic pathways were first repressed or upregulated to probe their effects on l-methionine accumulation. The l-methionine biosynthetic pathway was then modularized and iteratively genetic modifications were performed to uncover the multiple layers of limitations and stepwise improve the l-methionine titer. The metabolomics data further revealed a more evenly distributed metabolic flux in l-methionine biosynthesis pathway of the optimal strain and provided valuable suggestions for further optimization. The optimal strain produced 16.86 g/L of l-methionine in 48 h by fed-batch fermentation. This work is the first to our knowledge to systematically elucidate the molecular fundamentals of multilevel regulation of l-methionine biosynthesis. It also demonstrated that the systematic analysis strategy can boost our ability to identify the potential bottlenecks and optimize the metabolic flux in multibranched and multilevel regulated pathways for the production of corresponding chemicals.

RevDate: 2019-10-07
CmpDate: 2019-10-07

Penfold L, Woods A, Muckett P, et al (2018)

CAMKK2 Promotes Prostate Cancer Independently of AMPK via Increased Lipogenesis.

Cancer research, 78(24):6747-6761.

: New targets are required for treating prostate cancer, particularly castrate-resistant disease. Previous studies reported that calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) expression is increased in human prostate cancer. Here, we show that Camkk2 deletion or pharmacologic inhibition protects against prostate cancer development in a preclinical mouse model that lacks expression of prostate-specific Pten. In contrast, deletion of AMP-activated protein kinase (Ampk) β1 resulted in earlier onset of adenocarcinoma development. These findings suggest for the first time that Camkk2 and Ampk have opposing effects in prostate cancer progression. Loss of CAMKK2 in vivo or in human prostate cancer cells reduced the expression of two key lipogenic enzymes, acetyl-CoA carboxylase and fatty acid synthase. This reduction was mediated via a posttranscriptional mechanism, potentially involving a decrease in protein translation. Moreover, either deletion of CAMKK2 or activation of AMPK reduced cell growth in human prostate cancer cells by inhibiting de novo lipogenesis. Activation of AMPK in a panel of human prostate cancer cells inhibited cell proliferation, migration, and invasion as well as androgen-receptor signaling. These findings demonstrate that CAMKK2 and AMPK have opposing effects on lipogenesis, providing a potential mechanism for their contrasting effects on prostate cancer progression in vivo. They also suggest that inhibition of CAMKK2 combined with activation of AMPK would offer an efficacious therapeutic strategy in treatment of prostate cancer. SIGNIFICANCE: These findings show that CAMKK2 and its downstream target AMPK have opposing effects on prostate cancer development and raise the possibility of a new combined therapeutic approach that inhibits CAMKK2 and activates AMPK.

RevDate: 2019-10-07
CmpDate: 2019-10-07

Borsenberger V, Onésime D, Lestrade D, et al (2018)

Multiple Parameters Drive the Efficiency of CRISPR/Cas9-Induced Gene Modifications in Yarrowia lipolytica.

Journal of molecular biology, 430(21):4293-4306.

Yarrowia lipolytica is an oleaginous yeast of growing industrial interest for biotechnological applications. In the last few years, genome edition has become an easier and more accessible prospect with the world wild spread development of CRISPR/Cas9 technology. In this study, we focused our attention on the production of the two key elements of the CRISPR-Cas9 ribonucleic acid protein complex in this non-conventional yeast. The efficiency of NHEJ-induced knockout was measured by time-course monitoring using multiple parameters flow cytometry, as well as phenotypic and genotypic observations, and linked to nuclease production levels showing that its strong overexpression is unnecessary. Thus, the limiting factor for the generation of a functional ribonucleic acid protein complex clearly resides in guide expression, which was probed by testing different linker lengths between the transfer RNA promoter and the sgRNA. The results highlight a clear deleterious effect of mismatching bases at the 5' end of the target sequence. For the first time in yeast, an investigation of its maturation from the primary transcript was undertaken by sequencing multiple sgRNAs extracted from the host. These data provide insights into of the yeast small RNA processing, from synthesis to maturation, and suggests a pathway for their degradation in Y. lipolytica. Subsequently, a whole-genome sequencing of a modified strain detected no abnormal modification due to off-target effects, confirming CRISPR/Cas9 as a safe strategy for editing Y. lipolytica genome. Finally, the optimized system was used to promote in vivo directed mutagenesis via homology-directed repair with a ssDNA oligonucleotide.

RevDate: 2019-10-07
CmpDate: 2019-10-04

Wang Y, Wang S, Chen W, et al (2018)

CRISPR-Cas9 and CRISPR-Assisted Cytidine Deaminase Enable Precise and Efficient Genome Editing in Klebsiella pneumoniae.

Applied and environmental microbiology, 84(23):.

Klebsiella pneumoniae is a promising industrial microorganism as well as a major human pathogen. The recent emergence of carbapenem-resistant K. pneumoniae has posed a serious threat to public health worldwide, emphasizing a dire need for novel therapeutic means against drug-resistant K. pneumoniae Despite the critical importance of genetics in bioengineering, physiology studies, and therapeutic-means development, genome editing, in particular, the highly desirable scarless genetic manipulation in K. pneumoniae, is often time-consuming and laborious. Here, we report a two-plasmid system, pCasKP-pSGKP, used for precise and iterative genome editing in K. pneumoniae By harnessing the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 genome cleavage system and the lambda Red recombination system, pCasKP-pSGKP enabled highly efficient genome editing in K. pneumoniae using a short repair template. Moreover, we developed a cytidine base-editing system, pBECKP, for precise C→T conversion in both the chromosomal and plasmid-borne genes by engineering the fusion of the cytidine deaminase APOBEC1 and a Cas9 nickase. By using both the pCasKP-pSGKP and the pBECKP tools, the blaKPC-2 gene was confirmed to be the major factor that contributed to the carbapenem resistance of a hypermucoviscous carbapenem-resistant K. pneumoniae strain. The development of the two editing tools will significantly facilitate the genetic engineering of K. pneumoniaeIMPORTANCE Genetics is a key means to study bacterial physiology. However, the highly desirable scarless genetic manipulation is often time-consuming and laborious for the major human pathogen K. pneumoniae We developed a CRISPR-Cas9-mediated genome-editing method and a cytidine base-editing system, enabling rapid, highly efficient, and iterative genome editing in both industrial and clinically isolated K. pneumoniae strains. We applied both tools in dissecting the drug resistance mechanism of a hypermucoviscous carbapenem-resistant K. pneumoniae strain, elucidating that the blaKPC-2 gene was the major factor that contributed to the carbapenem resistance of the hypermucoviscous carbapenem-resistant K. pneumoniae strain. Utilization of the two tools will dramatically accelerate a wide variety of investigations in diverse K. pneumoniae strains and relevant Enterobacteriaceae species, such as gene characterization, drug discovery, and metabolic engineering.

RevDate: 2019-10-07
CmpDate: 2019-10-07

Oppel F, Schürmann M, Goon P, et al (2018)

Specific Targeting of Oncogenes Using CRISPR Technology.

Cancer research, 78(19):5506-5512.

In recent decades, tools of molecular biology have enabled researchers to genetically modify model organisms, including human cells. RNAi, zinc-finger nucleases, transcription activator-like effector nucleases, CRISPR-Cas9 (clustered regularly-interspaced short palindromic repeats and CRISPR-associated protein 9), retro- or lentiviral gene transfer, and many other methods can be utilized to remove genes, add genes, or change their expression. Within the same timeframe, survival rates for many highly malignant tumor diseases have not improved substantially. If modern medicine could apply even a subset of research methods in clinical management, which are already well established and controllable in basic research laboratories, this could strongly impact patients' prognosis. CRISPR-Cas9 is a method to precisely target and manipulate genomic loci and recent studies have attempted to use this method as a genetic treatment for Duchenne muscular dystrophy, blood disorders, autosomal-dominant hearing loss, and cancer. Some of these approaches target mutant genomic sequences specifically and try to avoid affecting the respective normal loci. Considering obvious genetic risks opposing the objected benefits, data are needed to show whether CRISPR technology is suitable as a future cancer therapy approach or not. Here, we develop strategies for the specific targeting of viral cancer drivers and oncogenes activated by mutation, using the latest CRISPR technology. Cancer Res; 78(19); 5506-12. ©2018 AACR.

RevDate: 2019-10-07
CmpDate: 2019-10-03

Bull SE, Seung D, Chanez C, et al (2018)

Accelerated ex situ breeding of GBSS- and PTST1-edited cassava for modified starch.

Science advances, 4(9):eaat6086.

Crop diversification required to meet demands for food security and industrial use is often challenged by breeding time and amenability of varieties to genome modification. Cassava is one such crop. Grown for its large starch-rich storage roots, it serves as a staple food and a commodity in the multibillion-dollar starch industry. Starch is composed of the glucose polymers amylopectin and amylose, with the latter strongly influencing the physicochemical properties of starch during cooking and processing. We demonstrate that CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)-mediated targeted mutagenesis of two genes involved in amylose biosynthesis, PROTEIN TARGETING TO STARCH (PTST1) or GRANULE BOUND STARCH SYNTHASE (GBSS), can reduce or eliminate amylose content in root starch. Integration of the Arabidopsis FLOWERING LOCUS T gene in the genome-editing cassette allowed us to accelerate flowering-an event seldom seen under glasshouse conditions. Germinated seeds yielded S1, a transgene-free progeny that inherited edited genes. This attractive new plant breeding technique for modified cassava could be extended to other crops to provide a suite of novel varieties with useful traits for food and industrial applications.

RevDate: 2019-10-07
CmpDate: 2019-10-03

De Luna N, Suarez-Calvet X, Garicano M, et al (2018)

Effect of MAPK Inhibition on the Differentiation of a Rhabdomyosarcoma Cell Line Combined With CRISPR/Cas9 Technology: An In Vitro Model of Human Muscle Diseases.

Journal of neuropathology and experimental neurology, 77(10):964-972.

The human rhabdomyosarcoma cell line TE671 has been used extensively to study different aspects of muscle biology. However, its ability to differentiate and form myotubes has not been explored. Here, we examined muscle differentiation when we specifically stopped proliferation of human TE671 (WT-TE671) cells by using 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126), an MAPK inhibitor. Our data show that treated cells initiated fusion, and myotube formation and that expression levels of dysferlin and myogenin were increased, whereas those of pax7 were decreased. Treatment of WT-TE671 cells with vitamin D3 alone and cotreatment with U0126 also promoted dysferlin expression. In addition, we knocked out the DYSF gene, which is involved in muscle differentiation, using CRISPR/Cas9 technology in WT-TE671 cells (Dysf-KO TE671). No dysferlin expression was observed before and after U0126 treatment. Although myogenin expression was absent in vehicle-treated Dysf-KO TE671 cells, after addition of U0126, myogenin reached levels similar to WT-TE671. This widely available source of human cells appropriately treated with U0126 may represent a useful model to study human muscle physiology in vitro. This dysferlin-deficient cell line should allow the study of pathophysiological pathways involved in dysferlin-deficient muscle and constitute a tool for high-throughput screening of therapeutic compounds for patients with dysferlinopathy and other muscle diseases.

RevDate: 2019-10-07
CmpDate: 2019-10-07

Koifman G, Shetzer Y, Eizenberger S, et al (2018)

A Mutant p53-Dependent Embryonic Stem Cell Gene Signature Is Associated with Augmented Tumorigenesis of Stem Cells.

Cancer research, 78(20):5833-5847.

Mutations in the tumor suppressor p53 are the most frequent alterations in human cancer. These mutations include p53-inactivating mutations as well as oncogenic gain-of-function (GOF) mutations that endow p53 with capabilities to promote tumor progression. A primary challenge in cancer therapy is targeting stemness features and cancer stem cells (CSC) that account for tumor initiation, metastasis, and cancer relapse. Here we show that in vitro cultivation of tumors derived from mutant p53 murine bone marrow mesenchymal stem cells (MSC) gives rise to aggressive tumor lines (TL). These MSC-TLs exhibited CSC features as displayed by their augmented oncogenicity and high expression of CSC markers. Comparative analyses between MSC-TL with their parental mutant p53 MSC allowed for identification of the molecular events underlying their tumorigenic properties, including an embryonic stem cell (ESC) gene signature specifically expressed in MSC-TLs. Knockout of mutant p53 led to a reduction in tumor development and tumorigenic cell frequency, which was accompanied by reduced expression of CSC markers and the ESC MSC-TL signature. In human cancer, MSC-TL ESC signature-derived genes correlated with poor patient survival and were highly expressed in human tumors harboring p53 hotspot mutations. These data indicate that the ESC gene signature-derived genes may serve as new stemness-based prognostic biomarkers as well as novel cancer therapeutic targets.Significance: Mesenchymal cancer stem cell-like cell lines express a mutant p53-dependent embryonic stem cell gene signature, which can serve as a potential prognostic biomarker and therapeutic target in cancer. Cancer Res; 78(20); 5833-47. ©2018 AACR.

RevDate: 2019-10-07
CmpDate: 2019-10-03

Quintero CM, Laursen KB, Mongan NP, et al (2018)

CARM1 (PRMT4) Acts as a Transcriptional Coactivator during Retinoic Acid-Induced Embryonic Stem Cell Differentiation.

Journal of molecular biology, 430(21):4168-4182.

Activation of the retinoic acid (RA) signaling pathway is important for controlling embryonic stem cell differentiation and development. Modulation of this pathway occurs through the recruitment of different epigenetic regulators at the retinoic acid receptors (RARs) located at RA-responsive elements and/or RA-responsive regions of RA-regulated genes. Coactivator-associated arginine methyltransferase 1 (CARM1, PRMT4) is a protein arginine methyltransferase that also functions as a transcriptional coactivator. Previous studies highlight CARM1's importance in the differentiation of different cell types. We address CARM1 function during RA-induced differentiation of murine embryonic stem cells (mESCs) using shRNA lentiviral transduction and CRISPR/Cas9 technology to deplete CARM1 in mESCs. We identify CARM1 as a novel transcriptional coactivator required for the RA-associated decrease in Rex1 (Zfp42) and for the RA induction of a subset of RA-regulated genes, including CRABP2 and NR2F1 (Coup-TF1). Furthermore, CARM1 is required for mESCs to differentiate into extraembryonic endoderm in response to RA. We next characterize the epigenetic mechanisms that contribute to RA-induced transcriptional activation of CRABP2 and NR2F1 in mESCs and show for the first time that CARM1 is required for this activation. Collectively, our data demonstrate that CARM1 is required for transcriptional activation of a subset of RA target genes, and we uncover changes in the recruitment of Suz12 and the epigenetic H3K27me3 and H3K27ac marks at gene regulatory regions for CRABP2 and NR2F1 during RA-induced differentiation.

RevDate: 2019-10-07
CmpDate: 2019-10-07

Vulinovic F, Krajka V, Hausrat TJ, et al (2018)

Motor protein binding and mitochondrial transport are altered by pathogenic TUBB4A variants.

Human mutation, 39(12):1901-1915.

Mutations in TUBB4A have been identified to cause a wide phenotypic spectrum of diseases ranging from hereditary generalized dystonia with whispering dysphonia (DYT-TUBB4A) and hereditary spastic paraplegia (HSP) to leukodystrophy hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC). TUBB4A encodes the brain-specific β-tubulin isotype, β-tubulin 4A. To elucidate the pathogenic mechanisms conferred by TUBB4A mutations leading to the different phenotypes, we functionally characterized three pathogenic TUBB4A variants (c.4C>G,p.R2G; c.745G>A,p.D249N; c.811G>A, p.A271T) as representatives of the mutational and disease spectrum) in human neuroblastoma cells and human induced pluripotent stem cell (iPSC)-derived neurons. We showed that mRNA stability was not affected by any of the TUBB4A variants. Although two mutations (p.R2G and p.D249N) are located at the α/β-tubulin interdimer interface, we confirmed incorporation of all TUBB4A mutants into the microtubule network. However, we showed that the mutations p.D249N and p.A271T interfered with motor protein binding to microtubules and impaired neurite outgrowth and microtubule dynamics. Finally, TUBB4A mutations, as well as heterozygous knockout of TUBB4A, disrupted mitochondrial transport in iPSC-derived neurons. Taken together, our findings suggest that functional impairment of microtubule-associated transport is a shared pathogenic mechanism by which the TUBB4A mutations studied here cause a spectrum of diseases.

RevDate: 2019-10-07
CmpDate: 2019-10-07

Ibarrola J, Sadaba R, Martinez-Martinez E, et al (2018)

Aldosterone Impairs Mitochondrial Function in Human Cardiac Fibroblasts via A-Kinase Anchor Protein 12.

Scientific reports, 8(1):6801.

Aldosterone (Aldo) contributes to mitochondrial dysfunction and cardiac oxidative stress. Using a proteomic approach, A-kinase anchor protein (AKAP)-12 has been identified as a down-regulated protein by Aldo in human cardiac fibroblasts. We aim to characterize whether AKAP-12 down-regulation could be a deleterious mechanism which induces mitochondrial dysfunction and oxidative stress in cardiac cells. Aldo down-regulated AKAP-12 via its mineralocorticoid receptor, increased oxidative stress and induced mitochondrial dysfunction characterized by decreased mitochondrial-DNA and Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) expressions in human cardiac fibroblasts. CRISPR/Cas9-mediated knock-down of AKAP-12 produced similar deleterious effects in human cardiac fibroblasts. CRISPR/Cas9-mediated activation of AKAP-12 blunted Aldo effects on mitochondrial dysfunction and oxidative stress in human cardiac fibroblasts. In Aldo-salt-treated rats, cardiac AKAP-12, mitochondrial-DNA and PGC-1α expressions were decreased and paralleled increased oxidative stress. In myocardial biopsies from patients with aortic stenosis (AS, n = 26), AKAP-12, mitochondrial-DNA and PGC-1α expressions were decreased as compared to Controls (n = 13). Circulating Aldo levels inversely correlated with cardiac AKAP-12. PGC-1α positively associated with AKAP-12 and with mitochondrial-DNA. Aldo decreased AKAP-12 expression, impairing mitochondrial biogenesis and increasing cardiac oxidative stress. AKAP-12 down-regulation triggered by Aldo may represent an important event in the development of mitochondrial dysfunction and cardiac oxidative stress.

RevDate: 2019-10-07
CmpDate: 2019-10-07

Santhakumar D, Rohaim MAMS, Hussein HA, et al (2018)

Chicken Interferon-induced Protein with Tetratricopeptide Repeats 5 Antagonizes Replication of RNA Viruses.

Scientific reports, 8(1):6794.

The intracellular actions of interferon (IFN)-regulated proteins, including IFN-induced proteins with tetratricopeptide repeats (IFITs), attribute a major component of the protective antiviral host defense. Here we applied genomics approaches to annotate the chicken IFIT locus and currently identified a single IFIT (chIFIT5) gene. The profound transcriptional level of this effector of innate immunity was mapped within its unique cis-acting elements. This highly virus- and IFN-responsive chIFIT5 protein interacted with negative sense viral RNA structures that carried a triphosphate group on its 5' terminus (ppp-RNA). This interaction reduced the replication of RNA viruses in lentivirus-mediated IFIT5-stable chicken fibroblasts whereas CRISPR/Cas9-edited chIFIT5 gene knockout fibroblasts supported the replication of RNA viruses. Finally, we generated mosaic transgenic chicken embryos stably expressing chIFIT5 protein or knocked-down for endogenous chIFIT5 gene. Replication kinetics of RNA viruses in these transgenic chicken embryos demonstrated the antiviral potential of chIFIT5 in ovo. Taken together, these findings propose that IFIT5 specifically antagonize RNA viruses by sequestering viral nucleic acids in chickens, which are unique in innate immune sensing and responses to viruses of both poultry and human health significance.

RevDate: 2019-10-07
CmpDate: 2019-10-03

Bhowmik P, Ellison E, Polley B, et al (2018)

Targeted mutagenesis in wheat microspores using CRISPR/Cas9.

Scientific reports, 8(1):6502.

CRISPR/Cas9 genome editing is a transformative technology that will facilitate the development of crops to meet future demands. However, application of gene editing is hindered by the long life cycle of many crop species and because desired genotypes generally require multiple generations to achieve. Single-celled microspores are haploid cells that can develop into double haploid plants and have been widely used as a breeding tool to generate homozygous plants within a generation. In this study, we combined the CRISPR/Cas9 system with microspore technology and developed an optimized haploid mutagenesis system to induce genetic modifications in the wheat genome. We investigated a number of factors that may affect the delivery of CRISPR/Cas9 reagents into microspores and found that electroporation of a minimum of 75,000 cells using 10-20 µg DNA and a pulsing voltage of 500 V is optimal for microspore transfection using the Neon transfection system. Using multiple Cas9 and sgRNA constructs, we present evidence for the seamless introduction of targeted modifications in an exogenous DsRed gene and two endogenous wheat genes, including TaLox2 and TaUbiL1. This study demonstrates the value and feasibility of combining microspore technology and CRISPR/Cas9-based gene editing for trait discovery and improvement in plants.

RevDate: 2019-10-07
CmpDate: 2019-10-04

Lin Y, Liu H, Klein M, et al (2018)

Efficient differentiation of cardiomyocytes and generation of calcium-sensor reporter lines from nonhuman primate iPSCs.

Scientific reports, 8(1):5907.

Nonhuman primate (NHP) models are more predictive than rodent models for developing induced pluripotent stem cell (iPSC)-based cell therapy, but robust and reproducible NHP iPSC-cardiomyocyte differentiation protocols are lacking for cardiomyopathies research. We developed a method to differentiate integration-free rhesus macaque iPSCs (RhiPSCs) into cardiomyocytes with >85% purity in 10 days, using fully chemically defined conditions. To enable visualization of intracellular calcium flux in beating cardiomyocytes, we used CRISPR/Cas9 to stably knock-in genetically encoded calcium indicators at the rhesus AAVS1 safe harbor locus. Rhesus cardiomyocytes derived by our stepwise differentiation method express signature cardiac markers and show normal electrochemical coupling. They are responsive to cardiorelevant drugs and can be successfully engrafted in a mouse myocardial infarction model. Our approach provides a powerful tool for generation of NHP iPSC-derived cardiomyocytes amenable to utilization in basic research and preclinical studies, including in vivo tissue regeneration models and drug screening.

RevDate: 2019-10-07
CmpDate: 2019-10-04

Wang X, He J, K Le (2018)

Making point mutations in Escherichia coli BL21 genome using the CRISPR-Cas9 system.

FEMS microbiology letters, 365(14):.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated protein 9 (Cas9) system is an efficient and rapid tool for genome editing. However, its utilization in bacteria suffers challenges such as the risk of repeated recognition and cutting by Cas9. Here we established a two-step genome editing strategy using the Streptococcus pyogenes CRISPR-Cas9 system to achieve a clean mutation with only the target sites into the Escherichia coli genome. This strategy can avoid the risk of repeated cutting by guide RNA (gRNA)/Cas9 without altering the protospacer-adjacent motif or inserting additional silent mutations into the genome. The principles and approaches we developed in this study can be applied to modify coding and non-coding sequences in essential and non-essential genes and can also be used for precise genome editing in other microorganisms.

RevDate: 2019-10-05

Nussenzweig PM, McGinn J, LA Marraffini (2019)

Cas9 Cleavage of Viral Genomes Primes the Acquisition of New Immunological Memories.

Cell host & microbe pii:S1931-3128(19)30469-X [Epub ahead of print].

Type II CRISPR-Cas systems defend prokaryotes from bacteriophage infection through the acquisition of short viral DNA sequences known as spacers, which are transcribed into short RNA guides to specify the targets of the Cas9 nuclease. To counter the potentially devastating propagation of escaper phages with mutations in the target sequences, the host population acquires many different spacers. Whether and how pre-existing spacers in type II systems affect the acquisition of new ones is unknown. Here, we demonstrate that previously acquired spacers promote additional spacer acquisition from the vicinity of the target DNA site cleaved by Cas9. Therefore, CRISPR immune cells acquire additional spacers at the same time as they destroy the infecting virus. This anticipates the rise of escapers or related viruses that could escape targeting by the first spacer acquired. Our results thus reveal Cas9's role in the generation of immunological memories.

RevDate: 2019-10-04

Calvo-Villamañán A, Bernheim A, D Bikard (2020)

Methods for the Analysis and Characterization of Defense Mechanisms Against Horizontal Gene Transfer: CRISPR Systems.

Methods in molecular biology (Clifton, N.J.), 2075:235-249.

CRISPR-Cas systems provide RNA-guided adaptive immunity to the majority of archaea and many bacteria. They are able to capture pieces of invading genetic elements in the form of novel spacers in an array of repeats. These elements can then be used as a memory to destroy incoming DNA through the action of RNA-guided nucleases. This chapter describes general procedures to determine the ability of CRISPR-Cas systems to capture novel sequences and to use them to block phages and horizontal gene transfer. All protocols are performed in Staphylococcus aureus using Type II-A CRISPR-Cas systems. Nonetheless, the protocols provided can be adapted to work with other bacteria and other types of CRISPR-Cas systems.

RevDate: 2019-10-04

Pfalz M, Gonzalo A, Christophorou N, et al (2020)

Identifying and Isolating Meiotic Mutants in a Polyploid Brassica Crop.

Methods in molecular biology (Clifton, N.J.), 2061:303-318.

This chapter provides a detailed description of TILLING and CRISPR-Cas9 approaches for the purpose of studying genes/factors involved in meiotic recombination in the polyploid species B. napus. The TILLING approach involves the screening and identification of EMS-mutagenized M2 B. napus plants. The strategy for high-throughput plant pooling, the set up for microfluidic PCR and sequencing is provided and the parameters for the analysis of sequence results and the detection of mutants are explained. The CRISPR-Cas system relies on the optimal design of guide RNAs and their efficient expression. The procedure for the generation and detection of knockout mutants is described with the aims to simultaneously target homologous genes.

RevDate: 2019-10-04

Theodorou I, Courtin P, Palussière S, et al (2019)

A dual-chain assembly pathway generates the high structural diversity of cell-wall polysaccharides in Lactococcus lactis.

The Journal of biological chemistry pii:RA119.009957 [Epub ahead of print].

In Lactococcus lactis, cell wall polysaccharides (CWPSs) act as receptors for many bacteriophages, and their structural diversity among strains explains, at least partially, the narrow host range of these viral predators. Previous studies have reported that lactococcal CWPS consists of two distinct components, a variable chain exposed at the bacterial surface, named polysaccharide pellicle (PSP), and a more conserved rhamnan chain anchored to, and embedded inside, peptidoglycan. These two chains appear to be covalently linked to form a large heteropolysaccharide. The molecular machinery for biosynthesis of both components is encoded by a large gene cluster, named cwps. In this study, using a CRISPR/Cas-based method, we performed a mutational analysis of the cwps genes. MALDI-TOF MS-based structural analysis of the mutant CWPS combined with sequence homology, TEM and phage sensitivity analyses enabled us to infer a role for each protein encoded by the cwps cluster. We propose a comprehensive CWPS biosynthesis scheme in which the rhamnan and PSP chains are independently synthesized from two distinct lipid-sugar precursors and are joined at the extracellular side of the cytoplasmic membrane by a mechanism involving a membrane-embedded glycosyltransferase with a GT-C fold. The proposed scheme encompasses a system that allows extracytoplasmic modification of rhamnan by complex substituting oligo-/polysaccharides. It accounts for the extensive diversity of CWPS structures observed among lactococci and may also have relevance to the biosynthesis of complex rhamnose-containing CWPSs in other Gram-positive bacteria.

RevDate: 2019-10-03

de Jesus Silva F, Ferreira LC, Campos VP, et al (2019)

Complete genome sequence of the biocontrol agent Bacillus velezensis UFLA258 and its comparison with related species: diversity within the commons.

Genome biology and evolution pii:5580496 [Epub ahead of print].

In this study, the full genome sequence of Bacillus velezensis strain UFLA258, a biological control agent of plant pathogens was obtained, assembled and annotated. With a comparative genomics approach, in silico analyses of all complete genomes of B. velezensis and closely related species available in the database were performed. The genome of B. velezensis UFLA258 consisted of a single circular chromosome of 3.95 Mbp in length, with a mean GC content of 46.69%. It contained 3,949 genes encoding proteins and 27 RNA genes. Analyses based on ANI and dDDH and a phylogeny with complete sequences of the rpoB gene confirmed that 19 strains deposited in the database as B. amyloliquefaciens were in fact B. velezensis. In total, 115 genomes were analyzed and taxonomically classified as follows: 105 were B. velezensis, nine were B. amyloliquefaciens and one was B. siamensis. Although these species are phylogenetically close, the combined analyses of several genomic characteristics, such as the presence of biosynthetic genes encoding secondary metabolites, CRISPr/Cas arrays, ANI and dDDH, and other information on the strains, including isolation source, allowed their unequivocal classification. This genomic analysis expands our knowledge about the closely related species, B. velezensis, B. amyloliquefaciens and B. siamensis, with emphasis on their taxonomical status.

RevDate: 2019-10-03

Chen JJ, Nathaniel DL, Raghavan P, et al (2019)

Compromised function of the ESCRT pathway promotes endolysosomal escape of tau seeds and propagation of tau aggregation.

The Journal of biological chemistry pii:RA119.009432 [Epub ahead of print].

Intercellular propagation of protein aggre-gation is emerging as a key mechanism in the progression of several neurodegenerative diseases, including Alzheimer's disease and frontotemporal dementia (FTD). However, we lack a systematic understanding of the cellular path-ways controlling prion-like propagation of aggregation. To un-cover such pathways, here we performed CRISPR interference (CRISPRi) screens in a human cell-based model of propagation of tau aggregation monitored by FRET. Our screens uncovered that knockdown of several compo-nents of the endosomal sorting complexes required for transport (ESCRT) machinery, including charged multivesicular body protein 6 (CHMP6), or CHMP2A in combination with CHMP2B (whose gene is linked to familial FTD), promote propagation of tau aggregation. We found that knocking down the genes encoding these proteins also causes damage to endolysosomal mem-branes, consistent with a role for the ESCRT pathway in endolysosomal membrane repair. Leakiness of the endolysosomal compartment significantly enhanced prion-like propagation of tau aggregation, likely by making tau seeds more available to pools of cytoplasmic tau. To-gether, these findings suggest that endolysoso-mal escape is a critical step in tau propagation in neurodegenerative diseases.

RevDate: 2019-10-01

Toro N, Mestre MR, Martínez-Abarca F, et al (2019)

Recruitment of Reverse Transcriptase-Cas1 Fusion Proteins by Type VI-A CRISPR-Cas Systems.

Frontiers in microbiology, 10:2160.

Type VI CRISPR-Cas systems contain a single effector nuclease (Cas13) that exclusively targets single-stranded RNA. It remains unknown how these systems acquire spacers. It has been suggested that type VI systems with adaptation modules can acquire spacers from RNA bacteriophages, but sequence similarities suggest that spacers may provide immunity to DNA phages. We searched databases for Cas13 proteins with linked RTs. We identified two different type VI-A systems with adaptation modules including an RT-Cas1 fusion and Cas2 proteins. Phylogenetic reconstruction analyses revealed that these adaptation modules were recruited by different effector Cas13a proteins, possibly from RT-associated type III-D systems within the bacterial classes Alphaproteobacteria and Clostridia. These type VI-A systems are predicted to acquire spacers from RNA molecules, paving the way for future studies investigating their role in bacterial adaptive immunity and biotechnological applications.

RevDate: 2019-10-01

Hanlon KS, Kleinstiver BP, Garcia SP, et al (2019)

High levels of AAV vector integration into CRISPR-induced DNA breaks.

Nature communications, 10(1):4439 pii:10.1038/s41467-019-12449-2.

Adeno-associated virus (AAV) vectors have shown promising results in preclinical models, but the genomic consequences of transduction with AAV vectors encoding CRISPR-Cas nucleases is still being examined. In this study, we observe high levels of AAV integration (up to 47%) into Cas9-induced double-strand breaks (DSBs) in therapeutically relevant genes in cultured murine neurons, mouse brain, muscle and cochlea. Genome-wide AAV mapping in mouse brain shows no overall increase of AAV integration except at the CRISPR/Cas9 target site. To allow detailed characterization of integration events we engineer a miniature AAV encoding a 465 bp lambda bacteriophage DNA (AAV-λ465), enabling sequencing of the entire integrated vector genome. The integration profile of AAV-465λ in cultured cells display both full-length and fragmented AAV genomes at Cas9 on-target sites. Our data indicate that AAV integration should be recognized as a common outcome for applications that utilize AAV for genome editing.

RevDate: 2019-10-01

Amoako DG, Somboro AM, Abia ALK, et al (2019)

Genome Mining and Comparative Pathogenomic Analysis of An Endemic Methicillin-Resistant Staphylococcus Aureus (MRSA) Clone, ST612-CC8-t1257-SCCmec_IVd(2B), Isolated in South Africa.

Pathogens (Basel, Switzerland), 8(4): pii:pathogens8040166.

This study undertook genome mining and comparative genomics to gain genetic insights into the dominance of the methicillin-resistant Staphylococcus aureus (MRSA) endemic clone ST612-CC8-t1257-SCCmec_IVd(2B), obtained from the poultry food chain in South Africa. Functional annotation of the genome revealed a vast array of similar central metabolic, cellular and biochemical networks within the endemic clone crucial for its survival in the microbial community. In-silico analysis of the clone revealed the possession of uniform defense systems, restriction-modification system (type I and IV), accessory gene regulator (type I), arginine catabolic mobile element (type II), and type 1 clustered, regularly interspaced, short palindromic repeat (CRISPR)Cas array (N = 7 ± 1), which offer protection against exogenous attacks. The estimated pathogenic potential predicted a higher probability (average Pscore ≈ 0.927) of the clone being pathogenic to its host. The clone carried a battery of putative virulence determinants whose expression are critical for establishing infection. However, there was a slight difference in their possession of adherence factors (biofilm operon system) and toxins (hemolysins and enterotoxins). Further analysis revealed a conserved environmental tolerance and persistence mechanisms related to stress (oxidative and osmotic), heat shock, sporulation, bacteriocins, and detoxification, which enable it to withstand lethal threats and contribute to its success in diverse ecological niches. Phylogenomic analysis with close sister lineages revealed that the clone was closely related to the MRSA isolate SHV713 from Australia. The results of this bioinformatic analysis provide valuable insights into the biology of this endemic clone.

RevDate: 2019-10-01

Gale GAR, Schiavon Osorio AA, Mills LA, et al (2019)

Emerging Species and Genome Editing Tools: Future Prospects in Cyanobacterial Synthetic Biology.

Microorganisms, 7(10): pii:microorganisms7100409.

Recent advances in synthetic biology and an emerging algal biotechnology market have spurred a prolific increase in the availability of molecular tools for cyanobacterial research. Nevertheless, work to date has focused primarily on only a small subset of model species, which arguably limits fundamental discovery and applied research towards wider commercialisation. Here, we review the requirements for uptake of new strains, including several recently characterised fast-growing species and promising non-model species. Furthermore, we discuss the potential applications of new techniques available for transformation, genetic engineering and regulation, including an up-to-date appraisal of current Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein (CRISPR/Cas) and CRISPR interference (CRISPRi) research in cyanobacteria. We also provide an overview of several exciting molecular tools that could be ported to cyanobacteria for more advanced metabolic engineering approaches (e.g., genetic circuit design). Lastly, we introduce a forthcoming mutant library for the model species Synechocystis sp. PCC 6803 that promises to provide a further powerful resource for the cyanobacterial research community.

RevDate: 2019-10-02
CmpDate: 2019-10-02

Xu L, Wang J, Liu Y, et al (2019)

CRISPR-Edited Stem Cells in a Patient with HIV and Acute Lymphocytic Leukemia.

The New England journal of medicine, 381(13):1240-1247.

The safety of CRISPR (clustered regularly interspaced short palindromic repeats)-based genome editing in the context of human gene therapy is largely unknown. CCR5 is a reasonable but not absolutely protective target for a cure of human immunodeficiency virus type 1 (HIV-1) infection, because CCR5-null blood cells are largely resistant to HIV-1 entry. We transplanted CRISPR-edited CCR5-ablated hematopoietic stem and progenitor cells (HSPCs) into a patient with HIV-1 infection and acute lymphoblastic leukemia. The acute lymphoblastic leukemia was in complete remission with full donor chimerism, and donor cells carrying the ablated CCR5 persisted for more than 19 months without gene editing-related adverse events. The percentage of CD4+ cells with CCR5 ablation increased by a small degree during a period of antiretroviral-therapy interruption. Although we achieved successful transplantation and long-term engraftment of CRISPR-edited HSPCs, the percentage of CCR5 disruption in lymphocytes was only approximately 5%, which indicates the need for further research into this approach. (Funded by the Beijing Municipal Science and Technology Commission and others; number, NCT03164135.).

RevDate: 2019-10-02
CmpDate: 2019-10-02

Anonymous (2018)

How to respond to CRISPR babies.

Nature, 564(7734):5.

RevDate: 2019-10-01
CmpDate: 2019-10-01

Kostyusheva AP, Kostyushev DS, Brezgin SA, et al (2019)

[Small Molecular Inhibitors of DNA Double Strand Break Repair Pathways Increase the ANTI-HBV Activity of CRISPR/Cas9].

Molekuliarnaia biologiia, 53(2):311-323.

The CRISPR/Cas9 nuclease system can effectively suppress the replication of the hepatitis B virus (HBV), while covalently closed circular DNA (cccDNA), a highly resistant form of the virus, persists in the nuclei of infected cells. The most common outcome of DNA double-strand breaks (DSBs) in cccDNA caused by CRISPR/Cas9 is double-strand break repair by nonhomologous end-joining, which results in insertion/deletion mutations. Modulation of the DNA double-strand break repair pathways by small molecules was shown to stimulate CRISPR/Cas9 activity and may potentially be utilized to enhance the elimination of HBV cccDNA. In this work, we used inhibitors of homologous (RI-1) and nonhomologous (NU7026) end-joining and their combination to stimulate antiviral activity of CRISPR/Cas9 on two cell models of HBV in vitro, i.e., the HepG2-1.1merHBV cells containing the HBV genome under the tet-on regulated cytomegalovirus promoter and the HepG2-1.5merHBV cells containing constitutive expression of HBV RNA under the wild-type promoter. The treatment of the cells with RI-1 or NU7026 after lentiviral transduction of CRISPR/Cas9 drops the levels of cccDNA compared to the DMSO-treated control. RI-1 and NU7026 resulted in 5.0-6.5 times more significant reduction in the HBV cccDNA level compared to the mock-control. In conclusion, the inhibition of both homologous and nonhomologous DNA double-strand break repair pathways increases the elimination of HBV cccDNA by CRISPR/Cas9 system in vitro, which may potentially be utilized as a therapeutic approach to treat chronic hepatitis B.

RevDate: 2019-10-01
CmpDate: 2019-10-01

Lam CK, JC Wu (2018)

Disease modelling and drug discovery for hypertrophic cardiomyopathy using pluripotent stem cells: how far have we come?.

European heart journal, 39(43):3893-3895.

RevDate: 2019-10-01
CmpDate: 2019-10-01

Lin SS, JL Bowman (2018)

MicroRNAs in Marchantia polymorpha.

The New phytologist, 220(2):409-416.

Contents Summary 409 I. Introduction 409 II. RNA silencing machinery in Marchantia polymorpha 410 III. miRNA prediction by integrating omics approach 410 IV. miRNAs and their targets in Marchantia polymorpha 410 V. Mpo-miR390-mediated MpTAS3 tasiRNA biogenesis and potential tasiARF target MpARF2 414 VI. Artificial miRNA and CRISPR-CAS9 edited MIR gene in Marchantia polymorpha 414 VII. Conclusions 415 Acknowledgements 415 References 415 SUMMARY: The liverwort Marchantia polymorpha occupies an important phylogenetic position for comparative studies of land plant gene regulation. Multiple gene regulatory pathways mediated by small RNAs, including microRNAs (miRNAs), trans-acting short-interfering RNAs, and heterochromatic siRNAs often associated with RNA-dependent DNA methylation, have been characterized in flowering plants. Genes for essential components for all of these small RNA-mediated gene silencing pathways are found in M. polymorpha as well as the moss Phsycomitrella patens, indicating that these pathways existed in the ancestral land plant. However, only seven miRNAs are conserved across land plants, with both ancestral and novel targets identified in M. polymorpha. There is little or no evidence that any of these conserved miRNAs are present in algae. As with other plants investigated, most miRNAs in M. polypmorpha exhibit lineage-specific evolution. Application of artificial miRNA and CRISPR-Cas9 technologies in genetic studies of M. polymorpha provide avenues to further investigate miRNA biology.

RevDate: 2019-10-01
CmpDate: 2019-10-01

Jiang W, Feng S, Huang S, et al (2018)

BE-PLUS: a new base editing tool with broadened editing window and enhanced fidelity.

Cell research, 28(8):855-861.

Base editor (BE), containing a cytidine deaminase and catalytically defective Cas9, has been widely used to perform base editing. However, the narrow editing window of BE limits its utility. Here, we developed a new editing technology named as base editor for programming larger C to U (T) scope (BE-PLUS) by fusing 10 copies of GCN4 peptide to nCas9(D10A) for recruiting scFv-APOBEC-UGI-GB1 to the target sites. The new system achieves base editing with a broadened window, resulting in an increased genome-targeting scope. Interestingly, the new system yielded much fewer unwanted indels and non-C-to-T conversions. We also demonstrated its potential use in gene disruption across the whole genome through induction of stop codons (iSTOP). Taken together, the BE-PLUS system offers a new editing tool with increased editing window and enhanced fidelity.

RevDate: 2019-10-01
CmpDate: 2019-10-01

Lee BL, Mirrashidi KM, Stowe IB, et al (2018)

ASC- and caspase-8-dependent apoptotic pathway diverges from the NLRC4 inflammasome in macrophages.

Scientific reports, 8(1):3788.

The NLRC4 inflammasome recognizes bacterial flagellin and components of the type III secretion apparatus. NLRC4 stimulation leads to caspase-1 activation followed by a rapid lytic cell death known as pyroptosis. NLRC4 is linked to pathogen-free auto-inflammatory diseases, suggesting a role for NLRC4 in sterile inflammation. Here, we show that NLRC4 activates an alternative cell death program morphologically similar to apoptosis in caspase-1-deficient BMDMs. By performing an unbiased genome-wide CRISPR/Cas9 screen with subsequent validation studies in gene-targeted mice, we highlight a critical role for caspase-8 and ASC adaptor in an alternative apoptotic pathway downstream of NLRC4. Furthermore, caspase-1 catalytically dead knock-in (Casp1 C284A KI) BMDMs genetically segregate pyroptosis and apoptosis, and confirm that caspase-1 does not functionally compete with ASC for NLRC4 interactions. We show that NLRC4/caspase-8-mediated apoptotic cells eventually undergo plasma cell membrane damage in vitro, suggesting that this pathway can lead to secondary necrosis. Unexpectedly, we found that DFNA5/GSDME, a member of the pore-forming gasdermin family, is dispensable for the secondary necrosis that follows NLRC4-mediated apoptosis in macrophages. Together, our data confirm the existence of an alternative caspase-8 activation pathway diverging from the NLRC4 inflammasome in primary macrophages.

RevDate: 2019-09-30

Zhou LY, Qin Z, Zhu YH, et al (2019)

Current RNA-based Therapeutics in Clinical Trials.

Current gene therapy, 19(3):172-196.

Long-term research on various types of RNAs has led to further understanding of diverse mechanisms, which eventually resulted in the rapid development of RNA-based therapeutics as powerful tools in clinical disease treatment. Some of the developing RNA drugs obey the antisense mechanisms including antisense oligonucleotides, small interfering RNAs, microRNAs, small activating RNAs, and ribozymes. These types of RNAs could be utilized to inhibit/activate gene expression or change splicing to provide functional proteins. In the meantime, some others based on different mechanisms like modified messenger RNAs could replace the dysfunctional endogenous genes to manage some genetic diseases, and aptamers with special three-dimensional structures could bind to specific targets in a high-affinity manner. In addition, the recent most popular CRISPR-Cas technology, consisting of a crucial single guide RNA, could edit DNA directly to generate therapeutic effects. The desired results from recent clinical trials indicated the great potential of RNA-based drugs in the treatment of various diseases, but further studies on improving delivery materials and RNA modifications are required for the novel RNA-based drugs to translate to the clinic. This review focused on the advances and clinical studies of current RNA-based therapeutics, analyzed their challenges and prospects.

RevDate: 2019-09-30

Bhoobalan-Chitty Y, Johansen TB, Di Cianni N, et al (2019)

Inhibition of Type III CRISPR-Cas Immunity by an Archaeal Virus-Encoded Anti-CRISPR Protein.

Cell pii:S0092-8674(19)31009-8 [Epub ahead of print].

Bacteria and archaea possess a striking diversity of CRISPR-Cas systems divided into six types, posing a significant barrier to viral infection. As part of the virus-host arms race, viruses encode protein inhibitors of type I, II, and V CRISPR-Cas systems, but whether there are natural inhibitors of the other, mechanistically distinct CRISPR-Cas types is unknown. Here, we present the discovery of a type III CRISPR-Cas inhibitor, AcrIIIB1, encoded by the Sulfolobus virus SIRV2. AcrIIIB1 exclusively inhibits CRISPR-Cas subtype III-B immunity mediated by the RNase activity of the accessory protein Csx1. AcrIIIB1 does not appear to bind Csx1 but, rather, interacts with two distinct subtype III-B effector complexes-Cmr-α and Cmr-γ-which, in response to protospacer transcript binding, are known to synthesize cyclic oligoadenylates (cOAs) that activate the Csx1 "collateral" RNase. Taken together, we infer that AcrIIIB1 inhibits type III-B CRISPR-Cas immunity by interfering with a Csx1 RNase-related process.

RevDate: 2019-09-30
CmpDate: 2019-09-30

Zhou S, Yu H, Zhao X, et al (2018)

Generation of gene-edited sheep with a defined Booroola fecundity gene (FecBB) mutation in bone morphogenetic protein receptor type 1B (BMPR1B) via clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9.

Reproduction, fertility, and development, 30(12):1616-1621.

Since its emergence, the clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) 9 system has been increasingly used to generate animals for economically important traits. However, most CRISPR/Cas9 applications have been focused on non-homologous end joining, which results in base deletions and insertions, leading to a functional knockout of the targeted gene. The Booroola fecundity gene (FecBB) mutation (p.Q249R) in bone morphogenetic protein receptor type 1B (BMPR1B) has been demonstrated to exert a profound effect on fecundity in many breeds of sheep. In the present study, we successfully obtained lambs with defined point mutations resulting in a p.249Q>R substitution through the coinjection of Cas9 mRNA, a single guide RNA and single-stranded DNA oligonucleotides into Tan sheep zygotes. In the newborn lambs, the observed efficiency of the single nucleotide exchange was as high as 23.8%. We believe that our findings will contribute to improved reproduction traits in sheep, as well as to the generation of defined point mutations in other large animals.

RevDate: 2019-09-30
CmpDate: 2019-09-30

Wang X, Chen X, Sun L, et al (2019)

Canonical cytosolic iron-sulfur cluster assembly and non-canonical functions of DRE2 in Arabidopsis.

PLoS genetics, 15(4):e1008094 pii:PGENETICS-D-18-01975.

As a component of the Cytosolic Iron-sulfur cluster Assembly (CIA) pathway, DRE2 is essential in organisms from yeast to mammals. However, the roles of DRE2 remain incompletely understood largely due to the lack of viable dre2 mutants. In this study, we successfully created hypomorphic dre2 mutants using the CRISPR/Cas9 technology. Like other CIA pathway mutants, the dre2 mutants have accumulation of DNA lesions and show constitutive DNA damage response. In addition, the dre2 mutants exhibit DNA hypermethylation at hundreds of loci. The mutant forms of DRE2 in the dre2 mutants, which bear deletions in the linker region of DRE2, lost interaction with GRXS17 but have stronger interaction with NBP35, resulting in the CIA-related defects of dre2. Interestingly, we find that DRE2 is also involved in auxin response that may be independent of its CIA role. DRE2 localizes in both the cytoplasm and the nucleus and nuclear DRE2 associates with euchromatin. Furthermore, DRE2 directly associates with multiple auxin responsive genes and maintains their normal expression. Our study highlights the importance of the linker region of DRE2 in coordinating CIA-related protein interactions and identifies the canonical and non-canonical roles of DRE2 in maintaining genome stability, epigenomic patterns, and auxin response.

RevDate: 2019-09-30
CmpDate: 2019-09-30

Machado-Pineda Y, Cardeñes B, Reyes R, et al (2018)

CD9 Controls Integrin α5β1-Mediated Cell Adhesion by Modulating Its Association With the Metalloproteinase ADAM17.

Frontiers in immunology, 9:2474.

Integrin α5β1 is a crucial adhesion molecule that mediates the adherence of many cell types to the extracellular matrix through recognition of its classic ligand fibronectin as well as to other cells through binding to an alternative counter-receptor, the metalloproteinase ADAM17/TACE. Interactions between integrin α5β1 and ADAM17 may take place both in trans (between molecules expressed on different cells) or in cis (between molecules expressed on the same cell) configurations. It has been recently reported that the cis association between α5β1 and ADAM17 keeps both molecules inactive, whereas their dissociation results in activation of their adhesive and metalloproteinase activities. Here we show that the tetraspanin CD9 negatively regulates integrin α5β1-mediated cell adhesion by enhancing the cis interaction of this integrin with ADAM17 on the cell surface. Additionally we show that, similarly to CD9, the monoclonal antibody 2A10 directed to the disintegrin domain of ADAM17 specifically inhibits integrin α5β1-mediated cell adhesion to its ligands fibronectin and ADAM17.

RevDate: 2019-09-30
CmpDate: 2019-09-30

Ylä-Herttuala S (2018)

CRISPR/Cas9 and p53: An Odd Couple Requiring Relationship Management.

Molecular therapy : the journal of the American Society of Gene Therapy, 26(12):2711.

RevDate: 2019-09-30
CmpDate: 2019-09-30

Pan X, Philippen L, Lahiri SK, et al (2018)

In Vivo Ryr2 Editing Corrects Catecholaminergic Polymorphic Ventricular Tachycardia.

Circulation research, 123(8):953-963.

RATIONALE: Autosomal-dominant mutations in ryanodine receptor type 2 (RYR2) are responsible for ≈60% of all catecholaminergic polymorphic ventricular tachycardia. Dysfunctional RyR2 subunits trigger inappropriate calcium leak from the tetrameric channel resulting in potentially lethal ventricular tachycardia. In vivo CRISPR/Cas9-mediated gene editing is a promising strategy that could be used to eliminate the disease-causing Ryr2 allele and hence rescue catecholaminergic polymorphic ventricular tachycardia.

OBJECTIVE: To determine if somatic in vivo genome editing using the CRISPR/Cas9 system delivered by adeno-associated viral (AAV) vectors could correct catecholaminergic polymorphic ventricular tachycardia arrhythmias in mice heterozygous for RyR2 mutation R176Q (R176Q/+).

METHODS AND RESULTS: Guide RNAs were designed to specifically disrupt the R176Q allele in the R176Q/+ mice using the SaCas9 (Staphylococcus aureus Cas9) genome editing system. AAV serotype 9 was used to deliver Cas9 and guide RNA to neonatal mice by single subcutaneous injection at postnatal day 10. Strikingly, none of the R176Q/+ mice treated with AAV-CRISPR developed arrhythmias, compared with 71% of R176Q/+ mice receiving control AAV serotype 9. Total Ryr2 mRNA and protein levels were significantly reduced in R176Q/+ mice, but not in wild-type littermates. Targeted deep sequencing confirmed successful and highly specific editing of the disease-causing R176Q allele. No detectable off-target mutagenesis was observed in the wild-type Ryr2 allele or the predicted putative off-target site, confirming high specificity for SaCas9 in vivo. In addition, confocal imaging revealed that gene editing normalized the enhanced Ca2+ spark frequency observed in untreated R176Q/+ mice without affecting systolic Ca2+ transients.

CONCLUSIONS: AAV serotype 9-based delivery of the SaCas9 system can efficiently disrupt a disease-causing allele in cardiomyocytes in vivo. This work highlights the potential of somatic genome editing approaches for the treatment of lethal autosomal-dominant inherited cardiac disorders, such as catecholaminergic polymorphic ventricular tachycardia.

RevDate: 2019-09-30
CmpDate: 2019-09-30

Zhang G, Isaji T, Xu Z, et al (2019)

N-acetylglucosaminyltransferase-I as a novel regulator of epithelial-mesenchymal transition.

FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 33(2):2823-2835.

N-Glycans are involved in numerous biologic processes, such as cell adhesion, migration, and invasion. To distinguish the functions of complex high-mannose types of N-glycans, we used the clustered, regularly interspaced, short palindromic repeats/Cas9 system to establish N-acetylglucosaminyltransferase (GnT)-I-knockout (KO) cells. Loss of GnT-I greatly induced cell-cell adhesion and decreased cell migration. In addition, the expression levels of epithelial-mesenchymal transition (EMT) markers such as α-SMA, vimentin, and N-cadherin were suppressed, whereas the expression of claudin-1 was promoted, suggesting a mesenchymal-epithelial transition-like phenotype, an opposite process to the EMT, was occurred in the KO cells. The phosphorylation levels of Smad-2, epidermal growth factor receptor, and integrin-mediated focal adhesion kinase (FAK) were consistently suppressed. Furthermore, the restoration of GnT-I in the KO cells suppressed the cell-cell adhesion and augmented the expression of EMT markers as well as that of FAK activation. The expression levels of integrins were upregulated in the KO cells, although their functions were decreased, whereas their expression levels were downregulated in the rescued cells, which suggests a negative feedback loop between function and expression. Finally, we also found that the expression of GnT-I was important for cell survival, resistance to cancer drugs, and increased colony formation. The results of the present study demonstrate that GnT-I works as a switch to turn on/off EMT, which further supports the notion that on most surface receptors, the N-glycans differentially play essential roles in biologic functions.-Zhang, G., Isaji, T., Xu, Z., Lu, X., Fukuda, T., Gu, J. N-acetylglucosaminyltransferase-I as a novel regulator of epithelial-mesenchymal transition.

RevDate: 2019-09-30
CmpDate: 2019-09-30

Lopes R, Korkmaz G, Revilla SA, et al (2018)

CUEDC1 is a primary target of ERα essential for the growth of breast cancer cells.

Cancer letters, 436:87-95.

Breast cancer is the most prevalent type of malignancy in women with ∼1.7 million new cases diagnosed annually, of which the majority express ERα (ESR1), a ligand-dependent transcription factor. Genome-wide chromatin binding maps suggest that ERα may control the expression of thousands of genes, posing a great challenge in identifying functional targets. Recently, we developed a CRISPR-Cas9 functional genetic screening approach to identify enhancers required for ERα-positive breast cancer cell proliferation. We validated several candidates, including CUTE, a putative ERα-responsive enhancer located in the first intron of CUEDC1 (CUE-domain containing protein). Here, we show that CUTE controls CUEDC1 expression, and that this interaction is essential for ERα-mediated cell proliferation. Moreover, ectopic expression of CUEDC1, but not a CUE-domain mutant, rescues the defects in CUTE activity. Finally, CUEDC1 expression correlates positively with ERα in breast cancer. Thus, CUEDC1 is a functional target gene of ERα and is required for breast cancer cell proliferation.

RevDate: 2019-09-30
CmpDate: 2019-09-30

Liu X, Cheng Y, Abraham JM, et al (2018)

Modeling Wnt signaling by CRISPR-Cas9 genome editing recapitulates neoplasia in human Barrett epithelial organoids.

Cancer letters, 436:109-118.

Primary organoid cultures generated from patient biopsies comprise a novel improved platform for disease modeling, being genetically stable and closely recapitulating in vivo scenarios. Barrett esophagus (BE) is the major risk factor for esophageal adenocarcinoma. There has been a dearth of long-term in vitro expansion models of BE neoplastic transformation. We generated a long-term virus-free organoid expansion model of BE neoplasia from patient biopsies. Both wild-type and paired APC-knockout (APCKO) BE organoids genome-edited by CRISPR-Cas9 showed characteristic goblet cell differentiation. Autonomous Wnt activation was confirmed in APCKO organoids by overexpression of Wnt target genes and nuclear-translocated β-catenin expression after withdrawal of Wnt-3A and R-spondin-1. Wnt-activated organoids demonstrated histologic atypia, higher proliferative and replicative activity, reduced apoptosis, and prolonged culturability. Wnt-activated organoids also showed sustained protrusive migration ability accompanied by disrupted basement membrane reorganization and integrity. This CRISPR-Cas9 editing human-derived organoid model recapitulates the critical role of aberrant Wnt/β-catenin signaling activation in BE neoplastic transformation. This system can be used to study other 'driver' pathway alterations in BE-associated neoplasia, avoiding signaling noise present in immortalized or cancer-derived cell lines.

RevDate: 2019-09-30
CmpDate: 2019-09-30

Wang D, Ma D, Han J, et al (2018)

CRISPR RNA Array-Guided Multisite Cleavage for Gene Disruption by Cas9 and Cpf1.

Chembiochem : a European journal of chemical biology, 19(20):2195-2205.

To achieve multisite-targeting-based DNA cleavage simultaneously, we designed two kinds of CRISPR RNA arrays by fusing four single guide RNAs (sgRNAs for Cas9 or crRNAs for Cpf1) with uncleavable RNA linkers (CRISPRay). The CRISPRay could operate on four adjacent target sites to cleave target DNA in a collaborative manner. Two CRISPR RNA arrays demonstrated robust inactivation of the firefly luciferase gene in living cells. In vitro DNA cleavage and DNA sequencing also verified that sgRNA arrays directed SpCas9 nuclease to cut target DNA at four cleavage sites simultaneously whereas crRNA-array-guided FnCpf1 nuclease showed target-activated, nonspecific DNase activity on both target DNA and nontarget DNA at random sites. Through optimization of the ratio of nuclease and CRIPSR RNAs, CRISPRay should further enhance gene interference in cells. This work presents a simple approach through which to improve multisite-directed gene disruption by fusing four guide RNAs (sgRNAs or crRNAs) into a CRISPR RNA string.

RevDate: 2019-09-30
CmpDate: 2019-09-30

Lin YC, Pecetta S, Steichen JM, et al (2018)

One-step CRISPR/Cas9 method for the rapid generation of human antibody heavy chain knock-in mice.

The EMBO journal, 37(18):.

Here, we describe a one-step, in vivo CRISPR/Cas9 nuclease-mediated strategy to generate knock-in mice. We produced knock-in (KI) mice wherein a 1.9-kb DNA fragment bearing a pre-arranged human B-cell receptor heavy chain was recombined into the native murine immunoglobulin locus. Our methodology relies on Cas9 nuclease-induced double-stranded breaks directed by two sgRNAs to occur within the specific target locus of fertilized oocytes. These double-stranded breaks are subsequently repaired via homology-directed repair by a plasmid-borne template containing the pre-arranged human immunoglobulin heavy chain. To validate our knock-in mouse model, we examined the expression of the KI immunoglobulin heavy chains by following B-cell development and performing single B-cell receptor sequencing. We optimized this strategy to generate immunoglobulin KI mice in a short amount of time with a high frequency of homologous recombination (30-50%). In the future, we envision that such knock-in mice will provide much needed vaccination models to evaluate immunoresponses against immunogens specific for various infectious diseases.


ESP Quick Facts

ESP Origins

In the early 1990's, Robert Robbins was a faculty member at Johns Hopkins, where he directed the informatics core of GDB — the human gene-mapping database of the international human genome project. To share papers with colleagues around the world, he set up a small paper-sharing section on his personal web page. This small project evolved into The Electronic Scholarly Publishing Project.

ESP Support

In 1995, Robbins became the VP/IT of the Fred Hutchinson Cancer Research Center in Seattle, WA. Soon after arriving in Seattle, Robbins secured funding, through the ELSI component of the US Human Genome Project, to create the original ESP.ORG web site, with the formal goal of providing free, world-wide access to the literature of classical genetics.

ESP Rationale

Although the methods of molecular biology can seem almost magical to the uninitiated, the original techniques of classical genetics are readily appreciated by one and all: cross individuals that differ in some inherited trait, collect all of the progeny, score their attributes, and propose mechanisms to explain the patterns of inheritance observed.

ESP Goal

In reading the early works of classical genetics, one is drawn, almost inexorably, into ever more complex models, until molecular explanations begin to seem both necessary and natural. At that point, the tools for understanding genome research are at hand. Assisting readers reach this point was the original goal of The Electronic Scholarly Publishing Project.

ESP Usage

Usage of the site grew rapidly and has remained high. Faculty began to use the site for their assigned readings. Other on-line publishers, ranging from The New York Times to Nature referenced ESP materials in their own publications. Nobel laureates (e.g., Joshua Lederberg) regularly used the site and even wrote to suggest changes and improvements.

ESP Content

When the site began, no journals were making their early content available in digital format. As a result, ESP was obliged to digitize classic literature before it could be made available. For many important papers — such as Mendel's original paper or the first genetic map — ESP had to produce entirely new typeset versions of the works, if they were to be available in a high-quality format.

ESP Help

Early support from the DOE component of the Human Genome Project was critically important for getting the ESP project on a firm foundation. Since that funding ended (nearly 20 years ago), the project has been operated as a purely volunteer effort. Anyone wishing to assist in these efforts should send an email to Robbins.

ESP Plans

With the development of methods for adding typeset side notes to PDF files, the ESP project now plans to add annotated versions of some classical papers to its holdings. We also plan to add new reference and pedagogical material. We have already started providing regularly updated, comprehensive bibliographies to the ESP.ORG site.

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By delivering the Cas9 nuclease, complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be precisely cut at any desired location, allowing existing genes to be removed and/or new ones added. That is, the CRISPR-Cas system provides a tool for the cut-and-paste editing of genomes. Welcome to the brave new world of genome editing. R. Robbins

Electronic Scholarly Publishing
961 Red Tail Lane
Bellingham, WA 98226

E-mail: RJR8222 @

Papers in Classical Genetics

The ESP began as an effort to share a handful of key papers from the early days of classical genetics. Now the collection has grown to include hundreds of papers, in full-text format.

Digital Books

Along with papers on classical genetics, ESP offers a collection of full-text digital books, including many works by Darwin (and even a collection of poetry — Chicago Poems by Carl Sandburg).


ESP now offers a much improved and expanded collection of timelines, designed to give the user choice over subject matter and dates.


Biographical information about many key scientists.

Selected Bibliographies

Bibliographies on several topics of potential interest to the ESP community are now being automatically maintained and generated on the ESP site.

ESP Picks from Around the Web (updated 07 JUL 2018 )