scholarly journals Enhanced bacterial immunity and mammalian genome editing via RNA polymerase-mediated dislodging of Cas9 from double strand DNA breaks

2018 ◽  
Author(s):  
Ryan Clarke ◽  
Robert Heler ◽  
Matthew S. MacDougall ◽  
Nan Cher Yeo ◽  
Alejandro Chavez ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Genome Editing ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Strand Break ◽  
Dna Breaks ◽  
Simple Method ◽  
Guide Rna ◽  
Highly Active ◽  
Double Strand Dna Breaks

SUMMARYThe ability to target the Cas9 nuclease to DNA sequences via Watson-Crick base pairing with a single guide RNA (sgRNA) has provided a dynamic tool for genome editing and an essential component of adaptive immune systems in bacteria. After generating a double strand break (DSB), Cas9 remains stably bound to it. Here we show persistent Cas9 binding blocks access to DSB by repair enzymes, reducing genome editing efficiency. Cas9 can be dislodged by translocating RNA polymerases, but only if the polymerase approaches one direction towards the Cas9-DSB complex. By exploiting these RNA polymerase-Cas9 interactions, Cas9 can be conditionally converted into a multi-turnover nuclease, mediating increased mutagenesis frequencies in mammalian cells and enhancing bacterial immunity to bacteriophages. These consequences of a stable Cas9-DSB complex provide insights into the evolution of PAM sequences and a simple method of improving selection of highly active sgRNA for genome editing.

scholarly journals Enhanced Bacterial Immunity and Mammalian Genome Editing via RNA-Polymerase-Mediated Dislodging of Cas9 from Double-Strand DNA Breaks

2018 ◽  
Vol 71 (1) ◽  
pp. 42-55.e8 ◽  
Author(s):  
Ryan Clarke ◽  
Robert Heler ◽  
Matthew S. MacDougall ◽  
Nan Cher Yeo ◽  
Alejandro Chavez ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Genome Editing ◽  
Mammalian Genome ◽  
Dna Breaks ◽  
Double Strand Dna Breaks ◽  
Double Strand Dna

A Simple Heat Treatment Increases SpCas9-Mediated Mutation Efficiency in Arabidopsis

2021 ◽  
Author(s):  
Shuta Kurokawa ◽  
Hafizur Rhaman ◽  
Naoshi Yamanaka ◽  
Chisato Ishizaki ◽  
Shaikhul Islam ◽  
...  
Keyword(s):  
Heat Treatment ◽  
Genome Editing ◽  
Target Sequence ◽  
Simple Method ◽  
Guide Rna ◽  
Highly Active ◽  
Seed Population ◽  
Target Sites ◽  
Mutation Efficiency ◽  
Rna Genes

Abstract The CRISPR/Cas9 system is now commonly employed for genome editing in various plants such as Arabidopsis, rice, and tobacco. In general, in genome editing of the Arabidopsis genome, the SpCas9 and guide RNA genes are introduced into the genome by the floral dip method. Mutations induced in the target sequence by SpCas9 are confirmed after selecting transformants by screening the T1 seed population. The advantage of this method is that genome-edited plants can be isolated easily. However, mutation efficiency in Arabidopsis using SpCas9 is not as high as that achieved in rice and tobacco, which are subjected to a tissue culture step. In this study, we compared four promoters and found that the parsley UBIQITIN promoter is highly active in Arabidopsis meristem tissue. Furthermore, we examined whether a simple heat treatment could improve mutation efficiency in Arabidopsis. Just one heat treatment at 37 °C for 24 hours increased the mutation efficiency at all four target sites from 3% to 42%, 43% to 62%, 54% to 75%, and 89 to 91%, respectively, without detectable off-target mutations. We recommend heat treatment of plate-grown plants at 37 °C for 24 hours as a simple method to increase the efficiency of CRISPR/Cas9-mediated mutagenesis in Arabidopsis.

scholarly journals CRISPAltRations: a validated cloud-based approach for interrogation of double-strand break repair mediated by CRISPR genome editing

2020 ◽  
Author(s):  
Gavin Kurgan ◽  
Rolf Turk ◽  
Heng Li ◽  
Nathan Roberts ◽  
Garrett R. Rettig ◽  
...  
Keyword(s):  
Genome Editing ◽  
Amplicon Sequencing ◽  
Strand Break ◽  
Analysis Tool ◽  
Sequencing Data ◽  
Dna Breaks ◽  
End Joining ◽  
Double Strand Dna Breaks ◽  
Non Homologous End Joining ◽  
Security Standards

AbstractCRISPR systems enable targeted genome editing in a wide variety of organisms by introducing single- or double-strand DNA breaks, which are repaired using endogenous molecular pathways. Characterization of on- and off-target editing events from CRISPR proteins can be evaluated using targeted genome resequencing. We characterized DNA repair footprints that result from non-homologous end joining (NHEJ) after double stranded breaks (DSBs) were introduced by Cas9 or Cas12a for >500 paired treatment/control experiments. We found that building our understanding into a novel analysis tool (CRISPAltRations) improved results’ quality. We validated our software using simulated rhAmpSeq™ amplicon sequencing data (11 gRNAs and 603 on- and off-target locations) and demonstrate that CRISPAltRations outperforms other publicly available software tools in accurately annotating CRISPR-associated indels and homology directed repair (HDR) events. We enable non-bioinformaticians to use CRISPAltRations by developing a web-accessible, cloud-hosted deployment, which allows rapid batch processing of samples in a graphical user-interface (GUI) and complies with HIPAA security standards. By ensuring that our software is thoroughly tested, version controlled, and supported with a UI we enable resequencing analysis of CRISPR genome editing experiments to researchers no matter their skill in bioinformatics.

scholarly journals RNA-programmed genome editing in human cells

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Martin Jinek ◽  
Alexandra East ◽  
Aaron Cheng ◽  
Steven Lin ◽  
Enbo Ma ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Cleavage ◽  
Human Cells ◽  
Limiting Factor ◽  
Dna Breaks ◽  
Genetic Changes ◽  
Rna Sequence ◽  
Guide Rna ◽  
Double Strand Dna Breaks

Type II CRISPR immune systems in bacteria use a dual RNA-guided DNA endonuclease, Cas9, to cleave foreign DNA at specific sites. We show here that Cas9 assembles with hybrid guide RNAs in human cells and can induce the formation of double-strand DNA breaks (DSBs) at a site complementary to the guide RNA sequence in genomic DNA. This cleavage activity requires both Cas9 and the complementary binding of the guide RNA. Experiments using extracts from transfected cells show that RNA expression and/or assembly into Cas9 is the limiting factor for Cas9-mediated DNA cleavage. In addition, we find that extension of the RNA sequence at the 3′ end enhances DNA targeting activity in vivo. These results show that RNA-programmed genome editing is a facile strategy for introducing site-specific genetic changes in human cells.

Prime-editors (nickases), hRad51–Cas9 nickase fusions and dCas9 have the same problem as conventional CRISPR-Cas9 of plasmid/Cas9 integration after making a double stranded break

2019 ◽  
Author(s):  
Sandeep Chakraborty
Keyword(s):  
Cellular Response ◽  
Sequencing Data ◽  
Dna Breaks ◽  
Precise Integration ◽  
Guide Rna ◽  
Double Strand Dna Breaks ◽  
Human Therapy ◽  
P53 Activation ◽  
Programmable Nucleases

‘Prime-editing’ proposes to replace traditional programmable nucleases (CRISPR-Cas9) using a catalytically impaired Cas9 (dCas9) connected to a engineered reverse transcriptase, and a guide RNA encoding both the target site and the desired change. With just a ‘nick’ on one strand, it is hypothe- sized, the negative, uncontrollable effects arising from double-strand DNA breaks (DSBs) - translocations, complex proteins, integrations and p53 activation - will be eliminated. However, sequencing data pro- vided (Accid:PRJNA565979) reveal plasmid integration, indicating that DSBs occur. Also, looking at only 16 off-targets is inadequate to assert that Prime-editing is more precise. Integration of plasmid occurs in all three versions (PE1/2/3). Interestingly, dCas9 which is known to be toxic in E. coli and yeast, is shown to have residual endonuclease activity. This also affects studies that use dCas9, like base- editors and de/methylations systems. Previous work using hRad51–Cas9 nickases also show significant integration in on-targets, as well as off-target integration [1]. Thus, we show that cellular response to nicking involves DSBs, and subsequent plasmid/Cas9 integration. This is an unacceptable outcome for any in vivo application in human therapy.

scholarly journals Double-strand DNA breaks recruit the centromeric histone CENP-A

2009 ◽  
Vol 106 (37) ◽  
pp. 15762-15767 ◽  
Author(s):  
Samantha G. Zeitlin ◽  
Norman M. Baker ◽  
Brian R. Chapados ◽  
Evi Soutoglou ◽  
Jean Y. J. Wang ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Dna Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Double Strand Dna Breaks ◽  
Histone H3 Variant ◽  
Radiation Induced ◽  
Non Homologous End Joining

The histone H3 variant CENP-A is required for epigenetic specification of centromere identity through a loading mechanism independent of DNA sequence. Using multiphoton absorption and DNA cleavage at unique sites by I-SceI endonuclease, we demonstrate that CENP-A is rapidly recruited to double-strand breaks in DNA, along with three components (CENP-N, CENP-T, and CENP-U) associated with CENP-A at centromeres. The centromere-targeting domain of CENP-A is both necessary and sufficient for recruitment to double-strand breaks. CENP-A accumulation at DNA breaks is enhanced by active non-homologous end-joining but does not require DNA-PKcs or Ligase IV, and is independent of H2AX. Thus, induction of a double-strand break is sufficient to recruit CENP-A in human and mouse cells. Finally, since cell survival after radiation-induced DNA damage correlates with CENP-A expression level, we propose that CENP-A may have a function in DNA repair.

scholarly journals Target-AID-Mediated Multiplex Base Editing in Porcine Fibroblasts

2021 ◽  
Author(s):  
Soo-Young Yum ◽  
Goo Jang ◽  
Okjae Koo
Keyword(s):  
Genome Editing ◽  
Cytidine Deaminase ◽  
Chromosomal Rearrangements ◽  
Dna Breaks ◽  
Base Editing ◽  
Multiple Loci ◽  
Double Strand Dna Breaks ◽  
Protospacer Adjacent Motif ◽  
Motif Site ◽  
Programmable Nucleases

Multiplex genome editing may induce genotoxicity and chromosomal rearrangements due to double-strand DNA breaks at multiple loci simultaneously induced by programmable nucleases, including CRISPR/Cas9. However, recently developed base-editing systems can directly substitute target sequences without double-strand breaks. Thus, the base-editing system is expected to be a safer method for multiplex genome-editing platforms for livestock. Target-AID is a base editing system composed of PmCDA1, a cytidine deaminase from sea lampreys, fused to Cas9 nickase. It can be used to substitute cytosine for thymine in 3-5 base editing windows, 18 bases upstream of the protospacer-adjacent motif site. In the current study, we demonstrated Target-AID-mediated base editing in porcine cells for the first time. We targeted multiple loci in the porcine genome using the Target-AID system and successfully induced target-specific base substitutions with up to 63.15% efficiency. This system can be used for the further production of various genome-engineered pigs.

scholarly journals Improving the Precision of Base Editing by Bubble Hairpin Single Guide RNA

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Zhiwei Hu ◽  
Yannan Wang ◽  
Qian Liu ◽  
Yan Qiu ◽  
Zhiyu Zhong ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Breaks ◽  
Single Nucleotide ◽  
Base Editing ◽  
Guide Rna ◽  
Guide Rnas ◽  
Double Stranded Dna ◽  
Target Sites ◽  
Guide Sequence ◽  
Sgrna Design

ABSTRACT Base editing is a powerful genome editing approach that enables single-nucleotide changes without double-stranded DNA breaks (DSBs). However, off-target effects as well as other undesired editings at on-target sites remain obstacles for its application. Here, we report that bubble hairpin single guide RNAs (BH-sgRNAs), which contain a hairpin structure with a bubble region on the 5′ end of the guide sequence, can be efficiently applied to both cytosine base editor (CBE) and adenine base editor (ABE) and significantly decrease off-target editing without sacrificing on-target editing efficiency. Meanwhile, such a design also improves the purity of C-to-T conversions induced by base editor 3 (BE3) at on-target sites. Our results present a distinctive and effective strategy to improve the specificity of base editing. IMPORTANCE Base editors are DSB-free genome editing tools and have been widely used in diverse living systems. However, it is reported that these tools can cause substantial off-target editings. To meet this challenge, we developed a new approach to improve the specificity of base editors by using hairpin sgRNAs with a bubble. Furthermore, our sgRNA design also dramatically reduced indels and unwanted base substitutions at on-target sites. We believe that the BH-sgRNA design is a significant improvement over existing sgRNAs of base editors, and our design promises to be adaptable to various base editors. We expect that it will make contributions to improving the safety of gene therapy.

Escherichia coli and colorectal cancer: Unfolding the enigmatic relationship

2021 ◽  
Vol 22 ◽  
Author(s):  
Rogayeh Nouri ◽  
Alka Hasani ◽  
Kourosh Masnadi Shirazi ◽  
Mohammad Reza Aliand ◽  
Bita Sepehri ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Escherichia Coli ◽  
Mammalian Cells ◽  
Chromosomal Rearrangements ◽  
Current Evidence ◽  
Dna Breaks ◽  
Colon Cells ◽  
E Coli ◽  
Dna Mismatch ◽  
Double Strand Dna Breaks

: Colorectal cancer (CRC) is one of the deadliest cancers in the world. Specific strains of intestinal Escherichia coli (E. coli) may influence the initiation and development of CRC by exploiting virulence factors and inflammatory pathways. Mucosa-associated E. coli strains are more prevalent in CRC biopsies in comparison to healthy controls. Moreover, these strains can survive and replicate within macrophages and induce a pro-inflammatory response. Chronic exposure to inflammatory mediators can lead to increased cell proliferation and cancer. Production of colobactin toxin by the majority of mucosa-associated E. coli isolated from CRC patients is another notable finding. Colibactin-producing E. coli strains, in particular, induce double-strand DNA breaks, stop the cell cycle, involve in chromosomal rearrangements of mammalian cells and are implicated in carcinogenic effects in animal models. Moreover, some enteropathogenic E. coli (EPEC) strains are able to survive and replicate in colon cells as chronic intracellular pathogens and may promote susceptibility to CRC by downregulation of DNA Mismatch Repair (MMR) proteins. In this review, we discuss current evidence and focus on the mechanisms by which E. coli can influence the development of CRC.

scholarly journals The Schizosaccharomyces pombe rad60 Gene Is Essential for Repairing Double-Strand DNA Breaks Spontaneously Occurring during Replication and Induced by DNA-Damaging Agents

2002 ◽  
Vol 22 (10) ◽  
pp. 3537-3548 ◽  
Author(s):  
Takashi Morishita ◽  
Yasuhiro Tsutsui ◽  
Hiroshi Iwasaki ◽  
Hideo Shinagawa
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Strand Break ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Multicopy Plasmid ◽  
Dna Breaks ◽  
Γ Irradiation ◽  
Synthetic Lethal ◽  
Double Strand Dna Breaks ◽  
Dna Double Strand Break

ABSTRACT To identify novel genes involved in DNA double-strand break (DSB) repair, we previously isolated Schizosaccharomyces pombe mutants which are hypersensitive to methyl methanesulfonate (MMS) and synthetic lethals with rad2. This study characterizes one of these mutants, rad60-1. The gene that complements the MMS sensitivity of this mutant was cloned and designated rad60. rad60 encodes a protein with 406 amino acids which has the conserved ubiquitin-2 motif found in ubiquitin family proteins. rad60-1 is hypersensitive to UV and γ rays, epistatic to rhp51, and defective in the repair of DSBs caused by γ-irradiation. The rad60-1 mutant is also temperature sensitive for growth. At the restrictive temperature (37°C), rad60-1 cells grow for several divisions and then arrest with 2C DNA content; the arrested cells accumulate DSBs and have a diffuse and often aberrantly shaped nuclear chromosomal domain. The rad60-1 mutant is a synthetic lethal with rad18-X, and expression of wild-type rad60 from a multicopy plasmid partially suppresses the MMS sensitivity of rad18-X cells. rad18 encodes a conserved protein of the structural maintenance of chromosomes (SMC) family (A. R. Lehmann, M. Walicka, D. J. Griffiths, J. M. Murray, F. Z. Watts, S. McCready, and A. M. Carr, Mol. Cell. Biol. 15:7067-7080, 1995). These results suggest that S. pombe Rad60 is required to repair DSBs, which accumulate during replication, by recombination between sister chromatids. Rad60 may perform this function in concert with the SMC protein Rad18.

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