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APOBEC3s induce mutations during the repair of CRISPR-Cas9-generated DNA breaks.

ABSTRACT: APOBEC3s induce mutations during the repair of CRISPR-Cas9-generated DNA breaks.

PROVIDER: PRJNA414960 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR6189580.fastq.gz	Fastqsanger.gz
	SRR6189590.fastq.gz	Fastqsanger.gz
	SRR6189600.fastq.gz	Fastqsanger.gz
	SRR6189610.fastq.gz	Fastqsanger.gz
	SRR6189620.fastq.gz	Fastqsanger.gz

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APOBEC3s induce mutations during the repair of CRISPR-Cas9-generated DNA breaks.

Project description:APOBEC-AID family of cytidine deaminase prefers single-stranded nucleic acids for cytidine to uracil deamination. Single-stranded nucleic acids are commonly involved in the DNA repair system for breaks generated by CRISPR-Cas9. Here, we show in human cells that APOBEC3s can trigger the cytidine deamination of single-stranded oligodeoxynucleotides, which ultimately results in base substitution mutations in genomic DNA through the homology-directed repair (HDR) of Cas9-generated double-strand breaks . In addition, the APOBEC3-catalyzed deamination in genomic single-stranded DNA formed during the repair of Cas9 nickase-generated single-strand breaks can be further processed to yield mutations mainly involving insertions or deletions (indels). Mechanistically, both APOBEC3-mediated deamination and DNA repair proteins play important roles in the generation of these indels. Correspondingly, optimizing conditions for the repair of CRISPR-Cas9-generated DNA breaks, such as using double-stranded donors in HDR or temporarily suppressing endogenous APOBEC3s, can substantially repress these unwanted mutations in genomic DNA.

2017-11-10 | GSE105146 | GEO

Precision genome editing using synthesis-dependent repair of Cas9-induced DNA breaks

Project description:The RNA-guided DNA endonuclease Cas9 has emerged as a powerful new tool for genome engineering. Cas9 creates targeted double-strand breaks (DSBs) in the genome. Knock-in of specific mutations (precision genome editing) requires homology-directed repair (HDR) of the DSB by synthetic donor DNAs containing the desired edits, but HDR has been reported to be variably efficient. Here, we report that linear DNAs (single and double-stranded) engage in a high-efficiency HDR mechanism that requires only ~35 nucleotides of homology with the targeted locus to introduce edits ranging from 1 to 1000 nucleotides. We demonstrate the utility of linear donors by introducing fluorescent protein tags in human cells and mouse embryos using PCR fragments. We find that repair is local, polarity-sensitive, and prone to template switching, characteristics that are consistent with gene conversion by synthesis-dependent strand-annealing (SDSA). Our findings enable rational design of synthetic donor DNAs for efficient genome editing.

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Precision genome editing using synthesis-dependent repair of Cas9-induced DNA breaks

Project description:Precision genome editing using synthesis-dependent repair of Cas9-induced DNA breaks

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SAMHD1 restrains aberrant nucleotide insertions at repair junctions generated by DNA end joining

Project description:Aberrant end joining of DNA double strand breaks leads to chromosomal rearrangements and to insertion of nuclear or mitochondrial DNA into breakpoints, which is commonly observed in cancer cells and constitutes a major threat to genome integrity. However, the mechanisms that are causative for these insertions are largely unknown. By monitoring end joining of different linear DNA substrates introduced into HEK293 cells, as well as by examining end joining of CRISPR/Cas9 induced DNA breaks in HEK293 and HeLa cells, we provide evidence that the dNTPase activity of SAMHD1 impedes aberrant DNA resynthesis at DNA breaks during DNA end joining. Hence, SAMHD1 expression or low intracellular dNTP levels lead to shorter repair joints and impede insertion of distant DNA regions prior end repair. Our results reveal a novel role for SAMHD1 in DNA end joining and provide new insights into how loss of SAMHD1 may contribute to genome instability and cancer development.

2021-01-20 | E-MTAB-8382 | biostudies-arrayexpress

Kinetics and fidelity of the repair of Cas9-induced double-strand DNA breaks

Project description:Kinetics and fidelity of the repair of Cas9-induced double-strand DNA breaks

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Kinetics and fidelity of the repair of Cas9-induced double-strand DNA breaks

Project description:The RNA-guided DNA endonuclease Cas9 is a powerful tool for genome editing. Little is known about the kinetics and fidelity of the double-strand break (DSB) repair process that follows a Cas9 cutting event in living cells. Here, we developed a strategy to measure the kinetics of DSB repair for single loci in human cells. Quantitative modeling of repaired DNA in time series after Cas9 activation reveals variable and often slow repair rates, with half-life times up to ~10 h. Furthermore, repair of the DSBs tends to be error-prone. Both classical and microhomology-mediated end-joining pathways contribute to the erroneous repair. Estimation of their individual rate constants indicates that the balance between these two pathways changes over time and can be altered by additional ionizing radiation. Our approach provides quantitative insights into DSB repair kinetics and fidelity in single loci, and indicates that Cas9-induced DSBs are repaired in an unusual manner.

2018-05-01 | GSE113129 | GEO

AAV vector-derived elements integrate into Cas9-generated double strand breaks and disrupt gene transcription

Project description:AAV vector-derived elements integrate into Cas9-generated double strand breaks and disrupt gene transcription

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Trabectedin derails transcription-coupled nucleotide excision repair to induce DNA breaks in highly transcribed genes

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Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair

Project description:Based on the hypothesis that, enhancing the local concentration of donor oligos could increase the correction rates, we generated and tested novel CRISPR-Cas9 systems, in which the DNA repair template is covalently conjugated to Cas9 (RNPD system). To validate our results from the HEK293T reporter cells, we here tested our approach at different endogenous genomic loci and in different cell types. We first targeted the human beta globin (HBB) locus in the K562 cell line, and analyzed correction- and editing frequencies using next generation sequencing (NGS). Next we targeted the Rosa26 and proprotein convertase subtilisin/kexin type 9 (Pcsk9) locus in mouse embryonic stem cells (mESCs). Here, RNPD system was always compared to Cas9 SNAP-tag fusion proteins with uncoupled donor oligos. To also directly compare the engineered RNPD system to the classical CRISPR-Cas9 system, we performed experiments where we used wild-type Cas9 with the uncoupled donor oligos as a control. We therefore targeted the fluorescent reporter locus as well as the endogenous loci HBB, empty spiracles homeobox 1 (EMX1), and C-X-C chemokine receptor type 4 (CXCR4) in HEK293T cells. Finally, we performed the analysis of three computationally predicted off-target sites of the reporter locus.

2018-06-04 | E-MTAB-6808 | biostudies-arrayexpress

p53 exon 11 mutants generated by CRISPR/Cas9 result in aberrant alternative splicing.

Project description:The Tp53 gene encodes the famous tumor suppressor protein p53. This gene is encoded in 11 exons and various alternative spliced versions of the transcripts have been detected. Here we generated targeted mutations in human colon cancer cell line HCT116 using CRISPR/Cas9 genome editing tools. The mutations were targeted to the 5' end of Tp53 gene exon 11. CRISPR/Cas9 transfected cells were allowed to repair double stranded breaks by non-homologous end joining. Mutant cell pools were single cell cloned and analyzed by Sanger DNA sequencing. Three mutant clones, mut4, mut5 and mut9 were found to contain biallelic homozygous mutations. Poly adenylated RNA was isolated from mutant cell pools was isolated and sequenced by Illumina RNASeq in triplicate. We find that exon 11 mutations result in the alternative splicing of earlier exons and cause the expression of the p53beta alternative splice product. This C-terminally deleted protein has been associated with the induction of senescence. We thus identify an exonic splicing silencer in exon 11 of the Tp53 gene which normally suppressed alternative splicing events that encode the p53beta variant. This sequence and the factors that associate with it are prime candidates for targeting, which may induce p53beta splicing and the induction of senescence in tumors.

2020-05-01 | GSE131416 | GEO

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