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Homo sapiens

ABSTRACT: Ectopic expression of RAD52 and dn53BP1 improves homology-directed repair during CRISPR/Cas9 genome editing

PROVIDER: PRJNA400601 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR5988540_1.fastq.gz	Fastqsanger.gz
	SRR5988540_2.fastq.gz	Fastqsanger.gz
	SRR5988550_1.fastq.gz	Fastqsanger.gz
	SRR5988550_2.fastq.gz	Fastqsanger.gz
	SRR5988560_1.fastq.gz	Fastqsanger.gz

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Ectopic expression of RAD52 and dn53BP1 improves homology-directed repair during CRISPR/Cas9 genome editing

Project description:Gene disruption by CRISPR/Cas9 is highly efficient and relies on the error-prone non-homologous end-joining (NHEJ) pathway. Conversely, precise gene editing requires homology-directed repair (HDR), which occurs at a lower frequency than NHEJ in mammalian cells. Here, by testing whether manipulation of DNA repair factors would improve HDR efficacy, we show that transient ectopic co-expression of RAD52 and a dominant-negative 53BP1 (dn53BP1) synergize to enable efficient HDR using a single-stranded oligonucleotide DNA donor template at multiple loci in human cells, including patient-derived induced pluripotent stem (iPS) cells. Co-expression of RAD52 and dn53BP1 improves multiplexed HDR-mediated editing, whereas expression of RAD52 alone enhances HDR with Cas9 nickase. Our data show that the frequency of NHEJ-mediated DSB repair in the presence of these two factors is not suppressed, and suggest that dn53BP1 competitively antagonizes 53BP1 to augment HDR in combination with RAD52. Importantly, co-expression of RAD52 and dn53BP1 does not alter Cas9 off-target activity. These findings support the use of RAD52 and dn53BP1 co-expression to overcome bottlenecks that limit HDR in precision genome editing.

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Gene editing in hematopoietic stem cells by co-delivery of Cas9/sgRNA ribonucleoprotein and templates for homology-directed repair in ‘all-in-one’ lentivirus-derived nanoparticles

Project description:Repair of double-strand DNA breaks generated by site-directed endonucleases, like Cas9, is the hallmark of gene editing based on homology-directed repair (HDR). HDR uses an exogenous DNA template to restore the cleaved DNA sequence and can facilitate specific gene corrections as well as insertion of genes or partial cDNA sequences. For CRISPR/Cas-directed gene editing, co-administration of the Cas9/single guide RNA (sgRNA) ribonucleoprotein (RNP) complex and a DNA template typically involves two different delivery strategies or different types of vehicles. This requires exquisite timing of delivery and may potentially challenge safety and therapeutic applicability. There is a need therefore for technologies that can ferry complete editing tool kits into cells. Here, we demonstrate the use of lentivirus-derived nanoparticles (LVNPs) to transport both RNP complexes and vector RNA, which upon reverse transcription serves as donor for HDR-directed repair. These ‘all-in-one’ LVNPs support targeted gene insertion with reduced off-target effects relative to nucleofection procedures. We show potent editing in the HBB gene in human erythroid progenitor cells as well as HDR-directed editing in hematopoietic stem and progenitor cells. Our findings mark a first step toward using a single virus-derived vehicle for delivering a full HDR gene editing kit.

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Project description:CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing

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Project description:Improved methods and optimized design for CRISPR Cas9 and Cas12a homology-directed repair

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

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

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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

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Project description:The clustered regularly interspaced short palindromic repeat (CRISPR)-associated enzyme Cas9 is an RNA-guided nuclease that has been widely adapted for genome editing in eukaryotic cells. However, the in vivo target specificity of Cas9 is poorly understood and most studies rely on in silico predictions to define the potential off-target editing spectrum. Using chromatin immunoprecipitation followed by sequencing (ChIP-seq), we delineate the genome-wide binding panorama of catalytically inactive Cas9 directed by two different single guide (sg) RNAs targeting the Trp53 locus. Cas9:sgRNA complexes are able to load onto multiple sites with short seed regions adjacent to 5’NGG3’ protospacer adjacent motifs (PAM). Examination of dmCas9 binding sites using two Trp53 targeting sgRNAs in Arf -/- MEF cell line (mouse).

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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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