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Detecting non-canonically-targeted SpCas9 generated DSBs in human cells by LAM-HTGTS

ABSTRACT: Detecting non-canonically-targeted SpCas9 generated DSBs in human cells by LAM-HTGTS

PROVIDER: PRJNA791602 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR17308335_1.fastq.gz	Fastqsanger.gz
	SRR17308335_2.fastq.gz	Fastqsanger.gz
	SRR17308336_1.fastq.gz	Fastqsanger.gz
	SRR17308336_2.fastq.gz	Fastqsanger.gz
	SRR17308337_1.fastq.gz	Fastqsanger.gz

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Detecting non-canonically-targeted SpCas9 generated DSBs in human cells by LAM-HTGTS

Project description:We identified robust cleavage by non-canonically-targeted SpCas9 that target protospacer with a few bases from the canonical NGG PAM in biochemistry assay. To determine whether this spaced SpCas9 cleavage exists in human cells, we employed high-throughput genome-wide translocation sequencing (LAM-HTGTS) using SpCas9 with three independent canonical sgRNAs served as bait (bait-A, bait-L and bait-D) to compared the cleavage activities of canonically-targeted and non-canonically-targeted SpCas9 in eight locus (RAG1B-D, RAG1K-O) in chromosome 11. We found that almost all 1bp-target shifted SpCas9 generated substantial DSBs as the canonical controls. While detected less frequently, 2bp-targeted shifted SpCas9 generated significant amounts of DSBs in RAG1D and RAG1L locus. These findings underscore the spaced PAM-mediated DSBs occurred in living cells.

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