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ABSTRACT: Polymerase on Cas9-induced deletions

PROVIDER: PRJNA758988 | ENA |

REPOSITORIES: ENA

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Dataset's files

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			Action	DRS
	SRR15672800_subreads.fastq.gz	Fastqsanger.gz
	SRR15672801_subreads.fastq.gz	Fastqsanger.gz
	SRR15672802_subreads.fastq.gz	Fastqsanger.gz
	SRR15672803_subreads.fastq.gz	Fastqsanger.gz
	SRR15672804_subreads.fastq.gz	Fastqsanger.gz

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Project description:CRISPR/Cas9-engineered human T cells hold great promise in improving T cell effector functions for adoptive T cell therapies. To ensure safety during CRISPR/Cas9-editing optimized gRNAs, novel variants or prediction tools for indel outcomes have been developed. Still, several reports describe the occurrence of CRISPR-induced chromosomal aberrations, such as loss of large DNA fragments or even aneuploidy. So far, the measures to increase the safety of T cell products focused on the CRISPR Cas9 system. However, T cell-intrinsic features, such as their massive expansion after TCR stimulation, have not been fully taken into consideration. Here, we describe driving forces of indel formation in primary human T cells. Increased T cell activation and proliferation speed correlate with larger deletions. Editing of non-activated T cells reduces the risk of unwanted large deletions but with the downside of reduced knock-out efficiencies. An alternative strategy to reduce the risk of large deletions is the addition of the small molecule pifithrin-α after CRISPR/Cas9 editing. Pifithrin-α treatment results in smaller, more defined deletions and reduces the risk of CRISPR-induced chromosomal translocations and aneuploidy in a p53-independent manner while maintaining the functionality of CRISPR-engineered T cells. Controlling T cell activation and the addition of pifithrin-α are easily accessible strategies for safer CRISPR/Cas9-engineering for adoptive T cell therapies.

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Project description:Polymerase on Cas9 deletion size

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