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CRaTER enrichment for on-target gene-editing enables generation of variant libraries in hiPSCs

ABSTRACT: CRaTER enrichment for on-target gene-editing enables generation of variant libraries in hiPSCs

PROVIDER: PRJNA881283 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR21604400_1.fastq.gz	Fastqsanger.gz
	SRR21604400_2.fastq.gz	Fastqsanger.gz
	SRR21604401_1.fastq.gz	Fastqsanger.gz
	SRR21604401_2.fastq.gz	Fastqsanger.gz
	SRR21604402_1.fastq.gz	Fastqsanger.gz

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CRaTER enrichment for on-target gene-editing enables generation of variant libraries in hiPSCs

Project description:Generation of transgenic cell lines is limited by inefficient gene editing requiring genotypic screening of hundreds to thousands of colonies to isolate correctly gene-edited cells. Here, we describe a novel method called CRISPRa On-Target Editing Retrieval (CRaTER) that enriches for cells with on-target knock-in of a promoterless cDNA-fluorescent reporter transgene by transiently overexpressing the targeted endogenous genetic locus and sorting for fluorescent cells. We use CRaTER to enrich for rare cells with heterozygous, biallelic-editing of the endogenous, transcriptionally-inactive MYH7 locus in human induced pluripotent stem cells (hiPSC), resulting in a 9-fold enrichment compared to antibiotics selection alone. We leveraged CRaTER to enrich for heterozygous knock-in of a library of single nucleotide variants (SNV) in MYH7, a gene encoding for sarcomeric MHC-β wherein autosomal dominant missense mutations cause cardiac and skeletal myopathies. CRaTER enabled 90% enrichment of heterozygous, biallelically-edited hiPSCs – a 38.6-fold enrichment compared to antibiotics selection alone – to generate 113 SNVs comprising 78 missense variants in MYH7. hiPSCs that have undergone CRaTER enrichment can differentiate to cardiomyocytes and exhibit expected localization and expression of MHC-β fusion proteins. Together, CRaTER substantially reduces the screening required for isolation of gene-edited cells, enabling the generation of transgenic cell lines at unprecedented scale.

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