CRaTER enrichment for on-target gene editing enables generation of variant libraries in hiPSCs.
J Mol Cell Cardiol
; 179: 60-71, 2023 06.
Article
em En
| MEDLINE
| ID: mdl-37019277
ABSTRACT
Standard transgenic cell line generation requires screening 100-1000s of colonies to isolate correctly edited cells. We describe CRISPRa On-Target Editing Retrieval (CRaTER) which enriches for cells with on-target knock-in of a cDNA-fluorescent reporter transgene by transient activation of the targeted locus followed by flow sorting to recover edited cells. We show CRaTER recovers rare cells with heterozygous, biallelic-editing of the transcriptionally-inactive MYH7 locus in human induced pluripotent stem cells (hiPSCs), enriching on average 25-fold compared to standard antibiotic selection. We leveraged CRaTER to enrich for heterozygous knock-in of a library of variants in MYH7, a gene in which missense mutations cause cardiomyopathies, and recovered hiPSCs with 113 different variants. We differentiated these hiPSCs to cardiomyocytes and show MHC-ß fusion proteins can localize as expected. Additionally, single-cell contractility analyses revealed cardiomyocytes with a pathogenic, hypertrophic cardiomyopathy-associated MYH7 variant exhibit salient HCM physiology relative to isogenic controls. Thus, CRaTER substantially reduces screening required for isolation of gene-edited cells, enabling generation of functional transgenic cell lines at unprecedented scale.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Cardiomiopatia Hipertrófica
/
Células-Tronco Pluripotentes Induzidas
/
Cardiomiopatias
Limite:
Humans
Idioma:
En
Revista:
J Mol Cell Cardiol
Ano de publicação:
2023
Tipo de documento:
Article