| Autor: |
Friedman CE, Fayer S, Pendyala S, Chien WM, Tran L, Chao L, Mckinstry A, Karbassi E, Fenix AM, Loiben A, Murry CE, Starita LM, Fowler DM, Yang KC |
| Jazyk: |
angličtina |
| Zdroj: |
BioRxiv : the preprint server for biology [bioRxiv] 2023 Jan 26. Date of Electronic Publication: 2023 Jan 26. |
| DOI: |
10.1101/2023.01.25.525582 |
| Abstrakt: |
Standard transgenic cell line generation requires screening 100-1000s of colonies to isolate correctly edited cells. We describe CR ISPR a On- T arget E diting R etrieval (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 single nucleotide variants (SNVs) in MYH7 , a gene in which missense mutations cause cardiomyopathies, and recovered hiPSCs with 113 different MYH7 SNVs. We differentiated these hiPSCs to cardiomyocytes and show MYH7 fusion proteins can localize as expected. Thus, CRaTER substantially reduces screening required for isolation of gene-edited cells, enabling generation of transgenic cell lines at unprecedented scale. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
|
