Article Impact Level: HIGH Data Quality: STRONG Summary of Journal of Molecular and Cellular Cardiology, 179, 60–71. https://doi.org/10.1016/j.yjmcc.2023.03.017 Dr. Clayton E. Friedman et al.
Points
- CRISPRa On-Target Editing Retrieval (CRaTER) enriches cells with desired gene editing by combining transient locus activation and flow sorting.
- Compared to traditional methods, CRaTER significantly reduces the screening required for isolating gene-edited cells.
- In a study using human induced pluripotent stem cells (hiPSCs), CRaTER achieves a 25-fold enrichment of cells with on-target knock-in of a cDNA-fluorescent reporter transgene.
- CRaTER enables the generation of a library of 113 variants in the MYH7 gene, known for causing cardiomyopathies in hiPSCs.
- Differentiated cardiomyocytes derived from CRaTER-enriched hiPSCs exhibit distinct physiological characteristics associated with pathogenic MYH7 variants, demonstrating the functional relevance of the gene editing process.
Summary
This research paper presents CRISPRa On-Target Editing Retrieval (CRaTER) as a novel approach to enrich cells with desired gene editing. The traditional method of generating transgenic cell lines involves screening numerous colonies to identify correctly edited cells. In contrast, CRaTER employs transient activation of the targeted locus and flow sorting to retrieve edited cells with on-target knock-in of a cDNA-fluorescent reporter transgene. The authors demonstrate the effectiveness of CRaTER by successfully enriching human induced pluripotent stem cells (hiPSCs) with heterozygous, biallelic-editing of the MYH7 locus, which is transcriptionally inactive. Compared to standard antibiotic selection, CRaTER achieves an average 25-fold enrichment.
Furthermore, the researchers leverage the capabilities of CRaTER to enrich for heterozygous knock-in of a library of variants in MYH7, a gene known for causing cardiomyopathies due to missense mutations. By applying CRaTER, they successfully recover hiPSCs with 113 different variants. These hiPSCs are subsequently differentiated into cardiomyocytes, and the study demonstrates that MHC-β fusion proteins can localize as expected. The authors also conduct single-cell contractility analyses, revealing that cardiomyocytes harboring a pathogenic MYH7 variant associated with hypertrophic cardiomyopathy exhibit distinct HCM physiology compared to isogenic controls. Thus, CRaTER significantly reduces the screening efforts required for isolating gene-edited cells, enabling the generation of functional transgenic cell lines on an unprecedented scale.
In summary, CRaTER represents a powerful method for enriching cells with desired gene editing by combining transient locus activation and flow sorting. The technique demonstrates its effectiveness in enriching hiPSCs with MYH7 locus editing, enabling the recovery of cells with heterozygous, biallelic modifications. Additionally, CRaTER facilitates the generation of an extensive library of MYH7 variants in hiPSCs, allowing the subsequent differentiation into disease-relevant cardiomyocytes. This study highlights the ability of CRaTER to streamline the screening process, reducing the time and effort required for the isolation of gene-edited cells and enabling the creation of functional transgenic cell lines at an unprecedented scale.
Link to the article: https://www.jmcc-online.com/article/S0022-2828(23)00063-9/fulltext
References Friedman, C. E., Fayer, S., Pendyala, S., Chien, W.-M., Loiben, A., Tran, L., Chao, L. S., Mckinstry, A., Ahmed, D., Karbassi, E., Fenix, A. M., Murry, C. E., Starita, L. M., Fowler, D. M., & Yang, K.-C. (2023). CRaTER enrichment for on-target gene editing enables generation of variant libraries in hiPSCs. Journal of Molecular and Cellular Cardiology, 179, 60–71. https://doi.org/10.1016/j.yjmcc.2023.03.017
