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. 2022 May:61:102754.
doi: 10.1016/j.scr.2022.102754. Epub 2022 Mar 18.

Generation of a dual edited human induced pluripotent stem cell Myl7-GFP reporter line with inducible CRISPRi/dCas9

Eyal Metzl-Raz  1 , Nike Bharucha  2 , Jennifer Arthur Ataam  2 , Alexandra A Gavidia  2 , William J Greenleaf  1 , Ioannis Karakikes  2
Affiliations

Generation of a dual edited human induced pluripotent stem cell Myl7-GFP reporter line with inducible CRISPRi/dCas9

Eyal Metzl-Raz et al. Stem Cell Res. 2022 May.

Abstract

Temporal regulation of CRISPRi activity is critical for genetic screens. Here, we present an inducible CRISPRi platform enabling selection of iPSC-derived cardiomyocytes and reversible gene knockdown. We targeted a doxycycline-inducible dCas9-KRAB-mCherry cassette into the AAVS1 locus in an MYL7-mGFP reporter iPSC line. A clone with bi-allelic integration displayed minimally leaky CRISPRi activity and strong expression upon addition of doxycycline in iPSCs, iPSC-derived cardiomyocytes, and multilineage differentiated cells. The CRISPRi activity was validated by targeting the MYOCD gene in iPSC cardiomyocytes. In summary, we developed a robust inducible CRISPRi platform to interrogate gene function in human iPSC-derived cardiomyocytes and other cells.

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Conflict of interest statement

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1.
Fig. 1.
Generation and characterization of inducible CRISPRi iPSCs.
See this image and copyright information in PMC

References

    1. Mandegar MA, et al. , 2016. CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs. Cell Stem Cell 18 (4), 541–553. - PMC - PubMed
    1. Corces MR, et al. , 2017. An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. Nat. Meth 14 (10), 959–962. - PMC - PubMed
    1. Feyen DAM, et al. , 2021. Unfolded Protein Response as a Compensatory Mechanism and Potential Therapeutic Target in PLN R14del Cardiomyopathy. Circulation 144 (5), 382–392. - PMC - PubMed