Heinson, Y. W. | Search bioRxiv

bioRxiv2023

Paradoxical anti-arrhythmic effects of Zim3, a CRISPRi effector, in human iPSC-cardiomyocytes

We show that Zim3, when used as Zim3-KRAB-dCas9 effector in interference CRISPR, without any guide RNAs, paradoxically upregulates key cardiac ion channel genes in human induced pluripotent stem-cell-derived cardiomyocytes, iPSC-CMs, responsible for healthy resting membrane potential, repolarization of the action potential and electrical transmission of signals. These were found to yield expected functional enhancements consistent with a more mature iPSC-CM phenotype, with potentially desirable properties.

molecular biology↗

bioRxiv2023

CRISPRi Gene Modulation and All-Optical Electrophysiology in Post-Differentiated Human iPSC-Cardiomyocytes

Uncovering gene-phenotype relationships can be enabled by precise gene modulation in human induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs) and follow up phenotyping using scalable all- optical electrophysiology platforms. Such efforts towards human functional genomics can be aided by recent CRISPR-derived technologies for reversible gene inhibition or activation (CRISPRi/a). We set out to characterize the performance of CRISPRi in post-differentiated iPSC-CMs, targeting key cardiac ion channel genes, KCNH2, KCNJ2, and GJA1, and providing a multiparametric quantification of the effects on cardiac repolarization, stability of the resting membrane potential and conduction properties using all- optical tools. More potent CRISPRi effectors, e.g. Zim3, and optimized viral delivery led to improved performance on par with the use of CRISPRi iPSC lines. Confirmed mild yet specific phenotype changes when CRISPRi is deployed in non-dividing differentiated heart cells is an important step towards more holistic pre-clinical cardiotoxicity testing and for future therapeutic use in vivo.

cell biology↗