Gene Modulation with CRISPR-based Tools in Human iPSC-Cardiomyocytes

Abstract

Precise control of gene expression (knock-out, knock-in, knockdown or overexpression) is at the heart of functional genomics - an approach to dissect the contribution of a gene/protein to the system's function. The development of a human in vitro system that can be patient-specific, induced pluripotent stem cells, iPSC, and the ability to obtain various cell types of interest, have empowered human disease modeling and therapeutic development. Scalable tools have been deployed for gene modulation in these cells and derivatives, including pharmacological means, DNA-based RNA interference and standard RNA interference (shRNA/siRNA). The CRISPR/Cas9 gene editing system, borrowed from bacteria and adopted for use in mammalian cells a decade ago, offers cell-specific genetic targeting and versatility. Outside genome editing, more subtle, time-resolved gene modulation is possible by using a catalytically "dead" Cas9 enzyme linked to an effector of gene transcription in combination with a guide RNA. The CRISPRi / CRISPRa (interference/activation) system evolved over the last decade as a scalable technology for performing functional genomics with libraries of gRNAs. Here, we review key developments of these approaches and their deployment in cardiovascular research. We discuss specific use with iPSC-cardiomyocytes and the challenges in further translation of these techniques.

Keywords: CRISPR; CRISPRa; CRISPRi; Gene knockdown; Gene modulation; Human iPSC-CMs; dCas9.

Fig. 1

Approaches for gene modulation. (A) Traditional methods to control gene function involve pharmacological compounds. (B) Targeting miRNAs and shRNA/siRNA in the naturally occurring RNAi pathways has been used to control post-transcriptional gene expression. (C) Basic mechanism of antisense oligonucleotides for targeting protein expression. (D) Epigenome editing with CRISPR. (E) DNA base editing tools introduce single nucleotide edits to the DNA. (F) RNA base editors include cytosine and adenine versions. (G) CRISPR-based approaches for gene activation and interference. Biorender was used for parts of this figure

Fig. 2

Timeline of CRISPRa and CRISPRi advancements. Developments of CRISPR-based tools for gene activation (CRISPRa) and inference (CRISPRi), and their expansion into genome-scale gRNA libraries and screens

Fig. 3

Human functional genomics by CRISPRi/a. CRISPR-based screening approaches, iPSC technology, and all-optical electrophysiology provide key elements for a high-throughput platform to perturb gene function and analyze respective genomic, protein, and functional changes for biological discovery of cardiac development and disease. Based on combined functional and transcriptomics data, one can build gene regulatory networks, GRNs, using machine learning techniques. The technology and the derived GRN models can be applied to cardiac development, guiding cell differentiation and maturation for regenerative medicine; disease modeling, drug development and cardiotoxicity testing. Biorender was used for parts of this figure