A multiplexed gRNA piggyBac transposon system facilitates efficient induction of CRISPRi and CRISPRa in human pluripotent stem cells

Abstract

CRISPR-Cas9-mediated gene interference (CRISPRi) and activation (CRISPRa) approaches hold promise for functional gene studies and genome-wide screens in human pluripotent stem cells (hPSCs). However, in contrast to CRISPR-Cas9 nuclease approaches, the efficiency of CRISPRi/a depends on continued expression of the dead Cas9 (dCas9) effector and guide RNA (gRNA), which can vary substantially depending on transgene design and delivery. Here, we design and generate new fluorescently labeled piggyBac (PB) vectors to deliver uniform and sustained expression of multiplexed gRNAs. In addition, we generate hPSC lines harboring AAVS1-integrated, inducible and fluorescent dCas9-KRAB and dCas9-VPR transgenes to allow for accurate quantification and tracking of cells that express both the dCas9 effectors and gRNAs. We then employ these systems to target the TCF4 gene in hPSCs and assess expression levels of the dCas9 effectors, individual gRNAs and targeted gene. We also assess the performance of our PB system for single gRNA delivery, confirming its utility for library format applications. collectively, our results provide proof-of-principle application of a stable, multiplexed pB gRnA delivery system that can be widely exploited to further enable genome engineering studies in hPSCs. Paired with diverse CRISPR tools including our dual fluorescence CRISPRi/a cell lines, this system can facilitate functional dissection of individual genes and pathways as well as larger-scale screens for studies of development and disease.

Parallelized engineering of mutational models using piggyBac transposon delivery of CRISPR libraries

“…Refinements to our protocol may facilitate genomic dosage alteration, including modifying transfection conditions to attain more PB integrations 35,41 , enhancing gRNA expression levels, harnessing other CRISPR systems 47,48 , and/or lengthening the duration of editing by prolonging Cas9 induction 49 . Although we found no relationship between PB DNA input and numbers of integrations per cell, multiple experiments exploring a wider range of PB input levels and alternative delivery techniques have reported higher per-cell PB integration counts [34][35][36] . Thus, PB integrations could be increased to provide more mutational opportunities for any fixed number of iPSC lines.…”

“…First, PB transposons can be scarlessly excised from cellular genomes 33 , such that final models would genetically differ only by CRISPR-induced mutations 34 . Second, the PB system has previously been utilized for genome engineering and more recently for CRISPR applications [35][36][37] , having demonstrated efficient editing in iPSCs with indel frequencies of 40-50% 34 . Third, adjusting the amounts of transposon and transposase plasmids transfected allows some control over how many PB integrations occur per cell 35,41 .…”

“…Given the relatively high efficiency of these CRISPR strategies, they should dovetail well with our PB system. Fourth, PB libraries increase multiplexing capacity for CRISPR activation/interference applications 36,37 . In all these ways, our PB method will promote systematic efforts to compile large collections of cellular models in isogenic backgrounds.…”

“…Here we leveraged the PB transposon, a mobile genetic element that can be scarlessly removed from the cellular genome 33 , to deliver Cas9 [34][35][36][37] together with a gRNA library to iPSCs and perform parallelized genome engineering. We explored both CNV and indel formation, balancing our interest in modeling reciprocal genomic disorders (RGDs) and dosage sensitivity with our desire to develop a method with broad applicability.…”

Fluorescent PSC-Derived Cardiomyocyte Reporter Lines: Generation Approaches and Their Applications in Cardiovascular Medicine

“…Unlike nodal and ventricular types, the atrial PSC-CM markers are not fully characterised. Further insights from novel fluorescent atrial PSC-CM reporter lines, such as PITX2 and KCNA5 , in combination with atrial-promoting differentiation approaches including retinoic acid treatment and CRISPRa/CRISPRi-mediated transcriptional activation/inhibition of atrial-regulating pathways [ 136 , 137 ], could assist the development of atrial cell isolation protocols.…”