Multiplex genome editing of human pluripotent stem cells using Cpf1

Abstract: Targeted genome editing of human pluripotent stem cells (hPSC) is critical for basic and translational research, and can be achieved with site-specific endonucleases. Class 2 clustered regularly interspaced short palindromic repeats (CRISPR) Cpf1 represents a class of RNA programmable DNA endonucleases with features that could be complementary to the widely used Class 1 CRISPR-Cas9 system. Here we engineered a single vector system to deliver both Cpf1 and guide RNA, and achieved efficient knockin in transformed human cells. We then adopted the system to function in hPSCs by increasing expression levels of Cpf1, and showed that Cpf1 efficiently mediate homology directed repair in multiple loci (including knockin of a 6 kb cassette at the INS) in multiple hPSC lines, resulting in clones with transgenic cassettes integrated only at the targeted loci in the genome. Furthermore, with delivery of Cpf1 and a single U6 promoter-driven guide RNA array composed of an AAVS1-targeting guide and a MAFB-targeting guide, we demonstrated efficient multiplex genome editing in hPSCs at the AAVS1 (knockin) and MAFB (knockout) loci. Targeted hPSCs expressed pluripotency markers, and could be directed to differentiate to pancreatic beta-like cells, neural progenitor cells, and neurons in vitro. Generated INS reporter cells differentiated into beta-like cells that express tdTomato and luciferase specifically in insulin-expressing cells, allowing for in vivo tracking of human beta-like cells in humanized mice. By targeted screening of potential off-target sequences with most homology to guide RNA targeted sequences, we could not detect off-target mutations for all guide RNAs at the condition of efficient genome editing with Cpf1 in hPSCs. This work provides a system complementary to Cas9 for potentially precise genome editing in hPSCs.