Simultaneous orthogonal cell engineering by a single CRISPR-Cas9 polyfunctional editor [bisulfite-Seq]

However, such orthogonal editing involves the induction of multiple DNA breaks, raising safety concerns related to the risks of chromosomal translocations. Here, we present a polyfunctional CRISPR-Cas9-based strategy that enables both transgene insertion and epigenetic silencing at distinct genomic loci in a single treatment without inducing reciprocal chromosomal translocations.

This is accomplished through an optimized all-in-one epigenome editor equipped with a catalytically active Cas9, whose endonuclease activity is selectively disabled at epigenetically silenced loci by the use truncated gRNAs. As a proof of concept, we demonstrated that this platform enables efficient multi-locus editing, including functional replacement of the endogenous TCR with a tumor-selective one, targeted insertion of a prototypic CAR with either a selectable marker or an immunomodulatory receptor into a TCR locus or a ubiquitously expressed gene, and durable, multiplexed epigenetic silencing of clinically relevant genes in primary human T cells.