Nature communicationsMay 8, 2026

One CRISPR-Cas9 tool enabling independent editing of different cell functions at the same time

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗OA ↗

Updated May 9, 2026

Abstract

A polyfunctional CRISPR-Cas9-based strategy enables efficient multi-locus editing without inducing chromosomal translocations.

The strategy allows for simultaneous transgene insertion and epigenetic silencing at multiple genomic locations.
Endogenous T cell receptors (TCRs) can be functionally replaced with tumor-selective receptors using this method.
Targeted insertion of a CAR with either a selectable marker or an immunomodulatory receptor can occur at TCR loci or other genes.
Durable epigenetic silencing of clinically relevant genes in primary human T cells is achievable through this approach.
The technique aims to enhance safety by avoiding reciprocal chromosomal translocations associated with traditional gene editing.

Simplified

The parallel disruption of multiple genes coupled with targeted transgene insertion offers a powerful strategy for more effective and precise cell engineering. 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 using truncated gRNAs. As a proof of concept, we demonstrate 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. Polyfunctional editing establishes a versatile and safe framework for orthogonal editing, broadening the scope of genome and epigenome engineering in cancer immunotherapy and beyond.

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Funding

Competing interests

Competing interests: A.L. is a co-founder, equity holder, and consultant of nChroma Bio, a company developing epigenome editing applications. P.S. was an employee of nChroma Bio at the time the work was conducted and holds an equity interest in the company. A.L., D.C., T.B., A.R., and M.A.C. are inventors on patent applications related to the development of the polyfunctional editing strategy and improved ETR design, filed by IRCCS Ospedale San Raffaele and Fondazione Telethon ETS or by nChroma Bio. All other authors have no competing interests.

PubMed

One CRISPR-Cas9 tool enabling independent editing of different cell functions at the same time

Abstract

Full Text

Funding

Competing interests

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