Molecular therapy : the journal of the American Society of Gene TherapyJul 18, 2026

One Base Editing System for Multipurpose Genetic Engineering of Immune Cells

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Jul 19, 2026

Abstract

The method achieves >95% quadplex gene knockout and successful integration of clinically relevant chimeric antigen receptors in human T cells.

A novel sgRNA design strategy and recombinant adeno-associated virus delivered DNA template enhance the efficiency of targeted gene editing.
The approach enables the simultaneous knockout of four genes while inserting specific CARs targeting CD19, CD33, or mesothelin.
No detectable translocations or significant off-target edits were observed during the engineering process.
The engineered CAR T cells demonstrated efficacy against multiple cancer cell lines and in a suppressive 3D spheroid culture model.
This method establishes a safe and simplified platform for advanced CAR T cell engineering.

Simplified

Current methods to engineer antigen-specific receptors rely on randomly integrating vectors or double-strand break induced targeted integration, both of which pose safety risks. To implement an all-in-one tool for multiplex knockout (KO) and knock in (KI), we expand the use of base editor nickase activity to stimulate homology-directed repair (HDR) and insert clinically-relevant chimeric antigen receptors (CARs) into specific loci. Through a novel sgRNA design strategy and a recombinant adeno-associated virus (rAAV) delivered DNA template, we enhanced the efficiency of ABE8e-stimulated HDR in human T cells. By combining KI of CD19, CD33, or mesothelin-targeting CARs with >95% quadplex gene KO (B2M/CD3ε/PDCD1/CISH), we achieve single-step generation of highly functional off-the-shelf CAR T cell products with enhanced function. Importantly, we found no detectable translocations or significant off-target edits and demonstrated efficacy against multiple cancer lines, and a suppressive 3D spheroid culture model. This efficient engineering process of Iterative Nicking for Synchronous Engineered Reprogramming of T cells (INSERT) establishes a safe, simplified platform for advanced therapeutic CAR T engineering.