Precise measurement of CRISPR genome editing outcomes through single-cell DNA sequencing

Nechama Kalter; Saurabh Gulati; Michael Rosenberg; Qawer Ayaz; Joanne Nguyen; Shu Wang; Benjamin Schroeder; Chieh-Yuan Li; Ayal Hendel

doi:10.1016/j.omtm.2025.101449

Precise measurement of CRISPR genome editing outcomes through single-cell DNA sequencing

Mol Ther Methods Clin Dev. 2025 Mar 14;33(2):101449. doi: 10.1016/j.omtm.2025.101449. eCollection 2025 Jun 12.

Authors

Nechama Kalter¹, Saurabh Gulati², Michael Rosenberg¹, Qawer Ayaz², Joanne Nguyen², Shu Wang², Benjamin Schroeder², Chieh-Yuan Li², Ayal Hendel¹

Affiliations

¹ The Institute for Advanced Materials and Nanotechnology, The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 529002, Israel.
² Mission Bio, 400 E Jamie Ct, Suite 100, South San Francisco, CA 94080, USA.

Abstract

Gene therapy for clinical applications necessitates a comprehensive, accurate, and precise measurement of gene-edited drug products. State-of-the-art pipelines for evaluating editing outcomes rely primarily on bulk sequencing approaches, which are limited to population-level assessment. Here, we leveraged Tapestri, a single-cell sequencing technology for an in-depth analysis of editing outcomes. Using this platform, we characterized the genotype of triple-edited cells simultaneously at more than 100 loci, including editing zygosity, structural variations, and cell clonality. Our findings revealed a unique editing pattern in nearly every edited cell, highlighting the importance of single-cell resolution measurement to ensure the highest safety standards.

Keywords: CRISPR-Cas9; DNA sequencing; cancer immunotherapy; genome editing; genomic instability; genotoxicity; off-target; safety; single-cell.