Evaluation protocol for CRISPR/Cas9-mediated CD19 knockout GM24385 cells by flow cytometry and Sanger sequencing

Sarah L Inwood; Linhua Tian; Kirsten Parratt; Samantha Maragh; Lili Wang

doi:10.2144/btn-2022-0015

Evaluation protocol for CRISPR/Cas9-mediated CD19 knockout GM24385 cells by flow cytometry and Sanger sequencing

Biotechniques. 2022 Jun;72(6):279-286. doi: 10.2144/btn-2022-0015.

Authors

Sarah L Inwood¹, Linhua Tian¹, Kirsten Parratt¹, Samantha Maragh¹, Lili Wang¹

Affiliation

¹ Biosystems and Biomaterials Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA.

PMID: 35703314
DOI: 10.2144/btn-2022-0015

Abstract

Although several genome editing options are available, CRISPR/Cas9 is one of the most commonly used systems for protein and advanced therapies. There are some long-term data regarding genomic and phenotypic stability, however, information is sparse. Flow cytometry can offer a method to characterize these edited cells for longitudinal studies. The objective of this work is to describe a protocol for using flow cytometry to measure the edits from CRISPR/Cas9 on a well-characterized B-lymphoblast cell line, GM24385, with the goal of supporting safe and effective CRISPR/Cas9-engineered therapies.

Keywords: CD19; CRISPR/Cas9; GM24385 cell line; flow cytometry; genome editing.

Publication types

Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

CRISPR-Cas Systems* / genetics
Cell Line
Flow Cytometry
Gene Editing* / methods