Rapid and Robust Generation of Homozygous Fluorescent Reporter Knock-In Cell Pools by CRISPR-Cas9

Yang, J; Guo, F; Chin, HS; Chen, GB; Zhang, Z; Williams, L; Kueh, AJ; Chow, PKH; Herold, MJ; Fu, NY

Author(s): Yang, J; Guo, F; Chin, HS; Chen, GB; Zhang, Z; Williams, L; Kueh, AJ; Chow, PKH; Herold, MJ; Fu, NY;
Details: Publication Year 2025-07-29,Volume 14,Issue #15,Page 1165
Journal Title: Cells
Abstract: Conventional methods for generating knock-out or knock-in mammalian cell models using CRISPR-Cas9 genome editing often require tedious single-cell clone selection and expansion. In this study, we develop and optimise rapid and robust strategies to engineer homozygous fluorescent reporter knock-in cell pools with precise genome editing, circumventing clonal variability inherent to traditional approaches. To reduce false-positive cells associated with random integration, we optimise the design of donor DNA by removing the start codon of the fluorescent reporter and incorporating a self-cleaving T2A peptide system. Using fluorescence-assisted cell sorting (FACS), we efficiently identify and isolate the desired homozygous fluorescent knock-in clones, establishing stable cell pools that preserve parental cell line heterogeneity and faithfully reflect endogenous transcriptional regulation of the target gene. We evaluate the knock-in efficiency and rate of undesired random integration in the electroporation method with either a dual-plasmid system (sgRNA and donor DNA in two separate vectors) or a single-plasmid system (sgRNA and donor DNA combined in one vector). We further demonstrate that coupling our single-plasmid construct with an integrase-deficient lentivirus vector (IDLV) packaging system efficiently generates fluorescent knock-in reporter cell pools, offering flexibility between electroporation and lentivirus transduction methods. Notably, compared to the electroporation methods, the IDLV system significantly minimises random integration. Moreover, the resulting reporter cell lines are compatible with most of the available genome-wide sgRNA libraries, enabling unbiased CRISPR screens to identify key transcriptional regulators of a gene of interest. Overall, our methodologies provide a powerful genetic tool for rapid and robust generation of fluorescent reporter knock-in cell pools with precise genome editing by CRISPR-Cas9 for various research purposes.
Publisher: MDPI
Keywords: CRISPR screen; CRISPR-Cas9; Tspan8; biallelic editing; fluorescent reporter knock-in cells; genetically engineered cells; genome editing; transcriptional regulation
Research Division(s): Cancer Biology and Stem Cells; Blood Cells and Blood Cancer
PubMed ID: 40801599
Publisher's Version: https://doi.org/10.3390/cells14151165
Open Access at Publisher's Site: https://doi.org/0.3390/cells14151165
Terms of Use/Rights Notice: Refer to copyright notice on published article.

Creation Date: 2025-08-19 01:46:03

Last Modified: 2025-08-19 01:46:21