An Efficient Genotyping Method for Genome-modified Animals and Human Cells Generated with CRISPR/Cas9 System

TLDR

CRISPR/Cas9 has accelerated the creation of diverse laboratory animals, yet genotyping has largely relied on the T7E1 endonuclease assay. The authors introduce a polyacrylamide gel electrophoresis (PAGE) method for genotyping mice with indel mutations. They applied PAGE to genotype six CRISPR/Cas9‑modified mouse strains from F0 to F2, including single and multiplexed edits, and to detect on‑ and off‑target effects in human 293T cells and iPSCs. PAGE detected mosaic DNA with a sensitivity of 0.5% and satisfies the growing need for rapid genotyping of genome‑modified animals and human cell lines generated by CRISPR/Cas9, TALEN, or ZFN.

Abstract

Abstract The rapid generation of various species and strains of laboratory animals using CRISPR/Cas9 technology has dramatically accelerated the interrogation of gene function in vivo . So far, the dominant approach for genotyping of genome-modified animals has been the T7E1 endonuclease cleavage assay. Here, we present a polyacrylamide gel electrophoresis-based (PAGE) method to genotype mice harboring different types of indel mutations. We developed 6 strains of genome-modified mice using CRISPR/Cas9 system and utilized this approach to genotype mice from F0 to F2 generation, which included single and multiplexed genome-modified mice. We also determined the maximal detection sensitivity for detecting mosaic DNA using PAGE-based assay as 0.5%. We further applied PAGE-based genotyping approach to detect CRISPR/Cas9-mediated on- and off-target effect in human 293T and induced pluripotent stem cells (iPSCs). Thus, PAGE-based genotyping approach meets the rapidly increasing demand for genotyping of the fast-growing number of genome-modified animals and human cell lines created using CRISPR/Cas9 system or other nuclease systems such as TALEN or ZFN.