Rapid generation of homozygous fluorescent knock-in human cells using CRISPR-Cas9 genome editing and validation by automated imaging and digital PCR screening.

Abstract

We previously described a protocol for genome engineering of mammalian cultured cells with clustered regularly interspaced short palindromic repeats and associated protein 9 (CRISPR-Cas9) to generate homozygous knock-ins of fluorescent tags into endogenous genes. Here we are updating this former protocol to reflect major improvements in the workflow regarding efficiency and throughput. In brief, we have improved our method by combining high-efficiency electroporation of optimized CRISPR-Cas9 reagents, screening of single cell-derived clones by automated bright-field and fluorescence imaging, rapidly assessing the number of tagged alleles and potential off-targets using digital polymerase chain reaction (PCR) and automated data analysis. Compared with the original protocol, our current procedure (1) substantially increases the efficiency of tag integration, (2) automates the identification of clones derived from single cells with correct subcellular localization of the tagged protein and (3) provides a quantitative and high throughput assay to measure the number of on- and off-target integrations with digital PCR. The increased efficiency of the new procedure reduces the number of clones that need to be analyzed in-depth by more than tenfold and yields to more than 26% of homozygous clones in polyploid cancer cell lines in a single genome engineering round. Overall, we were able to dramatically reduce the hands-on time from 30 d to 10 d during the overall ~10 week procedure, allowing a single person to process up to five genes in parallel, assuming that validated reagents-for example, PCR primers, digital PCR assays and western blot antibodies-are available.