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 have previously described a protocol for genome engineering of mammalian cultured cancer cells with CRISPR/Cas9 to generate homozygous knock-ins of fluorescent tags into endogenous genes 1. Here, we are updating this 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 PCR (dPCR) and automated data analysis. Compared to the original protocol 1, our current procedure (i) significantly increases the efficiency of tag integration, (ii) automates the identification of clones derived from single cells with correct subcellular localization of the tagged protein and (iii) provides a quantitative and high throughput assay to measure the number of on- and off-target integrations with dPCR. The increased efficiency of the new procedure reduces the number of clones that need to be analysed in- depth by more than ten-fold, and yields up to 20% 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 days to 10 days during the overall [~]10 weeks procedure, allowing a single person to process up to 5 genes in parallel, assuming that validated reagents - e.g. PCR-primers, dPCR-assays, Western Blot antibodies - are available.