Figure 1.

Genome editing by CHoP‐In. CHoP‐In genome editing relies utilises NHEJ‐mediated integration of a PCR‐generated donor into a CRISPR/Cas9‐induced DSB. A, To achieve this, two constructs must be prepared after identifying the genomic gRNA and PAM site. To introduce a DSB at the desired locus, a vector such as pX330 encoding both the gRNA and the Cas9 nuclease is constructed. Additionally, a CHoP‐In integration donor is produced by PCR, consisting of the desired integration fragment flanked by the same gene specific gRNA and PAM sites in the PCR primers. Importantly, the gRNA and PAM sites flanking the integration donor must be in the reverse orientation with respect to genomic locus as this prevents reconstitution and re‐cleaving of the sites following integration. B, The whole process can be completed in approximately 1 week, giving a mixed population of edited cells with minimal hands‐on time when compared with HDR mediated approaches