Traditional large genome editing techniques employ indirect cloning of large genomes into bacterial artificial chromosome (BAC) plasmid. This technique is rather difficult and protracted. Thus, bypassing the BAC plasmid stage in large genome editing would be a big win for the application.
The CRISPR-cas9 system, first discovered in Streptococcus pyogenes, is an important tool for targeting and modifying specific genomic sequences with high efficacy. The CRISPR-cas9 complex cleaves DNA upstream of the protospacer adjacent motif (PAM) in a sequence specific manner. Currently, the CRISPR system consists of two major components: RNA-guided nuclease (Cas9) and guide RNA (gRNA) complementary to the target sequence of interest. However, till date, only a few kilobases of DNA has been successfully edited.