Mathematical and computational analysis of CRISPR Cas9 sgRNA off-target homologies

The true power of genome editing mechanism known as RNA-programmable CRISPR Cas9 endonuclease system, lies in the fact that Cas9 can be guided to any loci complementary to a 20-nt RNA, single guide RNA (sgRNA), to cleave double stranded DNA, and therefore allows the introduction of wanted mutations. Unfortunately, sgRNA is prone to off-target homologous attachment, thus guiding Cas9 to cleave DNA sequences at unwanted sites. Using human genome and Streptococcus pyogenes Cas9 (SpCas9) as the example, this article analyzed the probabilities of off-target sites of sgRNAs and discovered that for large-size genomes such as human genome, off-target sites are nearly inevitable for sgRNA selection. Based on the mathematical analysis, it seems that the double nicking approach is currently the only feasible solution to promise genome editing specificity. An effective computational algorithm for off-target homology searching is also implemented to confirm the mathematical analysis.