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

Revolutionary in scope and application, the CRISPR Cas9 endonuclease system can be guided by 20-nt single guide RNA (sgRNA) to any complementary loci on the double-stranded DNA. Once the target site is located, Cas9 can then cleave the DNA and introduce mutations. Despite the power of this system, sgRNA is highly susceptible to off-target homologous attachment and can consequently cause Cas9 to cleave DNA at off-target sites. In order to better understand this flaw in the system, the human genome and Streptococcus pyogenes Cas9 (SpCas9) were used in a mathematical and computational study to analyze the probabilities of potential sgRNA off-target homologies. It has been concluded that off-target sites are nearly unavoidable for large-size genomes, such as the human genome. Backed by mathematical analysis, a viable solution is the double-nicking method which has the promise for genome editing specificity. Also applied in this study was a computational algorithm for off-target homology search that was implem...