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, Cas...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 implemented in Java to confirm the mathematical analysis.展开更多
文摘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 implemented in Java to confirm the mathematical analysis.
