Whole genome analysis of CRISPR Cas9 sgRNA off-target homologies via an efficient computational algorithm

BMC Genomics

Research

Ekaterina Lioutikova¹  Hong Zhou¹  Joseph Manthey¹  Daisy Li²  Michael Zhou²  Xiao Zeng³  Hong Wang⁴ 

[1] Department of Mathematical Science, University of Saint Joseph, 1678 Asylum Avenue, 06117, West Hartford, CT, USA;Hall High School, 975 N Main Street, 06117, West Hartford, CT, USA;PBSG, LLC, P. O. Box 771, 21714, Braddock Heights, MD, USA;Susan L. Cullman Laboratory for Cancer Research, Department of Chemical Biology and Centre for Cancer Prevention Research, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 164 Frelinghuysen Road, 08854, Piscataway, NJ, USA;

关键词: sgRNA;    Off-target homology;    Crispr;    Cas9;    Computational algorithm;    Genome wide;   

DOI  :  10.1186/s12864-017-4225-1

来源: Springer

【 摘 要 】

BackgroundThe beauty and power of the genome editing mechanism, CRISPR Cas9 endonuclease system, lies in the fact that it is RNA-programmable such that Cas9 can be guided to any genomic loci complementary to a 20-nt RNA, single guide RNA (sgRNA), to cleave double stranded DNA, allowing the introduction of wanted mutations. Unfortunately, it has been reported repeatedly that the sgRNA can also guide Cas9 to off-target sites where the DNA sequence is homologous to sgRNA.ResultsUsing human genome and Streptococcus pyogenes Cas9 (SpCas9) as an example, this article mathematically analyzed the probabilities of off-target homologies of sgRNAs and discovered that for large genome size such as human genome, potential off-target homologies are inevitable for sgRNA selection. A highly efficient computationl algorithm was developed for whole genome sgRNA design and off-target homology searches. By means of a dynamically constructed sequence-indexed database and a simplified sequence alignment method, this algorithm achieves very high efficiency while guaranteeing the identification of all existing potential off-target homologies. Via this algorithm, 1,876,775 sgRNAs were designed for the 19,153 human mRNA genes and only two sgRNAs were found to be free of off-target homology.ConclusionsBy means of the novel and efficient sgRNA homology search algorithm introduced in this article, genome wide sgRNA design and off-target analysis were conducted and the results confirmed the mathematical analysis that for a sgRNA sequence, it is almost impossible to escape potential off-target homologies. Future innovations on the CRISPR Cas9 gene editing technology need to focus on how to eliminate the Cas9 off-target activity.

【 授权许可】

CC BY   
© The Author(s). 2017

【 图 表 】

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