A Theoretical Study of the CRISPR-Cas9 Off-target EffectBased on Molecular Dynamics Simulations

摘要

Clustered regularly interspaced short palindromic repeat-CRISPR associated protein 9(CRISPR-Cas9) recognizes a target DNA strand by a guide RNA sequence of 20 bases in lengthand cleaves it.Although CRISPR is highly versatile as a novel genome-editing tool,this systemhas a major drawback of cutting non-target sequences (off-target effect).The past experimentalstudies have reported that some variants of mutants,for example,derived from streptococcuspyogenes Cas9 (spCas9),reduced the off-target effect and maintained high cleavage efficiencyfor a given target sequence.However,the mechanism of how these mutations improve DNArecognition has not been elucidated,and thus no rational design of mutants being free from theoff-target effect has been established.In the present study,we performed molecular dynamics(MD) simulations on wild-type and mutant spCas9 with target and non-target DNA sequences(PDB id:4un33 and modeled from it) at the DNA recognition stage in order to understand themechanism of the off-target effects.