Evaluation of CRISPR/Cas9 site-specific function and validation of sgRNA sequence by a Cas9/sgRNA-assisted reverse PCR technique.

ABSTRACT: The effective application of the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system in biology, medicine and other fields is hindered by the off-target effects and loci-affinity of Cas9-sgRNA, especially at a genome-wide scale. In order to eliminate the occurrence of off-target effects and evaluate loci-affinity by CRISPR/Cas9 site-specific detection and screening of high-affinity sgRNA sequences, respectively, we develop a CRISPR/Cas9-assisted reverse PCR method for site-specific detection and sgRNA sequence validation. The detection method based on PCR can be used directly in the laboratory with PCR reaction conditions, without the need for an additional detection system, and the whole process of detection can be completed within 2 h. Therefore, it can be easil

SUBMITTER: Zhang B 

PROVIDER: S-EPMC7929911 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature