Optimizing sgRNA length to improve target specificity and efficiency for the GGTA1 gene using the CRISPR/Cas9 gene editing system.

ABSTRACT: The CRISPR/Cas9 gene editing system has enhanced the development of genetically engineered animals for use in xenotransplantation. Potential limitations to the CRISPR/Cas9 system impacting the development of genetically engineered cells and animals include the creation of off-target mutations. We sought to develop a method to reduce the likelihood of off-target mutation while maintaining a high efficiency rate of desired genetic mutations for the GGTA1 gene. Extension of sgRNA length, responsible for recognition of the target DNA sequence for Cas9 cleavage, resulted in improved specificity for the GGTA1 gene and less off-target DNA cleavage. Three PAM sites were selected within exon 1 of the porcine GGTA1 gene and ten sgRNA of variable lengths were designed across these three sites. The sg

SUBMITTER: Matson AW 

PROVIDER: S-EPMC6903732 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature