Improving Stability and Specificity of CRISPR/Cas9 System by Selective Modification of Guide RNAs with 2'-fluoro and Locked Nucleic Acid Nucleotides.

ABSTRACT: The genome editing approach using the components of the CRISPR/Cas system has found wide application in molecular biology, fundamental medicine and genetic engineering. A promising method is to increase the efficacy and specificity of CRISPR/Cas-based genome editing systems by modifying their components. Here, we designed and chemically synthesized guide RNAs (crRNA, tracrRNA and sgRNA) containing modified nucleotides (2'-O-methyl, 2'-fluoro, LNA-locked nucleic acid) or deoxyribonucleotides in certain positions. We compared their resistance to nuclease digestion and examined the DNA cleavage efficacy of the CRISPR/Cas9 system guided by these modified guide RNAs. The replacement of ribonucleotides with 2'-fluoro modified or LNA nucleotides increased the lifetime of the crRNAs, while other t

SUBMITTER: Sakovina L 

PROVIDER: S-EPMC9655745 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature