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CRISPR-Cas9 Activities with Truncated 16-Nucleotide RNA Guides Are Tuned by Target Duplex Stability Beyond the RNA/DNA Hybrid.
Li, Yue; Cooper, Brendon H; Liu, Yukang; Wu, Difei; Zhang, Xiaojun; Rohs, Remo; Qin, Peter Z.
Afiliación
  • Li Y; Department of Chemistry, University of Southern California, Los Angeles, California 90089, United States.
  • Cooper BH; Department of Quantitative and Computational Biology, University of Southern California, Los Angeles, California 90089, United States.
  • Liu Y; Department of Chemistry, University of Southern California, Los Angeles, California 90089, United States.
  • Wu D; Department of Chemistry, University of Southern California, Los Angeles, California 90089, United States.
  • Zhang X; Department of Chemistry, University of Southern California, Los Angeles, California 90089, United States.
  • Rohs R; Department of Chemistry, University of Southern California, Los Angeles, California 90089, United States.
  • Qin PZ; Department of Quantitative and Computational Biology, University of Southern California, Los Angeles, California 90089, United States.
Biochemistry ; 62(17): 2541-2548, 2023 09 05.
Article en En | MEDLINE | ID: mdl-37552860
CRISPR-Cas9 has been adapted as a readily programmable genome manipulation agent, and continuing technological advances rely on an in-depth mechanistic understanding of Cas9 target discrimination. Cas9 interrogates a target by unwinding the DNA duplex to form an R-loop, where the RNA guide hybridizes with one of the DNA strands. It has been shown that RNA guides shorter than the normal length of 20-nucleotide (-nt) support Cas9 cleavage activity by enabling partial unwinding beyond the RNA/DNA hybrid. To investigate whether DNA segment beyond the RNA/DNA hybrid can impact Cas9 target discrimination with truncated guides, Cas9 double-stranded DNA cleavage rates (kcat) were measured with 16-nt guides on targets with varying sequences at +17 to +20 positions distal to the protospacer-adjacent-motif (PAM). The data reveal a log-linear inverse correlation between kcat and the PAM+(17-20) DNA duplex dissociation free energy (ΔGNN(17-20)0), with sequences having smaller ΔGNN(17-20)0 showing faster cleavage and a higher degree of unwinding. The results indicate that, with a 16-nt guide, "peripheral" DNA sequences beyond the RNA/DNA hybrid contribute to target discrimination by tuning the cleavage reaction transition state through the modulation of PAM-distal unwinding. The finding provides mechanistic insights for the further development of strategies that use RNA guide truncation to enhance Cas9 specificity.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: ARN / Sistemas CRISPR-Cas Idioma: En Revista: Biochemistry Año: 2023 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos
Texto completo
Añadir a Mi BVS
Imprimir
XML
PubMed Links
Buscar en Google

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: ARN / Sistemas CRISPR-Cas Idioma: En Revista: Biochemistry Año: 2023 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos