CRISPR/Cas9 gRNA activity depends on free energy changes and on the target PAM context
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CRISPR/Cas9 gRNA activity depends on free energy changes and on the target PAM context. / Corsi, Giulia I; Qu, Kunli; Alkan, Ferhat; Pan, Xiaoguang; Luo, Yonglun; Gorodkin, Jan.
I: Nature Communications, Bind 13, 3006, 2022.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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TY - JOUR
T1 - CRISPR/Cas9 gRNA activity depends on free energy changes and on the target PAM context
AU - Corsi, Giulia I
AU - Qu, Kunli
AU - Alkan, Ferhat
AU - Pan, Xiaoguang
AU - Luo, Yonglun
AU - Gorodkin, Jan
N1 - © 2022. The Author(s).
PY - 2022
Y1 - 2022
N2 - A major challenge of CRISPR/Cas9-mediated genome engineering is that not all guide RNAs (gRNAs) cleave the DNA efficiently. Although the heterogeneity of gRNA activity is well recognized, the current understanding of how CRISPR/Cas9 activity is regulated remains incomplete. Here, we identify a sweet spot range of binding free energy change for optimal efficiency which largely explains why gRNAs display changes in efficiency at on- and off-target sites, including why gRNAs can cleave an off-target with higher efficiency than the on-target. Using an energy-based model, we show that local gRNA-DNA interactions resulting from Cas9 "sliding" on overlapping protospacer adjacent motifs (PAMs) profoundly impact gRNA activities. Combining the effects of local sliding for a given PAM context with global off-targets allows us to better identify highly specific, and thus efficient, gRNAs. We validate the effects of local sliding on gRNA efficiency using both public data and in-house data generated by measuring SpCas9 cleavage efficiency at 1024 sites designed to cover all possible combinations of 4-nt PAM and context sequences of 4 gRNAs. Our results provide insights into the mechanisms of Cas9-PAM compatibility and cleavage activation, underlining the importance of accounting for local sliding in gRNA design.
AB - A major challenge of CRISPR/Cas9-mediated genome engineering is that not all guide RNAs (gRNAs) cleave the DNA efficiently. Although the heterogeneity of gRNA activity is well recognized, the current understanding of how CRISPR/Cas9 activity is regulated remains incomplete. Here, we identify a sweet spot range of binding free energy change for optimal efficiency which largely explains why gRNAs display changes in efficiency at on- and off-target sites, including why gRNAs can cleave an off-target with higher efficiency than the on-target. Using an energy-based model, we show that local gRNA-DNA interactions resulting from Cas9 "sliding" on overlapping protospacer adjacent motifs (PAMs) profoundly impact gRNA activities. Combining the effects of local sliding for a given PAM context with global off-targets allows us to better identify highly specific, and thus efficient, gRNAs. We validate the effects of local sliding on gRNA efficiency using both public data and in-house data generated by measuring SpCas9 cleavage efficiency at 1024 sites designed to cover all possible combinations of 4-nt PAM and context sequences of 4 gRNAs. Our results provide insights into the mechanisms of Cas9-PAM compatibility and cleavage activation, underlining the importance of accounting for local sliding in gRNA design.
KW - CRISPR-Cas Systems
KW - Genome
KW - RNA, Guide/genetics
U2 - 10.1038/s41467-022-30515-0
DO - 10.1038/s41467-022-30515-0
M3 - Journal article
C2 - 35637227
VL - 13
JO - Nature Communications
JF - Nature Communications
SN - 2041-1723
M1 - 3006
ER -
ID: 310503208