| Title: |
A positive, growth-based PAM screen identifies noncanonical motifs recognized by the S. pyogenes Cas9. |
| Authors: |
Collias, D; Leenay, R T; Slotkowski, R A; Zuo, Z; Collins, S P; McGirr, B A; Liu, J; Beisel, C L |
| Contributors: |
HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. |
| Source: |
6 ; 29 ; eabb4054 ; Science advances ; United States |
| Publisher Information: |
AAAS |
| Publication Year: |
2020 |
| Collection: |
Helmholtz Centre of Infection Research (HZI), Braunschweig: Repository |
| Description: |
CRISPR technologies have overwhelmingly relied on the Streptococcus pyogenes Cas9 (SpyCas9), with its consensus NGG and less preferred NAG and NGA protospacer-adjacent motifs (PAMs). Here, we report that SpyCas9 also recognizes sequences within an N(A/C/T)GG motif. These sequences were identified on the basis of preferential enrichment in a growth-based screen in Escherichia coli. DNA binding, cleavage, and editing assays in bacteria and human cells validated recognition, with activities paralleling those for NAG(A/C/T) PAMs and dependent on the first two PAM positions. Molecular-dynamics simulations and plasmid-clearance assays with mismatch-intolerant variants supported induced-fit recognition of an extended PAM by SpyCas9 rather than recognition of NGG with a bulged R-loop. Last, the editing location for SpyCas9-derived base editors could be shifted by one nucleotide by selecting between (C/T)GG and adjacent N(C/T)GG PAMs. SpyCas9 and its enhanced variants thus recognize a larger repertoire of PAMs, with implications for precise editing, off-target predictions, and CRISPR-based immunity. |
| Document Type: |
article in journal/newspaper |
| Language: |
English |
| Relation: |
Sci Adv. 2020 Jul 15;6(29):eabb4054. doi:10.1126/sciadv.abb4054.; https://hdl.handle.net/10033/622459; Science advances |
| DOI: |
10.1126/sciadv.abb4054 |
| Availability: |
https://hdl.handle.net/10033/622459; https://doi.org/10.1126/sciadv.abb4054 |
| Rights: |
Attribution-NonCommercial-ShareAlike 4.0 International ; http://creativecommons.org/licenses/by-nc-sa/4.0/ |
| Accession Number: |
edsbas.F1AAE6E9 |
| Database: |
BASE |