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PAMless SpRY exhibits a preference for the seed region for efficient targeting
- Source :
- Cell Reports, Vol 43, Iss 5, Pp 114225- (2024)
- Publication Year :
- 2024
- Publisher :
- Elsevier, 2024.
-
Abstract
- Summary: Protospacer-adjacent motif (PAM) recognition licenses Cas nucleases for genome engineering applications, thereby restricting gene targeting to PAM-containing regions. Protein engineering has led to PAM-relaxed SpCas9 variants like SpG and SpRY. Given the evolved role of PAMs in facilitating target-searching kinetics, it remains unclear how these variants quickly locate their targets. We show that SpG and SpRY exhibit a preference for the seed region. To compensate for the relaxed PAM recognition, SpRY has evolved a sequence preference for the seed region through interactions with A61R and A1322R. Furthermore, SpCas9 exhibits a significant decrease in target search kinetics on high-PAM-density DNA, slowing down up to three orders of magnitude compared to low-PAM-density DNA, suggesting the necessity for sequence recognition even in PAM-relaxed variants. This underscores the importance of considering Cas9 target-searching kinetics in SpCas9 PAMless engineering, providing valuable insights for further PAMless Cas9 protein engineering efforts.
- Subjects :
- CP: Molecular biology
Biology (General)
QH301-705.5
Subjects
Details
- Language :
- English
- ISSN :
- 22111247
- Volume :
- 43
- Issue :
- 5
- Database :
- Directory of Open Access Journals
- Journal :
- Cell Reports
- Publication Type :
- Academic Journal
- Accession number :
- edsdoj.99fc66d99734c8491b6715ec5329ba6
- Document Type :
- article
- Full Text :
- https://doi.org/10.1016/j.celrep.2024.114225