PAMless SpRY exhibits a preference for the seed region for efficient targeting
Chen Yang1, Zhiwei Zhou2, Xuanlong Sun3
1Key Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China; University of Chinese Academy of Sciences, Beijing 100049, China.
Cell Reports
|May 11, 2024
Summary
PAM-relaxed Cas9 variants like SpG and SpRY quickly find DNA targets by preferring the seed region. This finding is crucial for advancing Cas9 genome engineering and developing new PAMless Cas9 tools.
Area of Science:
- Genomics
- Molecular Biology
- Biochemistry
Background:
- Protospacer-adjacent motif (PAM) recognition is essential for Cas9 nuclease activity in genome engineering.
- Engineered Cas9 variants, such as SpG and SpRY, exhibit relaxed PAM recognition, expanding targeting capabilities.
- The mechanism by which these variants efficiently locate targets despite relaxed PAM specificity remains largely unexplored.
Purpose of the Study:
- To investigate the target-searching kinetics and DNA recognition mechanisms of PAM-relaxed SpCas9 variants (SpG and SpRY).
- To elucidate how SpRY compensates for relaxed PAM recognition to maintain efficient target identification.
- To understand the impact of DNA PAM density on Cas9 target search kinetics.
Main Methods:
- Utilized biochemical assays to analyze the target-searching behavior of SpG and SpRY variants.
- Investigated sequence preferences of SpG and SpRY, focusing on the seed region.
- Assessed the effect of varying PAM densities on Cas9 target search kinetics.
Main Results:
- SpG and SpRY variants demonstrate a preference for the DNA seed region.
- SpRY has evolved specific interactions with amino acids A61R and A1322R, contributing to its seed region preference.
- Cas9 target search kinetics significantly decrease (up to three orders of magnitude) on DNA with high PAM density compared to low PAM density.
Conclusions:
- PAM-relaxed Cas9 variants like SpG and SpRY utilize seed region preference to enhance target location efficiency.
- Understanding Cas9 target search kinetics is vital for optimizing PAMless Cas9 engineering strategies.
- These findings provide critical insights for the development of advanced and versatile Cas9-based genome engineering tools.
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