PAMless SpRY exhibits a preference for the seed region for efficient targeting

Chen Yang; Zhiwei Zhou; Xuanlong Sun; Haiyan Ju; Xinmin Yue; Shuquan Rao; Chaoyou Xue

doi:10.1016/j.celrep.2024.114225

PAMless SpRY exhibits a preference for the seed region for efficient targeting

Cell Rep. 2024 May 10;43(5):114225. doi: 10.1016/j.celrep.2024.114225. Online ahead of print.

Authors

Chen Yang¹, Zhiwei Zhou², Xuanlong Sun³, Haiyan Ju¹, Xinmin Yue³, Shuquan Rao⁴, Chaoyou Xue⁵

Affiliations

¹ Key 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.
² State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China; Tianjin Institutes of Health Science, Tianjin 301600, China.
³ Key Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
⁴ State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China; Tianjin Institutes of Health Science, Tianjin 301600, China. Electronic address: raoshuquan@ihcams.ac.cn.
⁵ Key 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. Electronic address: xuechy@tib.cas.cn.

PMID: 38733582
DOI: 10.1016/j.celrep.2024.114225

Abstract

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.

Keywords: CP: Molecular biology; PAMless; SpG; SpRY; target-searching kinetics.