Can SpRY recognize any PAM in human cells?

• 作者: Jinbin YE Haitao XI Yilu CHEN Qishu CHEN Xiaosheng LU Jineng LV Yamin CHEN Feng GU Junzhao ZHAO
• 来源期刊: 浙江大学学报（英文版）（B辑：生物医学和生物技术）
• 年，卷（期）: 2022,（5）
• 所属期刊栏目: Research Articles
• 页码范围: 382-391,中插5-中插11
• 页数: 1页
• 语种: 英文

摘要

The application of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) can be limited due to a lack of compatible protospacer adjacent motif (PAM) sequences in the DNA regions of interest. Recently, SpRY, a variant of Streptococcus pyogenes Cas9 (SpCas9), was reported, which nearly completely fulfils the PAM requirement. Meanwhile, PAMs for SpRY have not been well addressed. In our previous study, we developed the PAM Definition by Observable Sequence Excision (PAM-DOSE) and green fluorescent protein (GFP)-reporter systems to study PAMs in human cells. Herein, we endeavored to identify the PAMs of SpRY with these two methods. The results indicated that 5'-NRN-3', 5'-NTA-3', and 5'-NCK-3' could be considered as canonical PAMs. 5'-NCA-3' and 5'-NTK-3' may serve as non-priority PAMs. At the same time, PAM of 5'-NYC-3' is not recommended for human cells. These findings provide further insights into the application of SpRY for human genome editing.