Can SpRY recognize any PAM in human cells?

Jinbin YE; Haitao XI; Yilu Chen; Qishu Chen; Xiaosheng LU; Jineng LV; Yamin Chen; Feng GU; Junzhao Zhao

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Can SpRY recognize any PAM in human cells?

Author: Jinbin YE ¹ ; Haitao XI ¹ ; Yilu CHEN ¹ ; Qishu CHEN ¹ ; Xiaosheng LU ¹ ; Jineng LV ² ; Yamin CHEN ² ; Feng GU ³ ; Junzhao ZHAO ⁴
Author Information

1. Reproduction Center, Department of Obstetrics and Gynecology, the Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou 325000, China.
2. School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, State Key Laboratory and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou 325000, China.
3. School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, State Key Laboratory and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou 325000, China. z.joyce08@163.com, gufenguw@gmail.com.
4. Reproduction Center, Department of Obstetrics and Gynecology, the Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou 325000, China. z.joyce08@163.com.
Publication Type:Journal Article
Keywords: CRISPR/Cas; Protospacer adjacent motif (PAM); Recognize; SpRY
MeSH: CRISPR-Associated Protein 9/metabolism*; CRISPR-Cas Systems; DNA; Gene Editing/methods*; Humans; Streptococcus pyogenes/metabolism*
From: Journal of Zhejiang University. Science. B 2022;23(5):382-391
CountryChina
Language:English
Abstract: 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.