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On-line Access: 2024-08-27

Received: 2023-10-17

Revision Accepted: 2024-05-08

Crosschecked: 2022-05-13

Cited: 0

Clicked: 1602

Citations:  Bibtex RefMan EndNote GB/T7714

 ORCID:

Feng GU

https://orcid.org/0000-0002-9600-8407

Junzhao ZHAO

https://orcid.org/0000-0002-2385-205X

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Journal of Zhejiang University SCIENCE B 2022 Vol.23 No.5 P.382-391

http://doi.org/10.1631/jzus.B2100710


Can SpRY recognize any PAM in human cells?


Author(s):  Jinbin YE, Haitao XI, Yilu CHEN, Qishu CHEN, Xiaosheng LU, Jineng LV, Yamin CHEN, Feng GU, Junzhao ZHAO

Affiliation(s):  Reproduction Center, Department of Obstetrics and Gynecology, the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou 325000, China; more

Corresponding email(s):   z.joyce08@163.com, gufenguw@gmail.com

Key Words:  CRISPR/Cas, SpRY, Protospacer adjacent motif (PAM), Recognize


Jinbin YE, Haitao XI, Yilu CHEN, Qishu CHEN, Xiaosheng LU, Jineng LV, Yamin CHEN, Feng GU, Junzhao ZHAO. Can SpRY recognize any PAM in human cells?[J]. Journal of Zhejiang University Science B, 2022, 23(5): 382-391.

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author="Jinbin YE, Haitao XI, Yilu CHEN, Qishu CHEN, Xiaosheng LU, Jineng LV, Yamin CHEN, Feng GU, Junzhao ZHAO",
journal="Journal of Zhejiang University Science B",
volume="23",
number="5",
pages="382-391",
year="2022",
publisher="Zhejiang University Press & Springer",
doi="10.1631/jzus.B2100710"
}

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%A Yilu CHEN
%A Qishu CHEN
%A Xiaosheng LU
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A1 - Jineng LV
A1 - Yamin CHEN
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A1 - Junzhao ZHAO
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DOI - 10.1631/jzus.B2100710


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.

SpRY能识别人类细胞中前间区序列邻近基序吗?

目的:确定SpCas9突变体(SpRY)可以识别人类细胞中的前间区序列邻近基序(PAM),为SpRY在基因编辑中的应用提供参考。
创新点:利用可视序列切除定义PAM(PAM Definition by Observable Sequence Excision,PAM-DOSE)和绿色荧光报告系统这两种不同方法,确定了SpRY在人类细胞中介导有效切割时对PAM的要求。
方法:通过在PAM-DOSE法确定SpRY的PAM偏好性,并通过绿色荧光报告系统测试SpRY识别的PAM,同时总结SpRY在人类细胞中可被识别的PAM序列。
结论:5’-NRN-3’、5’-NTA-3’和5’-NCK-3’作为SpRY主要识别的PAMs,同时5’-NCA-3’和5’-NTK-3’可作为SpRY的次选PAM,且不推荐使用5’-NYC-3’。

关键词:CRISPR/Cas;SpRY;前间区序列邻近基序(PAM);识别

Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article

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