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.
概要
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′。
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Acknowledgments
This work was supported by Lin HE’s Academician Workstation of New Medicine and Clinical Translation (No. 18331105) and the Program for Basic Science and Technology Cooperation Projects of Wenzhou City (No. H22010011), China.
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Feng GU and Junzhao ZHAO conceived the idea and performed data analyses. Jinbin YE, Haitao XI, Yilu CHEN, Qishu CHEN, Xiaosheng LU, Jineng LV, Yamin CHEN, and Feng GU performed the experiments. Jinbin YE and Feng GU wrote the manuscript. All authors have read and approved the final manuscript, and therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data.
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Jinbin YE, Haitao XI, Yilu CHEN, Qishu CHEN, Xiaosheng LU, Jineng LV, Yamin CHEN, Feng GU, and Junzhao ZHAO declare that they have no conflict of interest.
This article does not contain any studies with human or animal subjects performed by any of the authors.
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Ye, J., Xi, H., Chen, Y. et al. Can SpRY recognize any PAM in human cells?. J. Zhejiang Univ. Sci. B 23, 382–391 (2022). https://doi.org/10.1631/jzus.B2100710
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DOI: https://doi.org/10.1631/jzus.B2100710