Efficient cleavage resolves PAM preferences of CRISPR-Cas in human cells

Lianchao Tang; Fayu Yang; Xiaoxue He; Haihua Xie; Xiaoyu Liu; Junhao Fu; Haitao Xi; Xiaosheng Lu; Changbao Liu; Zongming Song; Jia Qu; Junzhao Zhao; Feng Gu

doi:10.1016/j.cr.2019.08.002

Efficient cleavage resolves PAM preferences of CRISPR-Cas in human cells

Cell Regen. 2019 Oct 28;8(2):44-50. doi: 10.1016/j.cr.2019.08.002. eCollection 2019 Dec.

Authors

Lianchao Tang¹, Fayu Yang¹, Xiaoxue He¹, Haihua Xie¹, Xiaoyu Liu¹, Junhao Fu¹, Haitao Xi², Xiaosheng Lu², Changbao Liu², Zongming Song^{1

3}, Jia Qu¹, Junzhao Zhao², Feng Gu¹

Affiliations

¹ 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, Zhejiang 325027 China.
² The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325027 China.
³ Henan Provincial People's Hospital, Henan Eye Hospital, People's Hospital of Zhengzhou University, School of Clinical Medicine, Henan University, Zhengzhou, Henan 450003 China.

Abstract

Clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR-Cas) of bacterial adaptive immunity have been adopted as a powerful and versatile tool for manipulation of the genome. This paradigm has been widely applied in biological research and treatments of animal or cellular disease models. A critical feature of CRISPR-Cas is the protospacer adjacent motif (PAM), which dictates the DNA target recognition mechanism of Cas proteins. While, direct identifying functional PAM sequences in human cells remains a challenge. Here, we developed a positive screen system termed PAM-DOSE (PAM Definition by Observable Sequence Excision) to delineate the functional PAMs in human cells. Specifically, the PAM libraries for CRISPR-Cas (SpCas9, SpCas9-NG, FnCas12a, AsCas12a, LbCas12a and MbCas12a) were generated and the corresponding CRISPR-Cas mediated cleaved fragments with functional PAM in human cells were harvested for DNA sequencing, which could be tracked and visualized with either florescence microscopy or flow cytometry analysis. With this system, we identified the functional PAMs of CRISPR-Cas members. We also found that spacer sequence affects the PAM preference of Cas proteins. This method will facilitate identification of functional PAMs for Cas-mediated human genome editing applications.

Keywords: CRISPR-Cas; Human; Next-generation Sequencing; PAM.