Yu Miao, Ai Limei, Wang Bang, Lian Shifeng, Ip Lawrence, Liu James, Li Linxian, Tsai Shengdar Q, Kleinstiver Benjamin P, Zheng Zongli
Department of Biomedical Sciences and Tung Biomedical Sciences Centre, College of Biomedicine, City University of Hong Kong, Kowloon, Hong Kong SAR, China.
Ming Wai Lau Centre for Reparative Medicine, Karolinska Institutet, Shatin, Hong Kong SAR, China.
Nat Biomed Eng. 2025 Aug 13. doi: 10.1038/s41551-025-01464-y.
Characterizing the protospacer adjacent motif (PAM) requirements of different Cas enzymes is a bottleneck in the discovery of Cas proteins and their engineered variants in mammalian cell contexts. Here, to overcome this challenge and to enable more scalable characterization of PAM preferences, we develop a method named GenomePAM that allows for direct PAM characterization in mammalian cells. GenomePAM leverages genomic repetitive sequences as target sites and does not require protein purification or synthetic oligos. GenomePAM uses a 20-nt protospacer that occurs ~16,942 times in every human diploid cell and is flanked by nearly random sequences. We demonstrate that GenomePAM can accurately characterize the PAM requirement of type II and type V nucleases, including the minimal PAM requirement of the near-PAMless SpRY and extended PAM for CjCas9. Beyond PAM characterization, GenomePAM allows for simultaneous comparison of activities and fidelities among different Cas nucleases on thousands of match and mismatch sites across the genome using a single gRNA and provides insight into the genome-wide chromatin accessibility profiles in different cell types.
在哺乳动物细胞环境中，确定不同Cas酶的原间隔序列临近基序（PAM）要求是发现Cas蛋白及其工程变体的一个瓶颈。在此，为了克服这一挑战并实现对PAM偏好更具扩展性的表征，我们开发了一种名为GenomePAM的方法，该方法能够在哺乳动物细胞中直接对PAM进行表征。GenomePAM利用基因组重复序列作为靶位点，无需蛋白质纯化或合成寡核苷酸。GenomePAM使用一个20个核苷酸的原间隔序列，该序列在每个人类二倍体细胞中出现约16,942次，两侧是几乎随机的序列。我们证明，GenomePAM能够准确地表征II型和V型核酸酶的PAM要求，包括几乎无PAM的SpRY的最小PAM要求以及CjCas9的扩展PAM。除了PAM表征外，GenomePAM还允许使用单个gRNA同时比较不同Cas核酸酶在全基因组数千个匹配和错配位点上的活性和保真度，并深入了解不同细胞类型中的全基因组染色质可及性图谱。
