GenomePAM directs PAM characterization and engineering of CRISPR-Cas nucleases using mammalian genome repeats

Miao Yu; Limei Ai; Bang Wang; Shifeng Lian; Lawrence Ip; James Liu; Linxian Li; Shengdar Q. Tsai; Benjamin P. Kleinstiver; Zongli Zheng

doi:10.1038/s41551-025-01464-y

GenomePAM directs PAM characterization and engineering of CRISPR-Cas nucleases using mammalian genome repeats

Miao Yu
, Limei Ai
, Bang Wang
, Shifeng Lian
, Lawrence Ip
, James Liu
, Linxian Li
, Shengdar Q. Tsai
, Benjamin P. Kleinstiver
, Zongli Zheng^*

^*Corresponding author for this work

Research output: Journal Publications and Reviews › RGC 21 - Publication in refereed journal › peer-review

3 Citations (Scopus)

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Abstract

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. © The Author(s) 2025

Original language	English
Pages (from-to)	231-244
Number of pages	26
Journal	Nature Biomedical Engineering
Volume	10
Issue number	2
Online published	13 Aug 2025
DOIs	https://doi.org/10.1038/s41551-025-01464-y
Publication status	Published - Feb 2026

Funding

We thank support from Lau Grant LC230003 (Z.Z.), Swedish Research Council 202001418 (Z.Z.); Research Grants Council of the Hong Kong Special Administrative Region 11103024 and T12-101/23-N (Z.Z.); Shenzhen Medical Research Fund B2402002 (Z.Z.); the InnoHK initiative of the Innovation and Technology Commission of the Hong Kong Special Administrative Region Government (Z.Z.); Lau Grant LC230002 (L.L.); the Kayden–Lambert MGH Research Scholar Award 2023–2028 (B.P.K.); and National Institutes of Health (NIH) grant DP2-CA281401 (B.P.K.). We thank O. W. C. Leung for proofreading the manuscript.

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This full text is made available under CC-BY 4.0. https://creativecommons.org/licenses/by/4.0/

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title = "GenomePAM directs PAM characterization and engineering of CRISPR-Cas nucleases using mammalian genome repeats",

abstract = "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 \textasciitilde{}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. {\textcopyright} The Author(s) 2025",

author = "Miao Yu and Limei Ai and Bang Wang and Shifeng Lian and Lawrence Ip and James Liu and Linxian Li and Tsai, \{Shengdar Q.\} and Kleinstiver, \{Benjamin P.\} and Zongli Zheng",

year = "2026",

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doi = "10.1038/s41551-025-01464-y",

language = "English",

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AU - Yu, Miao

AU - Ai, Limei

AU - Wang, Bang

AU - Lian, Shifeng

AU - Ip, Lawrence

AU - Liu, James

AU - Li, Linxian

AU - Tsai, Shengdar Q.

AU - Kleinstiver, Benjamin P.

AU - Zheng, Zongli

PY - 2026/2

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N2 - 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. © The Author(s) 2025

AB - 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. © The Author(s) 2025

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JO - Nature Biomedical Engineering

JF - Nature Biomedical Engineering

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GenomePAM directs PAM characterization and engineering of CRISPR-Cas nucleases using mammalian genome repeats

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TBRS: Single-Cell Multi-Omics Study of Atherosclerotic Vascular Disease: Narrowing the Gap Between Bench and Bedside

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GenomePAM directs PAM characterization and engineering of CRISPR-Cas nucleases using mammalian genome repeats

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TBRS: Single-Cell Multi-Omics Study of Atherosclerotic Vascular Disease: Narrowing the Gap Between Bench and Bedside

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