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Scalable characterization of the PAM requirements of CRISPR–Cas enzymes using HT-PAMDA

Nature Protocols volume 16, pages 1511–1547 (2021)Cite this article

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Abstract

The continued expansion of the genome-editing toolbox necessitates methods to characterize important properties of CRISPR–Cas enzymes. One such property is the requirement for Cas proteins to recognize a protospacer-adjacent motif (PAM) in DNA target sites. The high-throughput PAM determination assay (HT-PAMDA) is a method that enables scalable characterization of the PAM preferences of different Cas proteins. Here, we provide a step-by-step protocol for the method, discuss experimental design considerations, and highlight how the method can be used to profile naturally occurring CRISPR–Cas9 enzymes, engineered derivatives with improved properties, orthologs of different classes (e.g., Cas12a), and even different platforms (e.g., base editors). A distinguishing feature of HT-PAMDA is that the enzymes are expressed in a cell type or organism of interest (e.g., mammalian cells), permitting scalable characterization and comparison of hundreds of enzymes in a relevant setting. HT-PAMDA does not require specialized equipment or expertise and is cost effective for multiplexed characterization of many enzymes. The protocol enables comprehensive PAM characterization of dozens or hundreds of Cas enzymes in parallel in <2 weeks.

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Fig. 1: The role of the PAM in host defense and genome editing.
Fig. 2: Overview of HT-PAMDA workflow.
Fig. 3: Detailed experimental workflow for in vitro cleavage reactions and library preparation.
Fig. 4: Representations of Cas enzyme PAM preference.
Fig. 5: Expected results of an HT-PAMDA experiment.

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Data availability

Source data for all figures are available in the NCBI Sequence Read Archive under BioProject ID PRJNA605711.

Code availability

Freely available open source code (under GNU General Public License v3.0) for HT-PAMDA analysis is available on the Kleinstiver Lab GitHub repository at https://github.com/kleinstiverlab/HT-PAMDA. The code in this protocol has been peer reviewed.

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Acknowledgements

We thank K. A. Christie for suggestions on the manuscript and A. A. Sousa for technical input on the development of the original PAMDA method. B.P.K. acknowledges support from NIH R00-CA218870, NIH P01-HL142494, a Career Development Award from the American Society of Gene & Cell Therapy, and the Margaret Q. Landenberger Research Foundation. J.K.J. acknowledges support from NIH R35 GM118158 and RM1 HG009490 for the development of the original PAMDA method.

Author information

Author notes

  1. Russell T. Walton

    Present address: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

  2. Russell T. Walton

    Present address: Broad Institute of MIT and Harvard, Cambridge, MA, USA

Authors and Affiliations

  1. Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA

    Russell T. Walton & Benjamin P. Kleinstiver

  2. Department of Pathology, Massachusetts General Hospital, Boston, MA, USA

    Russell T. Walton, Jonathan Y. Hsu, J. Keith Joung & Benjamin P. Kleinstiver

  3. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Jonathan Y. Hsu

  4. Molecular Pathology Unit, Center for Cancer Research and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA, USA

    Jonathan Y. Hsu & J. Keith Joung

  5. Department of Pathology, Harvard Medical School, Boston, MA, USA

    Jonathan Y. Hsu, J. Keith Joung & Benjamin P. Kleinstiver

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  4. Benjamin P. Kleinstiver
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Contributions

R.T.W. and B.P.K wrote the manuscript with input from all authors. R.T.W. developed the HT-PAMDA method in the B.P.K. laboratory; the original PAMDA method was developed by R.T.W. and B.P.K in the J.K.J. laboratory. All laboratory experiments for HT-PAMDA were performed by R.T.W. The original PAMDA software was developed by J.Y.H. and subsequently adapted for HT-PAMDA by R.T.W.

Corresponding author

Correspondence to Benjamin P. Kleinstiver.

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Competing interests

All authors are inventors on patent applications filed by Partners HealthCare that describe gene-editing and/or epigenome-editing technologies; R.T.W. and B.P.K. are inventors on a patent application related to the HT-PAMDA method. B.P.K. is an advisor to Acrigen Biosciences and consults for Avectas Inc. and ElevateBio. J.K.J. has financial interests in Beam Therapeutics, Chroma Medicine (formerly known as YKY, Inc.), Editas Medicine, Excelsior Genomics, Pairwise Plants, Poseida Therapeutics, SeQure Dx, Inc., Transposagen Biopharmaceuticals, and Verve Therapeutics (formerly known as Endcadia). J.K.J.’s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies.

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Key reference using this protocol

Walton, R. T., Christie, K. A., Whittaker, M. N. & Kleinstiver, B. P. Science 368, 290–296 (2020): https://doi.org/10.1126/science.aba8853

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Walton, R.T., Hsu, J.Y., Joung, J.K. et al. Scalable characterization of the PAM requirements of CRISPR–Cas enzymes using HT-PAMDA. Nat Protoc 16, 1511–1547 (2021). https://doi.org/10.1038/s41596-020-00465-2

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