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

Nat Protoc. 2021 Mar;16(3):1511-1547. doi: 10.1038/s41596-020-00465-2. Epub 2021 Feb 5.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Associated Protein 9 / metabolism
  • CRISPR-Cas Systems / genetics
  • CRISPR-Cas Systems / physiology*
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics
  • DNA / genetics
  • Gene Editing / methods*
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • Nucleotide Motifs / genetics
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • Research Design


  • RNA, Guide, CRISPR-Cas Systems
  • DNA
  • CRISPR-Associated Protein 9