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

## TL;DR

GenomePAM is a new method for characterizing and engineering CRISPR-Cas nucleases in mammalian cells using genomic repeats, enabling scalable and accurate PAM analysis.

## Contribution

GenomePAM introduces a scalable, mammalian cell-based method for PAM characterization without synthetic oligos or protein purification.

## Key findings

- GenomePAM accurately characterizes PAM requirements for type II and V nucleases, including SpRY and CjCas9.
- The method enables simultaneous comparison of Cas nuclease activities and fidelities across thousands of genomic sites.
- GenomePAM provides insights into genome-wide chromatin accessibility profiles in different cell types.

## 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. 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.