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Updated: Nov 20, 2025

Genome Editing in Mammalian Cell Lines using CRISPR-Cas
Published on: April 11, 2019
CRISPR technologies and the search for the PAM-free nuclease
Daphne Collias1, Chase L Beisel2,3,4
1Department of Chemical & Biomolecular Engineering, North Carolina State University, Raleigh, NC, 27695-7905, USA.
Abstract:
The ever-expanding set of CRISPR technologies and their programmable RNA-guided nucleases exhibit remarkable flexibility in DNA targeting. However, this flexibility comes with an ever-present constraint: the requirement for a protospacer adjacent motif (PAM) flanking each target. While PAMs play an essential role in self/nonself discrimination by CRISPR-Cas immune systems, this constraint has launched a far-reaching expedition for nucleases with relaxed PAM requirements. Here, we review ongoing efforts toward realizing PAM-free nucleases through natural ortholog mining and protein engineering. We also address potential consequences of fully eliminating PAM recognition and instead propose an alternative nuclease repertoire covering all possible PAM sequences.
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