Cracking the Code: A Rapid Way to Read CRISPR’s Targeting Requirements

To edit a specific gene, CRISPR-Cas enzymes must first recognise a short genetic tag known as a protospacer adjacent motif, or PAM. However, an enzyme’s PAM profile often changes depending on its working environment, showing intrinsic differences between bacterial, in vitro, and mammalian settings. Until now, methods for determining these profiles in mammalian cells have been technically complex and difficult to adopt broadly.

Researchers have now constructed a solution called PAM-readID. This novel method determines the PAM recognition profile of CRISPR-Cas nucleases specifically within mammalian cells by integrating double-stranded DNA fragments into breaks. The technique is described as rapid, simple, and accurate.

In tests involving various enzymes like SaCas9 and SpCas9, the method successfully produced accurate recognition profiles. Uniquely, it requires extremely low sequence depth to work effectively. It can even define profiles using standard Sanger sequencing, significantly lowering the cost and time compared to high-throughput alternatives. This easy-to-use tool promises to accelerate the advancement and exploitation of novel genome editing nucleases.