PAM-readID is a rapid, simple, and accurate PAM determination method for CRISPR-Cas enzymes in mammalian cells.

One CRISPR-Cas enzyme's recognized protospacer adjacent motif (PAM) profile always shows intrinsic differences between assays with different working environments, such as in vitro, in bacterial cells, or in mammalian cells. The developed methods in mammalian cells are technically complex and not readily amenable to be broadly adopted, highlighting the urgent need for a well-established PAM-determining method in mammalian cells. In this study, we construct a rapid, simple, and accurate method for determining the PAM recognition profile of CRISPR-Cas nucleases in mammalian cells. The developed method is termed PAM-readID, PAM REcognition-profile-determining Achieved by Double-stranded oligodeoxynucleotides Integration in DNA double-stranded breaks. Using PAM-readID, the PAM recognition profiles of SaCas9, SaHyCas9, Nme1Cas9, SpCas9, SpG, SpRY, and AsCas12a in mammalian cells are well produced. An accurate PAM preference for SpCas9 can be identified by analysis with extremely low sequence depth (500 reads). PAM-readID can also define a PAM recognition profile of Cas9 based on Sanger sequencing with a significantly lower cost of time and price than that of high-throughput sequencing. We present an easy-to-use method for comprehensively revealing functional PAM of CRISPR-Cas nucleases in mammalian cells, which can contribute towards accelerating the advancement of exploiting novel genome editing nucleases.