Genome expansion by a CRISPR trimmer-integrase

CRISPR-Cas adaptive immune systems capture DNA fragments from invading mobile genetic elements and integrate them into the host genome to provide a template for RNA-guided immunity. CRISPR systems maintain genome integrity and avoid autoimmunity by distinguishing between self and non-self, a process for which the CRISPR-Cas1:Cas2 integrase is necessary but not sufficient. In some microbes, the Cas4 endonuclease assists CRISPR adaptation, but many CRISPR-Cas systems lack Cas4. We show here that an elegant alternative pathway employs an internal exonuclease to select and process DNA for integration using the protospacer adjacent motif (PAM). A natural Cas1:Cas2-exonuclease fusion (trimmer-integrase) catalyzes coordinated DNA capture, trimming and integration. Five cryo-EM structures of the CRISPR trimmer-integrase, visualized both before and during DNA integration, show how asymmetric processing generates size-defined, PAM-containing substrates. Before genome integration, the PAM sequence is released by Cas1 and cleaved by the exonuclease, marking inserted DNA as self and preventing aberrant CRISPR targeting of the host. Together, these data support a model in which CRISPR systems lacking Cas4 use fused or recruited exonucleases for faithful acquisition of new CRISPR immune sequences. ### Competing Interest Statement The Regents of the University of California have filed patent applications for CRISPR-related molecules, compositions and methods on which J.A.D. is a co-inventor. J.A.D. is a co-founder of Caribou Biosciences, Editas Medicine, Intellia Therapeutics, Mammoth Biosciences and Scribe Therapeutics, and a director of Altos, Johnson & Johnson and Tempus. J.A.D. is a scientific advisor to Caribou Biosciences, Intellia Therapeutics, Mammoth Biosciences, Inari, Scribe Therapeutics, Felix Biosciences and Algen. J.A.D. also serves as Chief Science Advisor to Sixth Street and a Scientific Advisory Board member at The Column Group. J.A.D. conducts academic research projects sponsored by Roche and Apple Tree Partners.