Structural coordination between active sites of a Cas6-reverse transcriptase-Cas1—Cas2 CRISPR integrase complex

CRISPR-Cas systems provide adaptive immunity in bacteria and archaea by targeting foreign DNA for destruction using CRISPR RNA-guided enzymes. CRISPR immunity begins with integration of foreign sequences into the host CRISPR genomic locus, followed by transcription and maturation of CRISPR RNAs. In a few CRISPR systems, the Cas1 integrase and a Cas6 nuclease are fused to a reverse transcriptase that enables viral sequence acquisition from both DNA and RNA sources. To determine how these components work together, we determined a 3.7 Å resolution cryo-EM structure of a Cas6-RT-Cas1 protein complexed with Cas2, a subunit of the CRISPR integrase. The structure and accompanying mutagenesis experiments provide evidence of bidirectional crosstalk between the Cas1 and RT active sites and unidirectional crosstalk from Cas6 to the Cas1 and RT active sites. Together, these findings suggest regulated structural rearrangements that may coordinate the complex’s different enzymatic activities. ### Competing Interest Statement The Regents of the University of California have patents issued and pending for CRISPR technologies on which J.A.D. is an inventor. J.A.D. is a cofounder of Caribou Biosciences, Editas Medicine, Scribe Therapeutics, Intellia Therapeutics and Mammoth Biosciences. J.A.D. is a scientific advisory board member of Caribou Biosciences, Intellia Therapeutics, eFFECTOR Therapeutics, Scribe Therapeutics, Mammoth Biosciences, Synthego, Algen Biotechnologies, Felix Biosciences and Inari. J.A.D. is a Director at Johnson & Johnson and has research projects sponsored by Biogen, Pfizer, AppleTree Partners and Roche.