Extended Data Fig. 4: Cas1-2/3 predominantly recognizes IR motifs, CRISPR repeat and foreign DNA through non-sequence specific interactions.

a, Splayed 3′ ends of the foreign DNA are directed into the Cas1 transesterification active site. The product of the first strand-transfer reaction is shown in the Cas1a* active site (top), and the 3′ OH of the other end of the foreign DNA is positioned in the Cas1b* active site (bottom). The cryo-EM map is shown in transparent grey. b, Zoom-in on the Cas1-2/3 contacts to the CRISPR repeat (ChimeraX contacts command with default parameters). Most protein contacts occur to the DNA backbone and minor groove. Cas1 residue E184 appears to probe nucleotide G1 of the repeat. c, Zoom-in on the Cas1-2/3 contacts to the IR leader motifs. Most protein contacts occur to the DNA backbone and minor groove. d, DNAproDB analysis of Cas1-2/3 interactions with the 3′ ends of foreign DNA and the CRISPR leader-repeat junction. For clarity, only protein interactions to the nucleobases are shown⁹³. e, DNAproDB analysis of Cas1-2/3 interactions with the IR leader motifs⁹³. For clarity, only protein interactions to the nucleobases are shown. f, Zoom-in on the atomic fit of the base-pairs around the leader-repeat junction (-3 and +3 bps, coordinated by Cas1a*), to the cryo-EM map. Tension in the DNA loop at the leader-repeat junction has been released in the post-integration structure by a physical separation of base-pairs, as measured by an increase in base step rise. This tension may further pull the leaving 3′OH out of the Cas1 transesterification active site, to inhibit disintegration of the foreign DNA from the repeat. The approximate local base step rise was calculated using the http://web.x3dna.org/ webserver. g, A bioinformatic analysis of the first repeat from 24,940 CRISPR loci reveals that a 5′ GT dinucleotide is strongly conserved across most CRISPR subtypes. Similarly, a 5′ GT is present at the spacer-end of the repeat (seen as AC-3′ on the sense strand) within certain CRISPR subtypes (I-D, II-C, III-C, III-D, V-B, V-E, V-K, VI-A), but it is not broadly conserved.