Structure reveals why genome folding is necessary for site-specific integration of foreign DNA into CRISPR arrays

• Published in: Nature structural & molecular biology Vol. 30; no. 11; pp. 1675 - 1685
• Main Authors: Santiago-Frangos, Andrew, Henriques, William S., Wiegand, Tanner, Gauvin, Colin C., Buyukyoruk, Murat, Graham, Ava B., Wilkinson, Royce A., Triem, Lenny, Neselu, Kasahun, Eng, Edward T., Lander, Gabriel C., Wiedenheft, Blake
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.11.2023; Nature Publishing Group
• Subjects: 631/326; 631/45/147; 631/45/612; 631/535; 631/535/1258/1259; Archaea; Arrays; Binding Sites; Biochemistry; Biological Microscopy; Biomedical and Life Sciences; Clustered Regularly Interspaced Short Palindromic Repeats - genetics; CRISPR; CRISPR-Associated Proteins - metabolism; CRISPR-Cas Systems - genetics; Deoxyribonucleic acid; DNA; DNA - chemistry; Folding; Gene therapy; Genomes; Integrase; Integration host factor; Inverted repeat; Life Sciences; Membrane Biology; Plasmids; Protein Structure; Proteins
• ISSN: 1545-9993; 1545-9985; 1545-9985
• DOI: 10.1038/s41594-023-01097-2

Bacteria and archaea acquire resistance to viruses and plasmids by integrating fragments of foreign DNA into the first repeat of a CRISPR array. However, the mechanism of site-specific integration remains poorly understood. Here, we determine a 560-kDa integration complex structure that explains how Pseudomonas aeruginosa Cas (Cas1–Cas2/3) and non-Cas proteins (for example, integration host factor) fold 150 base pairs of host DNA into a U-shaped bend and a loop that protrude from Cas1–2/3 at right angles. The U-shaped bend traps foreign DNA on one face of the Cas1–2/3 integrase, while the loop places the first CRISPR repeat in the Cas1 active site. Both Cas3 proteins rotate 100 degrees to expose DNA-binding sites on either side of the Cas2 homodimer, which each bind an inverted repeat motif in the leader. Leader sequence motifs direct Cas1–2/3-mediated integration to diverse repeat sequences that have a 5′-GT. Collectively, this work reveals new DNA-binding surfaces on Cas2 that are critical for DNA folding and site-specific delivery of foreign DNA. Here, using cryo-EM, the authors show how Cas1–Cas2/3 and integration host factor, by means of a U-shaped bend that traps the invading DNA and a loop that positions it for the integrase, regulate integration of foreign DNA into the first repeat of the CRISPR array.