8X5V

BlCas9-sgRNA-target DNA complex

Summary for 8X5V

• Entry DOI: 10.2210/pdb8x5v/pdb
• Descriptor: BlCas9, RNA (110-mer), DNA (28-mer), ... (8 entities in total)
• Functional Keywords: crispr-cas, rna binding protein, rna binding protein-rna-dna complex, rna binding protein/rna/dna
• Biological source: Brevibacillus laterosporus; More
• Total number of polymer chains: 4
• Total formula weight: 156463.62

Authors

Nakane, T.,Nakagawa, R.,Yamashita, K.,Nishimasu, H.,Nureki, O. (deposition date: 2023-11-19, release date: 2024-07-10)

Primary citation

Nakane, T.,Nakagawa, R.,Ishiguro, S.,Okazaki, S.,Mori, H.,Shuto, Y.,Yamashita, K.,Yachie, N.,Nishimasu, H.,Nureki, O.
Structure and engineering of Brevibacillus laterosporus Cas9.
Commun Biol, 7:803-803, 2024

PubMed Abstract: The RNA-guided DNA endonuclease Cas9 cleaves double-stranded DNA targets complementary to an RNA guide, and is widely used as a powerful genome-editing tool. Here, we report the crystal structure of Brevibacillus laterosporus Cas9 (BlCas9, also known as BlatCas9), in complex with a guide RNA and its target DNA at 2.4-Å resolution. The structure reveals that the BlCas9 guide RNA adopts an unexpected architecture containing a triple-helix, which is specifically recognized by BlCas9, and that BlCas9 recognizes a unique NCNDN protospacer adjacent motif through base-specific interactions on both the target and non-target DNA strands. Based on the structure, we rationally engineered a BlCas9 variant that exhibits enhanced genome- and base-editing activities with an expanded target scope in human cells. This approach may further improve the performance of the enhanced BlCas9 variant to generate useful genome-editing tools that require only a single C PAM nucleotide and can be packaged into a single AAV vector for in vivo gene therapy.

PubMed: 38961195
DOI: 10.1038/s42003-024-06422-z

Experimental method

X-RAY DIFFRACTION (2 Å)