Structure and engineering of CRISPR-Cas9

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  • Eguma Ryuun
    Department of Biological Sciences, Graduate School of Science, The University of Tokyo
  • Nishimasu Hiroshi
    Department of Biological Sciences, Graduate School of Science, The University of Tokyo
  • Nureki Osamu
    Department of Biological Sciences, Graduate School of Science, The University of Tokyo

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Other Title
  • CRISPR-Cas9の立体構造と機能改変
  • CRISPR-Cas9 ノ リッタイ コウゾウ ト キノウ カイヘン

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<p>In the microbial type-II CRISPR-Cas （clustered regularly interspaced short palindromic repeat and CRISPR-associated）adaptive immune system, the RNA-guided DNA endonuclease Cas9 is responsible for the degradation of foreign genetic elements. Using dual RNA guides or a single chimeric RNA guide, Cas9 can recognize and cleave a double-stranded DNA target complementary to the RNA guide. Thus, Cas9 has been harnessed for numerous technologies, such as genome editing, epigenome editing and chromosome imaging. Recent structural studies provided mechanistic insights into the RNA-guided DNA recognition and cleavage by Cas9. The crystal structures revealed that Cas9 adopts a bilobed architecture, and recognizes the guide RNA-target DNA heteroduplex within the central channel between the two lobes in a non-sequence-specific manner, thereby explaining the RNA-guided DNA recognition by Cas9. In addition, the structures revealed that the HNH and RuvC endonuclease domains are located at positions suitable for cleaving the two strands in the unwound DNA target. Furthermore, the structural information facilitated the development of engineered Cas9 variants, such as those with enhanced cleavage fidelity or altered DNA-targeting specificities.</p>

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