DNA interference states of the hypercompact CRISPR–CasΦ effector
| Autor: | Jillian F. Banfield, Dominik A Herbst, Eva Nogales, Connor A. Tsuchida, Katarzyna M. Soczek, Jennifer A. Doudna, Patrick Pausch, Basem Al-Shayeb |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
Gene Editing
Chemistry Effector CRISPR-Associated Proteins Mutant Molecular Conformation RNA DNA Computational biology Protein engineering Article DNA-Binding Proteins chemistry.chemical_compound Genetic Techniques Genome editing Structural Biology Cleave CRISPR Bacteriophages Clustered Regularly Interspaced Short Palindromic Repeats CRISPR-Cas Systems DNA Cleavage Molecular Biology RNA Guide Kinetoplastida |
| Zdroj: | Nat Struct Mol Biol |
| ISSN: | 1545-9985 1545-9993 |
| DOI: | 10.1038/s41594-021-00632-3 |
| Popis: | CRISPR-CasΦ, a small RNA-guided enzyme found uniquely in bacteriophages, achieves programmable DNA cutting as well as genome editing. To investigate how the hypercompact enzyme recognizes and cleaves double-stranded DNA, we determined cryo-EM structures of CasΦ (Cas12j) in pre- and post-DNA-binding states. The structures reveal a streamlined protein architecture that tightly encircles the CRISPR RNA and DNA target to capture, unwind and cleave DNA. Comparison of the pre- and post-DNA-binding states reveals how the protein rearranges for DNA cleavage upon target recognition. On the basis of these structures, we created and tested mutant forms of CasΦ that cut DNA up to 20-fold faster relative to wild type, showing how this system may be naturally attenuated to improve the fidelity of DNA interference. The structural and mechanistic insights into how CasΦ binds and cleaves DNA should allow for protein engineering for both in vitro diagnostics and genome editing. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full text from SpringerLink