Structure of the mini-RNA-guided endonuclease CRISPR-Cas12j3
© 2021. The Author(s)..
CRISPR-Cas12j is a recently identified family of miniaturized RNA-guided endonucleases from phages. These ribonucleoproteins provide a compact scaffold gathering all key activities of a genome editing tool. We provide the first structural insight into the Cas12j family by determining the cryoEM structure of Cas12j3/R-loop complex after DNA cleavage. The structure reveals the machinery for PAM recognition, hybrid assembly and DNA cleavage. The crRNA-DNA hybrid is directed to the stop domain that splits the hybrid, guiding the T-strand towards the catalytic site. The conserved RuvC insertion is anchored in the stop domain and interacts along the phosphate backbone of the crRNA in the hybrid. The assembly of a hybrid longer than 12-nt activates catalysis through key functional residues in the RuvC insertion. Our findings suggest why Cas12j unleashes unspecific ssDNA degradation after activation. A site-directed mutagenesis analysis supports the DNA cutting mechanism, providing new avenues to redesign CRISPR-Cas12j nucleases for genome editing.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2021 |
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| Erschienen: |
2021 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:12 |
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| Enthalten in: |
Nature communications - 12(2021), 1 vom: 22. Juli, Seite 4476 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Carabias, Arturo [VerfasserIn] |
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| Links: |
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| Themen: |
CRISPR-Associated Proteins |
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| Anmerkungen: |
Date Completed 03.08.2021 Date Revised 04.01.2024 published: Electronic Citation Status MEDLINE |
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| doi: |
10.1038/s41467-021-24707-3 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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NLM328396303 |
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| 520 | |a © 2021. The Author(s). | ||
| 520 | |a CRISPR-Cas12j is a recently identified family of miniaturized RNA-guided endonucleases from phages. These ribonucleoproteins provide a compact scaffold gathering all key activities of a genome editing tool. We provide the first structural insight into the Cas12j family by determining the cryoEM structure of Cas12j3/R-loop complex after DNA cleavage. The structure reveals the machinery for PAM recognition, hybrid assembly and DNA cleavage. The crRNA-DNA hybrid is directed to the stop domain that splits the hybrid, guiding the T-strand towards the catalytic site. The conserved RuvC insertion is anchored in the stop domain and interacts along the phosphate backbone of the crRNA in the hybrid. The assembly of a hybrid longer than 12-nt activates catalysis through key functional residues in the RuvC insertion. Our findings suggest why Cas12j unleashes unspecific ssDNA degradation after activation. A site-directed mutagenesis analysis supports the DNA cutting mechanism, providing new avenues to redesign CRISPR-Cas12j nucleases for genome editing | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 7 | |a CRISPR-Associated Proteins |2 NLM | |
| 650 | 7 | |a Escherichia coli Proteins |2 NLM | |
| 650 | 7 | |a RNA, Guide, CRISPR-Cas Systems |2 NLM | |
| 650 | 7 | |a RNA, Viral |2 NLM | |
| 650 | 7 | |a ruvC protein, E coli |2 NLM | |
| 650 | 7 | |a Endodeoxyribonucleases |2 NLM | |
| 650 | 7 | |a EC 3.1.- |2 NLM | |
| 700 | 1 | |a Fuglsang, Anders |e verfasserin |4 aut | |
| 700 | 1 | |a Temperini, Piero |e verfasserin |4 aut | |
| 700 | 1 | |a Pape, Tillmann |e verfasserin |4 aut | |
| 700 | 1 | |a Sofos, Nicholas |e verfasserin |4 aut | |
| 700 | 1 | |a Stella, Stefano |e verfasserin |4 aut | |
| 700 | 1 | |a Erlendsson, Simon |e verfasserin |4 aut | |
| 700 | 1 | |a Montoya, Guillermo |e verfasserin |4 aut | |
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