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TitleA resurrected ancestor of Cas12a expands target access and substrate recognition for nucleic acid editing and detection.
Journal, issue, pagesNat Biotechnol, Vol. 43, Issue 10, Page 1663-1672, Year 2025
Publish dateOct 31, 2024
AuthorsYlenia Jabalera / Igor Tascón / Sara Samperio / Jorge P López-Alonso / Monika Gonzalez-Lopez / Ana M Aransay / Guillermo Abascal-Palacios / Chase L Beisel / Iban Ubarretxena-Belandia / Raul Perez-Jimenez /
PubMed AbstractThe properties of Cas12a nucleases constrict the range of accessible targets and their applications. In this study, we applied ancestral sequence reconstruction (ASR) to a set of Cas12a orthologs ...The properties of Cas12a nucleases constrict the range of accessible targets and their applications. In this study, we applied ancestral sequence reconstruction (ASR) to a set of Cas12a orthologs from hydrobacteria to reconstruct a common ancestor, ReChb, characterized by near-PAMless targeting and the recognition of diverse nucleic acid activators and collateral substrates. ReChb shares 53% sequence identity with the closest Cas12a ortholog but no longer requires a T-rich PAM and can achieve genome editing in human cells at sites inaccessible to the natural FnCas12a or the engineered and PAM-flexible enAsCas12a. Furthermore, ReChb can be triggered not only by double-stranded DNA but also by single-stranded RNA and DNA targets, leading to non-specific collateral cleavage of all three nucleic acid substrates with similar efficiencies. Finally, tertiary and quaternary structures of ReChb obtained by cryogenic electron microscopy reveal the molecular details underlying its expanded biophysical activities. Overall, ReChb expands the application space of Cas12a nucleases and underscores the potential of ASR for enhancing CRISPR technologies.
External linksNat Biotechnol / PubMed:39482449
MethodsEM (single particle)
Resolution3.08 - 3.75 Å
Structure data

18691

8qwd

EMDB-18691, PDB-8qwd:
Apo ReChb
Method: EM (single particle) / Resolution: 3.33 Å

EMDB-18692: Apo ReChb , Rec1 improved
Method: EM (single particle) / Resolution: 3.75 Å

18693

8qwe

EMDB-18693, PDB-8qwe:
Ternary complex of ReChb - crRNA - target dsDNA
Method: EM (single particle) / Resolution: 3.14 Å

18694

8qwf

EMDB-18694: Quaternary complex of ReChb - crRNA - target dsDNA - collateral dsDNA
PDB-8qwf: ReChb - crRNA - target dsDNA complex in the presence of collateral dsDNA
Method: EM (single particle) / Resolution: 3.08 Å

Chemicals

ChemComp-MG:
Unknown entry

ChemComp-HOH:
WATER

Source
  • synthetic construct (others)
KeywordsBIOSYNTHETIC PROTEIN / Cas / cryo-EM / ancestral sequence reconstruction / biotechnology / DNA / RNA

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