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TitleUnraveling the mechanisms of PAMless DNA interrogation by SpRY-Cas9.
Journal, issue, pagesNat Commun, Vol. 15, Issue 1, Page 3663, Year 2024
Publish dateApr 30, 2024
AuthorsGrace N Hibshman / Jack P K Bravo / Matthew M Hooper / Tyler L Dangerfield / Hongshan Zhang / Ilya J Finkelstein / Kenneth A Johnson / David W Taylor /
PubMed AbstractCRISPR-Cas9 is a powerful tool for genome editing, but the strict requirement for an NGG protospacer-adjacent motif (PAM) sequence immediately next to the DNA target limits the number of editable ...CRISPR-Cas9 is a powerful tool for genome editing, but the strict requirement for an NGG protospacer-adjacent motif (PAM) sequence immediately next to the DNA target limits the number of editable genes. Recently developed Cas9 variants have been engineered with relaxed PAM requirements, including SpG-Cas9 (SpG) and the nearly PAM-less SpRY-Cas9 (SpRY). However, the molecular mechanisms of how SpRY recognizes all potential PAM sequences remains unclear. Here, we combine structural and biochemical approaches to determine how SpRY interrogates DNA and recognizes target sites. Divergent PAM sequences can be accommodated through conformational flexibility within the PAM-interacting region, which facilitates tight binding to off-target DNA sequences. Nuclease activation occurs ~1000-fold slower than for Streptococcus pyogenes Cas9, enabling us to directly visualize multiple on-pathway intermediate states. Experiments with SpG position it as an intermediate enzyme between Cas9 and SpRY. Our findings shed light on the molecular mechanisms of PAMless genome editing.
External linksNat Commun / PubMed:38688943 / PubMed Central
MethodsEM (single particle)
Resolution2.79 - 3.69 Å
Structure data

EMDB-40681, PDB-8spq:
SpRY-Cas9:gRNA complex targeting TGG PAM DNA
Method: EM (single particle) / Resolution: 3.26 Å

EMDB-40705, PDB-8sqh:
SpRY-Cas9:gRNA complex targeting TTC PAM DNA
Method: EM (single particle) / Resolution: 3.69 Å

EMDB-40740, PDB-8srs:
SpRY-Cas9:gRNA complex targeting TAC PAM DNA
Method: EM (single particle) / Resolution: 2.79 Å

EMDB-41073, PDB-8t6o:
SpRY-Cas9:gRNA complex targeting TAC PAM DNA with 0 bp R-loop
Method: EM (single particle) / Resolution: 3.1 Å

EMDB-41074, PDB-8t6p:
SpRY-Cas9:gRNA complex targeting TAC PAM DNA with 2 bp R-loop
Method: EM (single particle) / Resolution: 3.15 Å

EMDB-41079, PDB-8t6s:
SpRY-Cas9:gRNA complex targeting TAC PAM DNA with 10 bp R-loop
Method: EM (single particle) / Resolution: 3.28 Å

EMDB-41080, PDB-8t6t:
SpRY-Cas9:gRNA complex targeting TAC PAM DNA with 13 bp R-loop
Method: EM (single particle) / Resolution: 3.04 Å

EMDB-41083, PDB-8t6x:
SpRY-Cas9:gRNA complex targeting TAC PAM DNA with 18 bp R-loop
Method: EM (single particle) / Resolution: 3.08 Å

EMDB-41084, PDB-8t6y:
SpRY-Cas9:gRNA complex bound to non-target DNA with 1 bp R-loop
Method: EM (single particle) / Resolution: 2.88 Å

EMDB-41085, PDB-8t76:
SpRY-Cas9:gRNA complex targeting TAC PAM DNA with 3 bp R-loop
Method: EM (single particle) / Resolution: 3.42 Å

EMDB-41086, PDB-8t77:
SpRY-Cas9:gRNA complex bound to non-target DNA with 6 bp R-loop
Method: EM (single particle) / Resolution: 3.25 Å

EMDB-41087, PDB-8t78:
SpRY-Cas9:gRNA complex bound to non-target DNA with 8 bp R-loop
Method: EM (single particle) / Resolution: 3.02 Å

EMDB-41088, PDB-8t79:
SpRY-Cas9:gRNA complex bound to non-target DNA with 10 bp R-loop
Method: EM (single particle) / Resolution: 3.04 Å

EMDB-41093, PDB-8t7s:
SpRYmer bound to NAC PAM DNA
Method: EM (single particle) / Resolution: 3.01 Å

EMDB-41775, PDB-8tzz:
SpG Cas9 with NGC PAM DNA target
Method: EM (single particle) / Resolution: 3.56 Å

EMDB-41867, PDB-8u3y:
SpG Cas9 with NGG PAM DNA target
Method: EM (single particle) / Resolution: 3.1 Å

Chemicals

ChemComp-MG:
Unknown entry

ChemComp-HOH:
WATER

Source
  • streptococcus pyogenes (bacteria)
  • streptococcus pyogenes serotype m1 (bacteria)
  • escherichia phage lambda (virus)
  • synthetic construct (others)
KeywordsIMMUNE SYSTEM / SpRY-Cas9 / CRISPR / Cas9 / IMMUNE SYSTEM/HYDROLASE / IMMUNE SYSTEM-HYDROLASE complex / PAM / R-loop / dimer / IMMUNE SYSTEM/RNA/DNA / SpG-Cas9 / IMMUNE SYSTEM-RNA-DNA complex / IMMUNE SYSTEM/DNA/RNA / IMMUNE SYSTEM-DNA-RNA complex

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