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CRISPR type II-A subgroups exhibit phylogenetically distinct mechanisms for prespacer insertion
- Source :
- J Biol Chem
- Publication Year :
- 2020
- Publisher :
- Elsevier BV, 2020.
-
Abstract
- CRISPR-Cas is an adaptive immune system that protects prokaryotes against foreign nucleic acids. Prokaryotes gain immunity by acquiring short pieces of the invading nucleic acid termed prespacers and inserting them into their CRISPR array. In type II-A systems, Cas1 and Cas2 proteins insert prespacers always at the leader–repeat junction of the CRISPR array. Among type II-A CRISPR systems, three distinct groups (G1, G2, and G3) exist according to the extent of DNA sequence conservation at the 3′ end of the leader. However, the mechanisms by which these conserved motifs interact with their cognate Cas1 and Cas2 proteins remain unclear. Here, we performed in vitro integration assays, finding that for G1 and G2, the insertion site is recognized through defined mechanisms, at least in members examined to date, whereas G3 exhibits no sequence-specific insertion. G1 first recognized a 12-bp sequence at the leader–repeat junction and performed leader-side insertion before proceeding to spacer-side insertion. G2 recognized the full repeat sequence and could perform independent leader-side or spacer-side insertions, although the leader-side insertion was faster than spacer-side. The prespacer morphology requirements for Cas1–Cas2 varied, with G1 stringently requiring a 5-nucleotide 3′ overhang and G2 being able to insert many forms of prespacers with variable efficiencies. These results highlight the intricacy of protein–DNA sequence interactions within the seemingly similar type II-A integration complexes and provide mechanistic insights into prespacer insertion. These interactions can be fine-tuned to expand the Cas1–Cas2 toolset for inserting small DNAs into diverse DNA targets.
- Subjects :
- 0301 basic medicine
CRISPR-Associated Proteins
Computational biology
Microbiology
Biochemistry
DNA sequencing
Insert (molecular biology)
03 medical and health sciences
chemistry.chemical_compound
CRISPR
Protein–DNA interaction
Molecular Biology
Phylogeny
Sequence (medicine)
Base Sequence
030102 biochemistry & molecular biology
biology
DNA
Cell Biology
Integrase
030104 developmental biology
chemistry
Nucleic acid
biology.protein
CRISPR-Cas Systems
Protein Binding
Subjects
Details
- ISSN :
- 00219258
- Volume :
- 295
- Database :
- OpenAIRE
- Journal :
- Journal of Biological Chemistry
- Accession number :
- edsair.doi.dedup.....f0664d8fbd1e721170bf7fb32dc54925