Structural and Mechanistic Basis of PAM-Dependent Spacer …
2015年11月5日 · We report on the structures of Cas1-Cas2-dual-forked DNA complexes in an effort toward understanding how the protospacer is sampled prior to insertion into the CRISPR locus. Our study reveals a protospacer DNA comprising a 23-bp duplex bracketed by tyrosine residues, together with anchored flanking 3′ overhang segments.
Unraveling the mechanisms of PAMless DNA interrogation by …
2024年4月30日 · 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...
Bacteriophage λ exonuclease and a 5′-phosphorylated DNA guide allow PAM ...
2024年9月18日 · Using single-molecule fluorescence resonance energy transfer analysis, we discover the enzymatic activity of bacteriophage λ exonuclease (λExo). We show binding of 5′ …
DNB-based on-chip motif finding: A high-throughput method to …
Here, we report a sensitive DocMF system that uses next-generation sequencing chips to profile protein-DNA interactions. Using DocMF, we successfully identified a variety of endonuclease recognition sites and the protospacer adjacent motif (PAM) sequences of …
PAM recognition by miniature CRISPR-Cas12f nucleases triggers ...
2020年5月21日 · Here, we present a collection of 10 exceptionally compact (422-603 amino acids) CRISPR-Cas12f nucleases that recognize and cleave dsDNA in a PAM dependent manner. Categorized as class 2 type V-F, they originate from the previously identified Cas14 family and distantly related type V-U3 Cas proteins found in bacteria.
We show that AceCas9 contacts the 5′-NNNCC-3′ PAM by forming a network of hydrogen bonds with both the cytosine and guanine bases. Mutation of the 5′-NNNCC-3′ to 5′-NNNTC-3′ severely impaired...
PAM-Dependent Target DNA Recognition and Cleavage by …
2016年12月15日 · The PAM-proximal region of the heteroduplex interacts with the OBD, Helical-I, Helical-II, and RuvC domains (Figures 2E and S2). The +1 phosphate group between dG(−1) and dG1 of the target DNA strand is stabilized by the side chain of Arg507 and the main-chain amide group of Gly478 in the OBD domain, which facilitates the unwinding of +1 ...
Cas4 Nucleases Define the PAM, Length, and Orientation of
Shiimori et al. demonstrate integral roles of Cas4 nucleases in DNA acquisition for CRISPR-Cas immunity in P. furiosus. The concerted action of two different Cas4 nucleases is required for protospacer adjacent motif (PAM) definition, pre-spacer processing, and integration of DNA fragments at the CRISPR array in a functional orientation.
Bio-informatic analysis of CRISPR protospacer adjacent motifs …
In this work, we did investigate some of the possible reasons for CRISPR selection of PAM sequences, specifically, the gene essentiality and function, surface exposure of amino acids, and whether the conservation of regulatory sequences in T4 Phages promoters raises their chance of being chosen as PAMs in CRISPR-Cas.
DNA topology regulates PAM-Cas9 interaction and DNA …
2022年11月3日 · CRISPR-Cas9-mediated genome editing depends on PAM recognition to initiate DNA unwinding. PAM mutations can abolish Cas9 binding and prohibit editing. Here, we identified a Cas9 from the thermophile Alicyclobacillus tengchongensis for which the PAM interaction can be robustly regulated by DNA topology.