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CIRSPR/Cas9 is a technology that uses Cas9 nuclease to specifically recognize and cleave target DNA under the guidance of a single guide RNA (gRNA)
.
This process involves the non-specific binding of Cas9/gRNA to double-stranded DNA (dsDNA), searching for and identifying PAM sites on dsDNA, and forming RNA/DNA heteroduplexes through homologous matches upstream of PAM to achieve on dsDNA.
On August 20, 2021, Chen Chunlai and Liu Junjie’s research group from the School of Life Sciences of Tsinghua University jointly published a titled "Non-specific interaction between spCas9 and PAM downstream DNA sites to accelerate its target" in Chemical Science.
Search process (Nonspecific interactions between SpCas9 and dsDNA sites located downstream of the PAM mediate facilitated diffusion to accelerate target search)" research paper
.
The paper reveals that under physiological salt concentration, spCas9 uses three-dimensional and one-dimensional diffusion to achieve efficient target search.
Combining biochemical experiments and single-molecule fluorescence technology, the study found that under physiological salt concentration, spCas9 uses three-dimensional and one-dimensional diffusion to quickly search for targets
.
The one-dimensional diffusion of spCas9 shows an asymmetric search area (from the upstream of PAM ~ 10 bp to the downstream of PAM ~ 30 bp) centered on the PAM site, which increases the target search efficiency by more than 10 times
Finally, the study proposed a detailed molecular model of the spCas9 search target (Figure 1): At physiological salt concentrations, the freely diffusing Cas9/gRNA complex in the solution interacts with dsDNA through three-dimensional random collisions
.
Once the lysine residues 1151-1156 non-specifically interact with dsDNA, spCas9 will instantaneously bind and perform a one-dimensional diffusion of ~ 20 bp along the dsDNA double helix to find PAM sites
Researcher Chen Chunlai and Researcher Liu Junjie from the School of Life Sciences of Tsinghua University are the co-corresponding authors of this article
.
Yang Mengyi, a PhD student who has graduated from the CLS program of the School of Life Sciences, Tsinghua University (now a research intern in the Nutrition and Development Laboratory of Beijing Children's Hospital, Capital Medical University), is the first author of this article
Paper link:
https://doi.
Links to related papers of the research group:
https://doi.
https://doi.
https://doi.
