Reasonable design of anti-CRISPR protein.

Introduction: CRISPR/Cas9 system is one of the most commonly used gene editing systems in experiments/clinicals.
CRISPR/Cas9 system is one of the most commonly used gene editing systems in experiments/clinicals, where SauCas9 is particularly widely used for its small size and easy loading.
background, how to regulate the activity of this gene editing tool accurately and effectively, and how to deal with the related off-target effect has become a hot research topic in this field.
, the study of the Acr protein (Anti-CRISPRproteins, a naturally occurring protein that is antagonists of the CRISPR/Cas9 gene editing effect) provides a viable direction for achieving these goals.
ACR proteins are used in a variety of ways to fight CRISPR, including competitive suppression of sgRNA loading (AcrIIC2), sgRNA and dsDNA hybridization (AcrIF1) ;PAM binding (AcrIIA4, AcrIIA2).
based on the functions of these ACR natural states, the modification is combined with protein engineering technology to achieve accurate regulation of CRISPR/Cas9 systems.
Recently, the Dominik team at Heidelberg University in Germany used the naturally occurring broad-spectrum Acr protein AcrIIC1 modification to obtain a protein that has a good regulatory effect on SauCas9, achieving precise regulation of the gene editing process mediated by SauCas9.
first used NmeCas9 as an example to explore ways to enhance the inhibitory effects of AcrIIC1.
Because AcrIIC1 can affect the activity of NmeCas9 nucleases by binding to the HNH domain of the NmeCas9 protein without interfering with DNA binding, the researchers suspect that using the "domaininsertion" method commonly used in protein engineering may be able to limit the composition/activity of AcrIIC1 and stabilize its HNH binding surface, thus enhancing inhibition.
by studying the HNH domain interoperability pattern between AcrIIC1 and NmeCas9, the researchers selected the loop5 structure located on the surface of AcrIIC1 and farthest from its HNH binding surface, and attempted to fuse the LOV2, mCherry, and PDZ domain structure fusions in this area.
further qualitative and quantitative experimental determination of the fusion gene editing ability confirmed that most AcrIIC1 fusions have stronger inhibition ability, of which Chimera.10 is the most significant.
after obtaining the AcrIIC1 fusion, which significantly enhanced the inhibition effect of NmeCas9, this study further studied the specific mechanism of the fusion inhibition NmeCas9 with Chimera.10 as an example.
By comparing the relative content of binding/unconsorted DNA and edited/uncliped nucleic acids, the researchers found that AcrIIC1 fusions also inhibited NmeCas9 by inhibiting nuclease activity without affecting DNA binding.
but this may also be related to an increase in the expression of AcrIIC1 fuses in cells.
same time, studies have found that AcrIIC1 can cause a decrease in in-cell expression in NmeCas9, which may be further associated with inhibition of gene editing mediated by NmeCas9.
After discovering a way to enhance the inhibitory effects of ACRIIC1 on NmeCas9, the researchers turned to the possibility of SauCas9 inhibition of ACRIIC1 pairs with NmeCas9 highly structurally ommosome HNH domains, thus providing an effective regulatory approach for clinical/laboratory gene editing.
researchers first demonstrated that AcrIIC1 was able to bind relatively weakly and inhibit gene editing mediated by SauCas9, and showed that one of the sources of this weak inhibition was the weakening of AcrIIC1's affinity with SauCas9.
this affinity difference, the researchers compared the structure of NmeCas9 with SauCas9, selected site points with significant differences in the HNH-AcrIIC1 interface (Site1, Site2) and The relevant residues on rIIC1 were bit mutations and combinations, and then the mutants were tested for gene clip activity, resulting in AcrIIC1X, a mutant with significant inhibitory effect on the gene clipping activity of SauCas9.
, however, further "domaininsertion" of AcrIIC1X did not significantly enhance its suppression of SauCas9, a phenomenon that partly explains the difference between SauCas9 and NmeCas9.
the study, the researchers used liver cell-specific miR-122 to design a SauCas9-edited molecular switch: the binding site of miR-122 was inserted into the 3' end of the ACRIIC1X gene and fed into the body with the CRISPR/Cas9 system.
makes it possible for Cas9 to function properly in cells with microRNA because the interference of miR-122 prevents ACRIIC1X expression, whereas in cells other than liver cells without miR-122, the gene editing mediated by SauCas9 is inhibited due to the expression of ACRIIC1X.
the study provides a means to regulate gene editing tools by transforming natural Acr proteins, making it possible to regulate the gene editing process precisely and efficiently.
addition, the idea of enhancing Acr inhibition through domaininsertion in experiments is also very instructive for other systems.
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