Most genome-editing methods rely on delivery of exogenous plasmid or viral DNA into cells to express the necessary editing tools intracellularly. However, since the expression is uncontrollable once the plasmid is expressed, sometimes the produced nucleases like Cas9 and sgRNA will lead to the excessive off-target editing after the on-target editing.
To solve this problem, cytidine deaminase was fused to the amino terminus of dCas9 nuclease. The complex can not only retain the ability of cytidine deaminase, but also don’t induce dsDNA breaks because of the inactivation of Cas9. When the complex is expressed, it can still bind to the target loci by following the sgRNA. When targeted, the fused cytidine deaminase will be able to mediate the direct conversion of cytidine to uridine, thereby effecting a C to T (or G to A) substitution. By introducing an uracil glycosylase inhibitor that inhibits base excision repair, and a nickase to manipulate cellular mismatch repair into replacing the G-containing DNA strand, the combination enables efficient and permanent C to T conversion with minimal indel formation.
When binding with the target DNA, Cas9 complex not only shows specific interactions(sgRNA: DNA strand), but also nonspecific interactions(residues N497, R661, Q695 and Q926). By changing these four amino acids into alanine which only has CH3 on the side chain, the nonspecific interaction between the backbone of the target DNA strand and the dCas9 was eliminated.
When comparing the on-target efficiency, even though HF-BE3 showed slightly reduced on-target editing compared with BE3(WT), the absolute value of off-target editing of HF-BE3 was reduced significantly. Both of these changes come from the elimination of unspecific interaction between dCas9 and the target DNA strand. It also proves that those four mutations modestly affect the binding between sgRNA and target DNA strand.
As stated above, gene editing is uncontrollable when it is accomplished with the transfection method because the gene expression on the plasmid is uncontrolled. To solve this problem, and edit the target gene quantitatively, deliver the protein into the cell is necessary. By engineering the target protein with a polyanionic molecule, the protein can be delivered by cationic lipid reagents. The lipid bilayer of the vehicle protects the protein from degradation and can prevent neutralization by antibodies.
By employing this delivery method, both BE3(WT) and HF-BE3 complexed with sgRNA can be transferred into the mammalian cells successfully and complete the gene editing task. Since HF-BE3 is less stable in cell, and there is no continuous expression of new protein produced, the on-target editing efficiency of HF-BE3 is reduced because of the degradation. Another advantage of the protein delivery method, compared with plasmid, is that it also increases the editing specificity that is independent of dosage.
Compared with HF-Cas9 gene editing system, the HF-BE3 showed higher specificity. That’s probably because that base editing, unlike Cas9-mediated indel formation, does not require DNA cleavage but only necessitates DNA-binding and R-loop formation. Therefore, in gene editing area, protein delivery of HF-BE3 like base editors can generate specific and precise modifications to genomic DNA without requiring exogenous DNA.
Last but not least, compared with dCas9-activator which can boost the expression of gene by recruiting more mediation proteins or domains that bind with RNA polymerase, the fusion between deaminase and dCas9 is another example about the application of dCas9 platform.
2017-12-19-1513661698