Singapore Researchers Design New C to G Base Editor to Correct Disease-Associated Mutations
A single nucleotide/base change in some genes can cause a genetic disease. An attractive therapeutic strategy would be the ability to reverse this single nucleotide. CRISPR has emerged as a powerful gene-editing tool to routinely edit DNA sequences and disrupt genes by adding or deleting DNA with minimal side effects. However, current base editors that can edit are not very good at effecting single base editing.
Two common base editors- CBEs (Cytidine Base Editors) and ABEs (Adenine Base Editors) are used to convert a C:G base pair to A:T and A:T base pair to C:G, respectively. These are widely studied and optimized since about 50% of disease-associated SNPs can be targeted with CBEs and ABEs. However, there are no robust editors to edit a C to G or G to C. Such an editor would enable us to develop therapies for diseases such as cystic fibrosis, sickle cell anemia, and a range of cardiovascular, neurological, and musculoskeletal diseases, which make up about 40% of genetic diseases with an SNP of C:G or G:C.
New Class of Base Editors
In a new study published in Nature Communications, a team of scientists from the Genome Institute of Singapore (GIS) report a new class of base editors- CGBEs (C to G Base Editor), which can specifically change a C to G or G to C. Using the endogenous base excision repair (BER) pathway in mammalian cells and linking it to nCas9 from a base editor which contains uracil DNA glycosylase, the authors developed a CGBE with CBE containing nCas9 fused with DNA ligase 3 and other components of the BER which can convert C to G.
Using this design, they developed 7 candidates with different orientations of the fusion of Cas9 with BER proteins and tested their C:G and G:C editing efficiency in HEK2 and HEK3 loci in HEK293 cells. Sequencing the loci showed that CGBE candidates indeed performed better in editing C:G to G:C compared to other editors with 13% efficiency against 4% from control editors. This efficiency was further improved in other genomic sites with up to 24% editing efficiency.
Next, the authors tested the CGBEs in disease-associated genomic sites and found that some tweaking with the editor design (removing the uracil glycosylase inhibitor) and analysis into the sequence context could improve C:G to G:C editing to about 16% efficiency in human cells within a three-nucleotide window. Off-target editing for CGBEs was lower compared to control BE3 editors. The CGBEs performed better in eHAP, HTB9 cell lines but were less efficient in human stem cells, suggesting their architecture needs to be optimized based on cell lines.
In a remarkable improvement on current base editors, the team has managed to expand the repertoire of editors available for genome editing. However, the authors concede that a detailed analysis of off-target effects and optimization of editing efficiency is required to translate the CGBEs to therapeutic strategies.
In a press release, Dr. Chew Wei Leong, Senior Research Scientist at GIS, said, “The CGBE gene editor is a ground-breaking invention that for the first time, directly converts C to G in genes, which potentially opens up treatment avenues for a substantial fraction of genetic disorders associated with single-nucleotide mutations.”
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