NEW YORK – Researchers in the US and China have developed a new, more precise cytosine base editor (CBE) that eliminates the problem of bystander mutations, while increasing the percentage of perfectly modified alleles more than 6,000-fold for disease correction and more than 600-fold for disease modeling compared with the currently available base editor, BE4max.
In a study published on Wednesday in Science Advances, the investigators said that although CBEs enable efficient cytidine-to-thymidine (C-to-T) substitutions at targeted loci without double-stranded breaks, current CBEs edit all Cs within their activity windows, generating undesired bystander mutations. When a bystander C is adjacent to the target C, existing base editors edit both Cs.
To improve the precision of CBE, the researchers engineered the human APOBEC3G (A3G) deaminase into three variants and fused them to a Cas9 nickase. The resulting A3G-BEs exhibited selective editing of the second C in the 5'-CC-3' motif in human cells to install a single disease-associated C-to-T substitution with high precision.
Various APOBEC enzymes in vertebrates mediate defense against infections from retroviruses or retrotransposons by deaminating C to U in the viral complementary DNA, suggesting that these cytosine deaminases could have unique preferences for particular sequence motifs to distinguish DNA sequences from the native host. The researchers identified human APOBEC3G (A3G) as a candidate for developing sequence-specific BEs in multiple C contexts. They characterized and engineered A3G-BE variants to efficiently edit a single C at various endogenous sites in HEK293T cells, ending up with three novel variants — A3G-BE4.4, A3G-BE5.13, and A3G-BE5.14 — that exhibited high editing efficiencies and precision in the context of the CC motif.
Further analyses showed that while A3G-BE4.4, A3G-BE5.13, and A3G-BE5.14 all selectively edited the second C within the CC motifs across all the sites the researchers tested, A3G-BE4.4 displayed a preference for a narrower editing window than the other two variants. Further testing revealed consistent and broad base-editing activity window for all three variants, but they differed mainly in their relative editing efficiencies — A3G-BE5.13 was the most efficient, followed by A3G-BE5.14 and A3G-BE4.4, the researchers said.
To test A3G-BEs in disease-relevant contexts, they then sought to precisely generate SNPs of reported human pathogenic diseases. The investigators selected three genetic variants caused by C-to-T substitution in which the wild-type sequences lie within the preferential 5′-CC-3′ motif of A3G-BEs, including cystic fibrosis, hypertonic myopathy, and transthyretin amyloidosis. They constructed individual sgRNAs targeted to these disease-associated sites, and cotransfected them into HEK293T cells with BE4max, A3G-BE4.4, A3G-BE5.13, and A3G-BE5.14.
Direct comparison with BE4max of the modified allele frequencies demonstrated that A3G-BEs induced a substantially higher proportion of perfectly modified alleles for all three models and that A3G-BE5.13 achieved the highest percentage of perfectly modified alleles for hypertonic myopathy (36 percent). For transthyretin amyloidosis, all A3G-BEs produced the desired allele with high efficiencies (more than 35 percent), while BE4max failed to edit the target C because of its inability to edit outside its activity window, the researchers said. As a result, A3G-BE5.14 accomplished 613-fold higher correct modeling of transthyretin amyloidosis than BE4max did. Similarly, for cystic fibrosis, all A3G-BEs induced more than 50 percent of the perfectly modified alleles, while BE4max averaged 0.6 percent.
The investigators also set out to examine the therapeutic applicability of the A3G-BEs. They selected three reported human pathogenic SNPs caused by T>C mutations, which can be preferentially targeted by A3G-BEs, including hereditary pyropoikilocytosis, cystic fibrosis, and holocarboxylase synthetase deficiency. They generated three HEK293T cell lines containing 200 base pairs of each disease-relevant sequence integrated into the genome, and codelivered the BEs and sgRNAs targeted to the disease-associated sites.
Their analyses showed that all A3G-BEs significantly outperformed BE4max by a minimum of threefold in correcting hereditary pyropoikilocytosis and cystic fibrosis. In addition, A3G-BE4.4 exclusively induced more than 50 percent of perfectly corrected alleles among other BEs and accomplished 6,496-fold higher correction than BE4max in holocarboxylase synthetase deficiency.
"We identified 540 human pathogenic single nucleotide polymorphisms that could be precisely correctable by our A3G-BEs. [They] also appears to decrease off-target edits at both the DNA and RNA levels," senior author and Rice University biomolecular engineer Xue Gao said in a statement. "There are three billion base pairs in humans. I believe this technology's level of precision is going to be a significant contributor toward treating genetic disease."