Targeted C-to-T and A-to-G dual mutagenesis system for RhtA transporter in vivo evolution.

T7 RNA polymerase (T7RNAP) has been fused with cytosine or adenine deaminase individually, enabling in vivo C-to-T or A-to-G transitions on DNA sequence downstream of T7 promoter, and greatly accelerated directed protein evolution. However, its base conversion type is limited. In this study, we created a dual-functional system for simultaneous C-to-T and A-to-G in vivo mutagenesis, called T7-DualMuta, by fusing T7RNAP with both cytidine deaminase (PmCDA1) and a highly active adenine deaminase (TadA-8e). The C-to-T and A-to-G mutagenesis frequencies of T7-DualMuta were 4.02 × 10-3 and 1.20 × 10-2, respectively, with 24 h culturing and distributed mutations evenly across the target gene. The T7-DualMuta system was used to in vivo directed evolution of L-homoserine transporter RhtA, resulting in efficient variants that carried the four types of base conversions by T7-DualMuta. The evolved variants greatly increased the host growth rates at L-homoserine concentrations of 8 g/L, which was not previously achieved, and demonstrated the great in vivo evolution capacity. The novel molecular device T7-DualMuta efficiently provides both C/G-to-T/A and A/T-to-G/C mutagenesis on target regions, making it useful for various applications and research in Enzymology and Synthetic Biology studies. It also represents an important expansion of the base editing toolbox.ImportanceA T7-DualMuta system for simultaneous C-to-T and A-to-G in vivo mutagenesis was created. The mutagenesis frequency was 4.02 × 107 fold higher than the spontaneous mutation, which was reported to be approximately 10-10 bases per nucleotide per generation. This mutant system can be utilized for various applications and research in Enzymology and Synthetic Biology studies.

Glycosylase-based base editors for efficient T-to-G and C-to-G editing in mammalian cells.

Base editors show promise for treating human genetic diseases, but most current systems use deaminases, which cause off-target effects and are limited in editing type. In this study, we constructed deaminase-free base editors for cytosine (DAF-CBE) and thymine (DAF-TBE), which contain only a cytosine-DNA or a thymine-DNA glycosylase (CDG/TDG) variant, respectively, tethered to a Cas9 nickase. Multiple rounds of mutagenesis by directed evolution in Escherichia coli generated two variants with enhanced base-converting activity-CDG-nCas9 and TDG-nCas9-with efficiencies of up to 58.7% for C-to-A and 54.3% for T-to-A. DAF-BEs achieve C-to-G/T-to-G editing in mammalian cells with minimal Cas9-dependent and Cas9-independent off-target effects as well as minimal RNA off-target effects. Additional engineering resulted in DAF-CBE2/DAF-TBE2, which exhibit altered editing windows from the 5' end to the middle of the protospacer and increased C-to-G/T-to-G editing efficiency of 3.5-fold and 1.2-fold, respectively. Compared to prime editing or CGBEs, DAF-BEs expand conversion types of base editors with similar efficiencies, smaller sizes and lower off-target effects.