Obtaining the best igRNAs for bystander-less correction of all ABE-reversible pathogenic SNVs using high-throughput...

Imperfect -gRNA (igRNA) provides a simple strategy for single-base editing of a base editor. However, a significant number of igRNAs need to be generated and tested for each target locus to achieve efficient single-base reversion of pathogenic single nucleotide variations (SNVs), which hinders the direct application of this technology. To provide ready-to-use igRNAs for single-base and bystander-less correction of all the adenine base editor (ABE)-reversible pathogenic SNVs, we employed a high-throughput method to edit all 5,253 known ABE-reversible pathogenic SNVs, each with multiple systematically designed igRNAs, and two libraries of 96,000 igRNAs were tested. A total of 1,988 SNV loci could be single-base reversed by igRNA with a >30% efficiency. Among these 1,988 loci, 378 SNV loci exhibited an efficiency of more than 90%. At the same time, the bystander editing efficiency of 76.62% of the SNV loci was reduced to 0%, while remaining below 1% for another 18.93% of the loci. These ready-to-use igRNAs provided the best solutions for a substantial portion of the 4,657 pathogenic/likely pathogenic SNVs. In this work, we overcame one of the most significant obstacles of base editors and provide a ready-to-use platform for the genetic treatment of diseases caused by ABE-reversible SNVs.