Although single nucleotide variants (SNVs) are the most common genetic alteration associated with human diseases and the driver for protein functional evolution, an efficient method is still lacking to create and screen a large cohort of functional SNVs in mammalian cells. Almost all the existing technologies for genetic screening, including CRISPR, CRISPR interference, and shRNAs, require disruption of a gene or alteration of its expression. None of these methods is able to introduce de novo functions to a protein, which requires amino acid substitutions. Thus, our understanding of SNVs and their functional significance to human diseases are still very limited. Previous strategies using CRISPR to generate point mutations rely on the creation of double-strand DNA breaks (DSB) and subsequent homologous recombination (HR). Given the low efficiency of HR, it’s unpractical to create a large cohort of point mutations in vivo suitable for screening.
In this study, a team led by Dr. CHANG Xing from the Institute of Health Sciences (IHS), Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences
describes TAM, a novel genetic diversification method that directs somatic hypermutation to a given genomic locus by fusing AID (a cytidine deaminase) with catalytic inactive Cas9 protein.
Somatic hypermutation is a unique molecular mechanism in B cells to diversify immunoglobulin proteins in order to cope with the extremely diversified pathogens from environment. Guided by sgRNAs, dCas9-AID fusion protein enables programmable genetic diversification at the sgRNA-targeted loci, providing a novel forward genetic approach to study functional SNVs and facilitate protein evolution in a dish. Fused with dCas9, AID directly subverts cytidines targeted by sgRNAs into the other three bases (Thymidine, Guanosine, Adenosine), creating a large repertoire of sequence variants at desired loci and eliminating the need to create DSB and invoke HR.
Moreover, combined with a uracil DNA glycosylases inhibitor, dCas9-AIDx efficiently converts cytidines specifically to thymidines within a narrow window, allowing precise correction (or recreation) of a SNV for in-depth analysis.
Thus, dCas9-AIDx is highly efficient for high-complexity screening of functional variants, which is demonstrated by a screening to identify Imatinib-resistant mutations in the ABL kinase within 4-5 weeks.
Guided by sgRNAs, dCas9-AID fusion protein efficiently converts cytosines and guanines in the targeted region to the other three bases, thus enabling programmable genetic diversification at the sgRNA-targeted loci. (Image provided by Dr. CHANG Xing's group)
Prof. CHANG Xing
Institute of Health sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Shanghai Jiao Tong University School of Medicine,
Shanghai 200031, China.