Genome Editing by Targeted Deaminase

DNA deamination is the process by which DNA nucleotides are deaminated and turn into other type of nucleotides (ex. cytosine to uracil). Although most deaminated nucleotides are repaired properly, excess deamination or compromised repair may results in mutagenesis. The activation-induced cytidine deaminase (AID, also known as AICDA) is responsible for generating genetic variation at the immunoglobulin locus, an essential process for the adaptive immune system in vertebrates, although precise mechanism by which AID activity is specifically localized remains elusive.

By tethering the DNA deaminase activity to nuclease-deficient CRISPR/Cas9 system, we have developed a genome editing tool that enable targeted point mutagenesis. An AID ortholog PmCDA1 from sea lamprey was attached to nuclease-deficient mutant of Cas9 (D10A and H840A) as a fusion protein or by protein-protein interaction. This hybrid system, termed Target-AID, induced cytosine point mutation in 3-5 bases range at the distal site within target sequence. Major mutations were C to T point mutations, while C to G was also observed at substantial rate in yeast. Use of nickase Cas9 (D10A), which retains single-strand cleaving activity, greatly increase the efficiency, although it also occasionally induce insertion/deletion (indel) in animal and plant cells. Uracil-DNA glycosylase inhibitor (UGI) further increased the efficiency and reduced the indel formation. The toxicity associated with the nuclease activity has been greatly diminished, enabling application of this technique to wider range of organisms. Direct introduction of herbicide-tolerant mutation was performed in rice. Multiplex editing in combination with the herbicide-tolerant mutagenesis enabled leveraging selection of simultaneously edited plants.