Target-AID base editor used in fungi
Since its beginnings, CRISPR technology was used mainly to disrupt genes – to create so-called knock-outs. “Precise editions” were usually random insertions and deletions, which could move reading frame of the gene and lead to expression of completely different, nonfunctional protein. Later developments introduced CRISPR base editors, allowing to truly precise edit base pairs in the genome, paving the new way to knock out genes – by converting existing amino acid codons to stop codons, essentially cutting short the protein.
New work uses base editor called Target-AID to introduce stop codons by converting cytosine (C) to thymine (T). Codons such as CAA (glutamine), CAG (glutamine), CGA (arginine) were converted to TAA, TAG, TGA (all of them – stop codons), respectively. That intervention had to happen in very narrow window, that is from -20 to -15 bases from PAM recognized by the base editor. In effect, only three genes were targeted in Yarrowia lipolytica: TRP1, PEX10, HIS3.
Scientists achieved high efficiency of disruption in two cases, up to 94% for TRP1 and up to 84% for PEX10. However, they were not so successful with HIS3, reaching 0-3% efficiency.
Mechanism of base editing was more efficient after deletion of KU70 gene, which is involved in one of the DNA repair pathways (NHEJ).
The study was motivated by issues with previous genetic engineering attempts in Y. lipolytica. As authors concluded, they provided genetic engineering landscape for the organism:
It is a simpler method than traditional genome-editing methods currently being used in Y. lipolytica.
Publication: Bae, S., Park, B. G., Kim, B., & Hahn, J. (2019). Multiplex Gene Disruption by Targeted Base Editing of Yarrowia lipolytica Genome Using Cytidine Deaminase Combined with the CRISPR/Cas9 System. Biotechnology Journal, 1900238. doi:10.1002/biot.201900238
Photo: Trinh lab, University of Tennessee, Knoxville