(450f) High Efficiency Multiplexed Cytosine Base Editing in Yarrowia Lipolytica

Metabolic engineering involves manipulating gene functions such as knockouts, knock-in, and overexpression to produce the desired target molecule. Knockouts can be performed by CRISPR-Cas9. However, multiple dsDNA breaks are toxic to the host. To alleviate this problem, we have developed an efficient base editor guided by Cas9 nickase in the oleaginous yeast Yarrowia lipolytica. We observed indel-free base editing when we used dead Cas9 fused to PmCDA1 or the KU70 strain. We increased the editing efficiency by optimizing gRNA expression by removing intragenic nucleotides between tRNA and gRNA, using nCas9(H840A) to nick the non-edited strand of DNA, integrating base editor, and optimizing cell culture conditions. We observed that the editing window, PAM preference, efficiency, and indel percentage vary depending upon the type of Cas9, strain and DNA strand targeted. We extended the editing to multiplexed base editing of 5 genes simultaneously. An optimized golden gate assembly was used to assemble guide RNAs targeting multiple genes without pre-cloning. tRNA arrays were constructed to express multiple gRNAs without requiring individual promoters. tRNA arrays were optimized with one PolIII promoter driving two or three gRNA arrays. Finally, we achieved highest efficiency by using CAN1 co-selection mediated editing to disrupt multiple genes. We also observed cytosine base editing by fusing cytosine deaminase to LbCpf1 to target A/T rich sequences. To perform base editing, we used deaminase fused to LbCpf1 with truncated gRNA or dead Cpf1 fused to gRNA.