Design and function evaluation of cytosine base editors in Pichia pastoris

Abstract:

[Objective] A targeted mutation system was established in Pichia pastoris to provide an efficient editing tool for genetic engineering.[Methods] A cytosine base editor (CBE) was designed with nCas9 derived from the clustered regularly interspaced short palindromic repeats/Cas9 nuclease (CRISPR/Cas9) system and a cytosine deaminase PmCDA1. A cytosine-rich sequence in the yeast genome was selected as the target to validate the CBE activity. High-throughput sequencing was employed to analyze the editing efficiency and mode of the CBE. Furthermore, we explored how factors such as the length of linker peptides, the relative position of fusion proteins, and the length of gRNA targeting sequence affect the CBE function. [Results] The CBEs established by the fusion of nCas9 and PmCDA1 were capable of editing the cytosine in the P. pastoris genome. The highest editing efficiency was achieved with a linker peptide of (GGGGS)₁₀, and the editing window was located between C20 and C14 at the distal end of the protospacer adjacent motif. The window location at C18 achieved the maximum editing efficiency of 85.1%. Changes in the relative position of nCas9 and PmCDA1 had minor effects on the editing efficiency and modes of CBEs. However, the length of gRNA targeting sequence affected the editing efficiency of CBEs and cannot be shorter than 17 nt. The gRNA targeting sequences of 19-23 nt were capable of guiding CBEs to edit the genome. [Conclusion] This study establishes a set of CBEs with high editing efficiency in P. pastoris, which serves as a solid foundation for further genetic engineering.