The Establishment Of The CRISPR/Cas9 Gene Editing System In Rhodosporidium Parvum

Rhodosporidium toruloides is a typical oil-producing yeast,which can produce oil under the restriction of various nutrients,and can also be used to produce carotenoids.However,as a non-model strain,the convinent genetic tools for R.toruloides remains unavailable.This study aims to establish a CRISPR/Cas9 gene editing system for R.toruloides.Endogenous diacylglycerol acyltransferase gene was transformed into the strain Rt NP11 by Agrobacterium-mediated transformation and the changes of cell lipid content were compared and evaluated.On the basis of this transformation method,RNA polymerase III promoter U6 was cloned from the Rt NP11 genome and a tRNA^Glyly gene was identified from the Rt NP11 genome.The U6promoter and the tRNA^Gly gene were fused to form an artificial hybrid promoter U6-tRNAGly.A codon-optimized Cas9 gene was synthesized.The Cas9 expression cassette and the gRNA transcription cassette were connected to the plasmid framework by Gibson method.The key gene CRT1 in carotenoid biosynthesis pathway was used as the validation target for the CRISPR-Cas9 system.The 20 bp target gene sequence was introduced into the vector by PCR amplification,and the expression vector was constructed and transferred into Rt NP11 to get an engineered strain Rt NP11-U6-tRNAGly Target 1.In order to improve the efficiency of gene editing,the gRNA promoter was optimized first to obtain the transformed strains Rt NP11-SNR52-tRNAGly Target 1,Rt NP11-SCR1-tRNAGly Target 1,and Rt NP11-SCR1-tRNALys-tRNAGly Target 1.Then,new target sequences were designed on the CRT1 gene to obtain the transformed strains Rt NP11-SNR52-tRNAGly Target2,Rt NP11-SNR52-tRNAGly Target 3,Rt NP11-SCR1-tRNAGly Target 2,Rt NP11-SCR1-tRNAGly Target 3,Rt NP11-U6-tRNAGly Target 2,and Rt NP11-U6-tRNAGly Target 3.The gene mutation was analyzed by phenotypic observation and DNA sequencing.Agrobacterium-mediated transformation can effectively transform Rt NP11.Overexpression of DGAT2 can increase cell lipid content by 50%.Deletion and insertion of nucleotides near the site of cleavage from CRISPR-Cas9 editing was observed.Promoters affected the efficiency of gene editing.The gene editing efficiency of SCR1-tRNAGly was 20%.However,changing the target sequence did not improve the efficiency of gene editing.The CRISPR/Cas9gene editing system established in this study will facilitate the genetic manipulation of R.toruloides.