The cause of on-target point mutations generated by CRISPR-Cas9 treatment in the yeast Xanthophyllomyces dendrorhous

Abstract

Recognizing outcomes of DNA repair induced by CRISPR-Cas9 cutting is vital for precise genome editing. Reported DNA repair outcomes after Cas9 cutting include deletions/insertions and low frequency of genomic rearrangements and nucleotide substitutions. Thus far, substitution mutations caused by CRISPR-Cas9 has not attracted much attention. Here, we identified on-target point mutations induced by CRISPR-Cas9 treatment in the yeast Xanthophyllomyces dendrorhous by Sanger and Illumina sequencing. Different from previous studies, our findings suggested that the on-target mutations are not random and they cannot render the gRNA effective. Moreover, these point mutations showed strong sequence dependence that is not consistent with the observations in Hela cells, in which CRISPR-mediated substitutions were considered lacking sequence dependence and conversion preferences. Furthermore, this study demonstrated that the NHEJ components Ku70, Ku80, Mre11, or RAD50, and the overlapping roles of non-essential DNA polymerases were necessary for the emergence of point mutations, increasing the knowledge on CRISPR-Cas9 mediated DNA repair.

Generation of stable homozygous transformants of diploid yeasts such as Xanthophyllomyces dendrorhous

“…This technique has been used for integration of carotenogenic genes into Yarrowia lipolytica (Schwartz et al 2017). A first attempt applying this technique to X. dendrorhous has been successful (Hong et al 2021). Future developments may demonstrate its potential in pathway modifications of X. dendrorhous and other diploid yeasts.…”

The biological principles and advanced applications of DSB repair in CRISPR-mediated yeast genome editing

“…lipolytica – – AtoB, HMGR, HMGS α-farnesene production [ 80 ] Y . lipolytica CRISPR–Cas9, gRNA Random integration: 1.6 × 10 4 colonies/μg DNA Leu2 Enhance production of lipase and β‐carotene [ 81 ] Xanthophyllomyces dendrorhous CRISPR–Cas9, gRNA 64.3% of point mutants, 23.8 of deletion, 4.8% of chromosome rearrangement CrtE and CrtS – [ 89 , 90 ] …”

“…dendrorhous, were selected as target genes, and 283 color-identified mutants from 623,727 colonies were sent for Sanger sequencing. Among these mutants, 64.3% showed point mutations, 23.8% showed deletions, and 4.8% showed chromosome rearrangements [ 90 ].…”