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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.

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