The invention relates to a special safe and efficient
CRISPR / Cas (Clustered Regulatory Interspaced Short Palindromic Repeats /
CRISPR associated) 9
gene editing
system for
Kluyveromyces optimization, and belongs to the field of
biotechnology. In the prior art, pCAS plasmids used in
saccharomyces cerevisiae have Cas9
gene sequences and gRNA (
guide RNA) elements at the same time, and efficient genomemodification in the
saccharomyces cerevisiae can be realized through one-
time transformation, but stable
copying and expression cannot be realized in
Kluyveromyces. According to the
system, Cas9 / gRNAfusion plasmids are transformed in
Kluyveromyces cells, the plasmids are targetedly delivered to endogenous
DNA sequences of the Kluyveromyces cells, and
double strand breaks are generated; and donorDNA sequences are transformed in the Kluyveromyces cells, the sequences generate
homologous recombination at the
double strand breaks and target sites, and Tag sequences are inserted in the target sites. Through the modification, the new safe and efficient
CRISPR / Cas9
gene editing
system which is specially used for the Kluyveromyces and can perform stable
copying and expression in the Kluyveromyces and perform
gene modification is established.