The invention discloses a
CRISPR / Cas9-mediated large
DNA fragment assembling method. Specifically, the invention provides a
nucleic acid construct capable of carrying out constitutive expression of Cas9 in
yeast; the
nucleic acid construct contains a
yeast Tef1
promoter operationally connected with a Cas9
gene, a replication origin from pBR322 and a screening mark thereof, and a replication region from CEN6ARS4 and a screening mark thereof; and the
nucleic acid construct is in the form of single-copy replication in the
yeast and in the form of high-copy replication in
Escherichia coli. The invention also provides a nucleic acid construct capable of carrying out constitutive expression of sgRNA in yeast, a nucleic acid construct used as a
receptor vector and
a DNA assembling method. According to the invention, two or more to-be-assembled large
DNA fragments are successfully assembled in microzyme to form a large
plasmid in
one shot; so low-efficiency in-vitro
digestion recovery, genetic transformation and the like of large
DNA fragments are avoided, and the method provided by the invention is more convenient and efficient compared with traditional methods.