The invention relates to a method for rapidly obtaining a
CRISPR / Cas9 (clustered regularly interspersed short palindromic repeats / Cas9)
gene knockout
stable cell line through
monoclonal cell sorting and belongs to the technical field of
genetic engineering and genetic modification. According to the method, a
CRISPR / Cas9
system, single-
cell sorting by a flow cytometer and
fluorescent protein screening on an
expression vector are combined, positive
monoclonal cells can be obtained in short time, and
gene knockout work efficiency of the
cell line is greatly improved. Compared with a traditional cell line
gene knockout method, the flow cytometer is utilized for single-
cell sorting, on one hand, tedious work of antibiotic screening is omitted, so that a large quantity of single cells can be obtained in quite short time, and on the other hand, the condition that cells in each culture hole are single cells can be guaranteed and
false positive rate is reduced. Sorting is performed 40-80 h after cell
transfection, at the time point, the highest
survival rate of the cells after sorting can be guaranteed, and the screening efficiency is improved accordingly.