The aim of this study is to add bio-barcode in cells by CRISPR/Cas9 for differentially marking the cells. The bio-barcode sequence,conditional induced Cas9 sequence and the corresponding sgRNA sequence were integrated into the cell through the PB enzyme, and then the cell markers were analyzed by sequencing the bio-barcode after inducing the expression of Cas9. The designed bio-barcode sequence contained 6 sgRNA target sites overlapping each other,which allowed the bio-barcode sequence to be spliced only once by Cas9. As results,the designed bio-barcode sequence in N2a cells after Dox induction was efficiently spliced by Cas9,and there were 9 different genotypes among 9 clones. After Cre inducing the E14 embryonic stem cells containing Cas9 sequence regulated by loxp-stop-loxp and bio-barcode sequence,monoclonal sequencing showed that only 2 monoclonal cells of 21 cell lines remained the original genotype,and the rest 19 strains presented 18 different genotypes respectively. In summary,this study established a novel approach to spatio-temporally add bio-barcode markers with high specificity and relative stability into cells.%旨在利用CRISPR/Cas9对细胞添加不同的生物条形码(Barcode),实现对细胞进行不同的标记.将生物条形码序列、条件诱导性Cas9序列及相应的sgRNA序列通过PB酶整合到细胞中,诱导Cas9表达之后对生物条形码序列测序分析细胞的标记情况.所设计的生物条形码含有6个相互重叠的sgRNA识别位点,这种设计可以使条形码序列只会被Cas9切割一次.结果显示,所设计的生物条形码序列在N2a细胞中经Dox诱导之后能够高效地被Cas9切割,所挑的9个克隆中,生物条形码序列有9种不同的基因型.含有受loxp-stop-loxp调控表达Cas9的序列及生物条形码序列的E14胚胎干细胞经Cre诱导之后,挑单克隆测序分析显示,21株细胞中仅2株单克隆细胞生物条形码保持原来的基因型,另外19株有18种不同的基因型.成功建立了可进行时空调控地在细胞内高效添加特异且相对稳定的生物条形码标记的方法.
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