A Novel Approach to Establish Bio-barcode in Cells by CRISPR/Cas9

doi:10.13560/j.cnki.biotech.bull.1985.2017-0087

Abstract

Abstract: 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.

Key words: bio-barcode, CRISPR/Cas9, cell marking

WEI Yu, ZHOU Chang-yang, LI Yan-li. A Novel Approach to Establish Bio-barcode in Cells by CRISPR/Cas9[J]. Biotechnology Bulletin, 2017, 33(8): 40-45.

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