Biotechnology Bulletin ›› 2017, Vol. 33 ›› Issue (8): 40-45.doi: 10.13560/j.cnki.biotech.bull.1985.2017-0087

• Technique and method • Previous Articles     Next Articles

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

WEI Yu1, ZHOU Chang-yang2, LI Yan-li1   

  1. 1. College of Life Sciences,Shanghai University,Shanghai 200444;
    2. Institute of Neuroscience,Shanghai 200031
  • Received:2017-02-10 Online:2017-08-01 Published:2017-08-01

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