TY - JOUR
T1 - Conditional gene knockout and reconstitution in human iPSCs with an inducible Cas9 system
AU - Wu, Mengyao
AU - Liu, Senquan
AU - Gao, Yongxing
AU - Bai, Hao
AU - Machairaki, Vasiliki
AU - Li, Gang
AU - Chen, Tong
AU - Cheng, Linzhao
N1 - Publisher Copyright:
© 2018
PY - 2018/5
Y1 - 2018/5
N2 - Precise genome editing in human induced pluripotent stem cells (iPSCs) significantly enhances our capability to use human iPSCs for disease modeling, drug testing and screening as well as investigation of human cell biology. In this study, we seek to achieve conditional expression of the CD55 gene in order to interrogate its functions. We used two human iPSC lines that have unique genotypes, and constructed an inducible Cas9 gene expression system that is integrated at the AAVS1 safe harbor site in the human genome. Using paired guide RNAs, we observed efficient knock-out with an intended deletion in the coding region of several genes including CD55 and ETV6 genes. This paired guide RNA approach enabled us to efficiently identify homozygous iPSC clones with an intended deletion. Once an iPSC clone lacking CD55 expression was identified and characterized, we were able to use the same doxycycline system to induce expression of a CD55 transgene from a piggyBac vector, in both undifferentiated and differentiated iPSCs. This single cell line of gene knock-out complemented with an inducible transgene is sufficient to achieve conditional expression of the CD55 gene. The methodology described here is broadly applicable to other genes in order to interrogate their functions.
AB - Precise genome editing in human induced pluripotent stem cells (iPSCs) significantly enhances our capability to use human iPSCs for disease modeling, drug testing and screening as well as investigation of human cell biology. In this study, we seek to achieve conditional expression of the CD55 gene in order to interrogate its functions. We used two human iPSC lines that have unique genotypes, and constructed an inducible Cas9 gene expression system that is integrated at the AAVS1 safe harbor site in the human genome. Using paired guide RNAs, we observed efficient knock-out with an intended deletion in the coding region of several genes including CD55 and ETV6 genes. This paired guide RNA approach enabled us to efficiently identify homozygous iPSC clones with an intended deletion. Once an iPSC clone lacking CD55 expression was identified and characterized, we were able to use the same doxycycline system to induce expression of a CD55 transgene from a piggyBac vector, in both undifferentiated and differentiated iPSCs. This single cell line of gene knock-out complemented with an inducible transgene is sufficient to achieve conditional expression of the CD55 gene. The methodology described here is broadly applicable to other genes in order to interrogate their functions.
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U2 - 10.1016/j.scr.2018.03.003
DO - 10.1016/j.scr.2018.03.003
M3 - Article
C2 - 29554589
AN - SCOPUS:85043985382
SN - 1873-5061
VL - 29
SP - 6
EP - 14
JO - Stem Cell Research
JF - Stem Cell Research
ER -