Conditional gene knockout and reconstitution in human iPSCs with an inducible Cas9 system.
Stem Cell Res
; 29: 6-14, 2018 05.
Article
in En
| MEDLINE
| ID: mdl-29554589
ABSTRACT
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.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Gene Knockout Techniques
/
Induced Pluripotent Stem Cells
/
CRISPR-Cas Systems
Limits:
Humans
Language:
En
Journal:
Stem Cell Res
Year:
2018
Document type:
Article
Affiliation country:
Estados Unidos