Open AccessCRISPR‐Cas9‐Based Genome Editing of Human Induced Pluripotent Stem Cells
Author(s) -
Giacalone Joseph C.,
Sharma Tasneem P.,
Burnight Erin R.,
Fingert John F.,
Mullins Robert F.,
Stone Edwin M.,
Tucker Budd A.
Publication year - 2018
Publication title -
current protocols in stem cell biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.658
H-Index - 28
eISSN - 1938-8969
pISSN - 1941-7322
DOI - 10.1002/cpsc.46
Subject(s) - genome editing , crispr , biology , induced pluripotent stem cell , computational biology , cas9 , genome , genetics , selection (genetic algorithm) , gene , embryonic stem cell , computer science , artificial intelligence
Human induced pluripotent stem cells (hiPSCs) are the ideal cell source for autologous cell replacement. However, for patients with Mendelian diseases, genetic correction of the original disease‐causing mutation is likely required prior to cellular differentiation and transplantation. The emergence of the CRISPR‐Cas9 system has revolutionized the field of genome editing. By introducing inexpensive reagents that are relatively straightforward to design and validate, it is now possible to correct genetic variants or insert desired sequences at any location within the genome. CRISPR‐based genome editing of patient‐specific iPSCs shows great promise for future autologous cell replacement therapies. One caveat, however, is that hiPSCs are notoriously difficult to transfect, and optimized experimental design considerations are often necessary. This unit describes design strategies and methods for efficient CRISPR‐based genome editing of patient‐ specific iPSCs. Additionally, it details a flexible approach that utilizes positive selection to generate clones with a desired genomic modification, Cre‐ lox recombination to remove the integrated selection cassette, and negative selection to eliminate residual hiPSCs with intact selection cassettes. © 2018 by John Wiley & Sons, Inc.