Improvement of the knock-in effciency in the genome of human induced pluripotent stem cells using the CRISPR/Cas9 system
https://doi.org/10.18699/VJ18.446
Abstract
Human induced pluripotent stem (hiPS) cells are a powerful tool for biomedical research. The ability to create patient-specifc pluripotent cells and their subsequent differentiation into any somatic cell type makes hiPS cells a valuable object for creating in vitro models of human diseases, screening drugs and a future source of cells for regenerative medicine. To realize entirely a potential of hiPScells, effective and precise methods for their genome editing are needed. The CRISPR/Cas9 system is the most widely used method for introducing site-specifc double-stranded breaks into DNA. It allows genes of interest to be knocked out with high efciency. However, knock-in into the target site of the genome is a much more difcult task. Moreover, many researchers have noted a low efciency of introducing target constructs into the hiPS cells’ genome. In this review, I attempt to describe the currently known information regarding the matter of increasing efciency of targeted insertions into hiPS cells’ genome. Here I will describe the most effective strategies for designing the donor template for homology-directed repair, methods to manipulate the double-strand break repair pathways introduced by a nuclease, including control of CRISPR/Cas9 delivery time. A low survival rate of hiPS cells following genome editing experiments is another difculty on the way towards successful knock-in, and here several highly effective approaches addressing it are proposed. Finally, I describe the most promising strategies, one-step reprogramming and genome editing, which allows gene-modifed integration-free hiPS cells to be efciently generated directly from somatic cells.
About the Author
М. М. Gridina
Institute of Cytology and Genetics, SB RAS
Russian Federation
Novosibirsk
Russian Federation
Novosibirsk
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