High-Level Precise Knockin of iPSCs by Simultaneous Reprogramming and Genome Editing of Human Peripheral Blood Mononuclear Cells

Stem Cell Reports
Wei WenXiao-Bing Zhang

Abstract

We have developed an improved episomal vector system for efficient generation of integration-free induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells. More recently, we reported that the use of an optimized CRISPR-Cas9 system together with a double-cut donor increases homology-directed repair-mediated precise gene knockin efficiency by 5- to 10-fold. Here, we report the integration of blood cell reprogramming and genome editing in a single step. We found that expression of Cas9 and KLF4 using a single vector significantly increases genome editing efficiency, and addition of SV40LT further enhances knockin efficiency. After these optimizations, genome editing efficiency of up to 40% in the bulk iPSC population can be achieved without any selection. Most of the edited cells show characteristics of iPSCs and genome integrity. Our improved approach, which integrates reprogramming and genome editing, should expedite both basic research and clinical applications of precision and regenerative medicine.

Methods Mentioned

BETA
gene knockout
biopsy
fluorescence-activated cell sorting
gene knockin
flow cytometry
transfection
FACS
PCR
Illumina sequencing
density gradient centrifugation

Software Mentioned

Galaxy
CHOPCHOP

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