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

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. In this article, Dr. Zhang and colleagues describe an episomal vector system for one-step generation and genome editing (knockin) of integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). Up to 40% knockin editing efficiencies in bulk iPSCs can be achieved by co-expression of Cas9 and KLF4 in a single vector and with addition of SV40LT.