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

摘要

class="head no_bottom_margin" id="sec1title">IntroductionInduced pluripotent stem cells (iPSCs) have been recognized as an attractive cell source for stem cell therapy, drug discovery, and disease modeling (, , ). Since blood cells can be easily obtained through a minimally invasive process and have been widely applied in clinical diagnosis, we and other investigators have been working on reprogramming of peripheral blood (PB) mononuclear cells (MNCs) in recent years (, , , , , , , , , , , , href="#bib43" rid="bib43" class=" bibr popnode">Su et al., 2016, href="#bib49" rid="bib49" class=" bibr popnode">Wen et al., 2016, href="#bib48" rid="bib48" class=" bibr popnode">Wen et al., 2017). More recently, we reported an efficient system for PB MNC reprogramming using an optimized combination of episomal vectors that express five reprogramming factors, and found that thousands of integration-free iPSC colonies can be generated from 1 × 10⁶ PB MNCs (href="#bib49" rid="bib49" class=" bibr popnode">Wen et al., 2016, href="#bib48" rid="bib48" class=" bibr popnode">Wen et al., 2017). This seemingly simple but highly efficient system has been adopted by many other laboratories (href="#bib6" rid="bib6" class=" bibr popnode">Chou et al., 2015, href="#bib21" rid="bib21" class=" bibr popnode">Hu et al., 2015). The episomal vectors we used are plasmids carrying EBNA1 and oriP, which maintain the transgene expression for 1–2 weeks, allowing for successful reprogramming, and are gradually depleted from the cells during passage leading to generation of integration-free iPSCs.For clinical regenerative medicine applications, patient-specific iPSCs, which often carry a disease-causing gene(s), have to be genome edited before differentiation into functional cells for therapy. CRISPR-Cas9 is a powerful genome editing technology to achieve this goal (href="#bib26" rid="bib26" class=" bibr popnode">Li et al., 2015, href="#bib37" rid="bib37" class=" bibr popnode">Park et al., 2015, href="#bib51" rid="bib51" class=" bibr popnode">Xie et al., 2014). CRISPR-Cas9 is an adoptive immune system evolved in bacteria and Archaea to fight against invading agents such as bacteriophages or plasmids (href="#bib50" rid="bib50" class=" bibr popnode">Wright et al., 2016), and has been successfully engineered to target the human genome (href="#bib18" rid="bib18" class=" bibr popnode">Hou et al., 2013). Diverse CRISPR systems have been adapted for use in editing iPSCs (href="#bib18" rid="bib18" class=" bibr popnode">Hou et al., 2013, href="#bib39" rid="bib39" class=" bibr popnode">Ran et al., 2015, href="#bib57" rid="bib57" class=" bibr popnode">Zetsche et al., 2015), among which the most commonly used system is derived from Streptococcus pyogenes. In this system, single guide RNA (sgRNA) guides endonuclease Cas9 to cleave a double-stranded DNA sequence of ∼20 bp in length at 3 bp upstream of the protospacer adjacent motif (PAM) NGG. After double-stranded DNA cleavage, the damage is often repaired by error-prone non-homologous end joining (NHEJ) or precise homologous recombination (HR) pathway (href="#bib17" rid="bib17" class=" bibr popnode">Hockemeyer et al., 2011, href="#bib52" rid="bib52" class=" bibr popnode">Yang et al., 2013). If a donor template that harbors both left and right homology arms is provided, the cells can be tricked to use the donor template to repair the damage instead of searching for the sister chromatids. As a result, a DNA fragment of interest can be precisely knocked in. However, this homology-directed repair (HDR)-mediated knockin system is far less efficient than gene knockout. In attempt to break this bottleneck in precise genome editing, we and other investigators developed a novel double-cut HDR donor, which is flanked by sgRNA recognition sequence and is released after CRISPR-Cas9 cleavage (href="#bib23" rid="bib23" class=" bibr popnode">Irion et al., 2014, href="#bib58" rid="bib58" class=" bibr popnode">Zhang et al., 2017). After optimization, we observed an ∼5-fold increase in HDR efficiency using the double-cut donor with homology arms of 300–600 bp in length relative to circular plasmid donors (href="#bib58" rid="bib58" class=" bibr popnode">Zhang et al., 2017). Shortly after publication of our discovery, a similar study also reported the unprecedented editing efficiency of homology-mediated end joining compared with HR and microhomology-mediated end joining (href="#bib53" rid="bib53" class=" bibr popnode">Yao et al., 2017).The conventional strategy of regenerative medicine is generation of iPSCs first followed by genome editing. However, this is a time-consuming and labor-intensive procedure that requires ∼3 months of cell culture and two clone selections (href="#bib13" rid="bib13" class=" bibr popnode">Ding et al., 2013, href="#bib16" rid="bib16" class=" bibr popnode">Hockemeyer et al., 2009, href="#bib17" rid="bib17" class=" bibr popnode">Hockemeyer et al., 2011, href="#bib19" rid="bib19" class=" bibr popnode">Howden et al., 2011, href="#bib41" rid="bib41" class=" bibr popnode">Soldner et al., 2011, href="#bib56" rid="bib56" class=" bibr popnode">Yusa et al., 2011, href="#bib61" rid="bib61" class=" bibr popnode">Zou et al., 2009). To cut back on the time spent, one-step simultaneous reprogramming and CRISPR-Cas9 genome editing to generate gene-modified iPSCs from somatic cells has been proposed (href="#bib20" rid="bib20" class=" bibr popnode">Howden et al., 2015, href="#bib46" rid="bib46" class=" bibr popnode">Tidball et al., 2017). href="#bib20" rid="bib20" class=" bibr popnode">Howden et al. (2015) have reported generation of gene edited iPSCs from fibroblasts by nucleofection of episomal vectors expressing reprogramming factors and CRISPR-Cas9 vectors. They targeted a GFP reporter to the DNMT3A locus during reprogramming. They observed up to 5% GFP-positive edited cells in bulk cells, which is five times higher than that achieved by direct editing of iPSCs. These data provide the first evidence for the benefit of combining somatic cell reprogramming and genome editing in a single step. However, the use of fibroblasts from human skin biopsy is problematic because of the high mutation rate of skin cells after long-term exposure to UV light radiation and the invasive procedure used to procure the cells (href="#bib1" rid="bib1" class=" bibr popnode">Abyzov et al., 2012). In contrast to fibroblasts, PB cells are a preferable cell source for reprogramming (href="#bib60" rid="bib60" class=" bibr popnode">Zhang, 2013). As such, we attempted to generate gene edited iPSCs from PB MNCs by simultaneously reprogramming and gene editing.In this study, we designed double-cut donors for HDR knockin of fluorescent reporters (href="#bib58" rid="bib58" class=" bibr popnode">Zhang et al., 2017). The knockin efficiency can be precisely determined by fluorescence-activated cell sorting (FACS) analysis of fluorescence-positive cells. A simple combination of reprogramming vectors and genome editing plasmids led to a nearly 10% knockin efficiency. Further improvements, including combining Cas9 and KLF4 expression in one vector and addition of SV40LT, increased HDR efficiency to up to 40%. Thus, in this study, we have established an optimized reprogramming and CRISPR-Cas9 system to efficiently generate gene-modified integration-free iPSCs directly from PB.