Highly efficient genome editing via CRISPR-Cas9 in human pluripotent stem cells is achieved by transient BCL-XL overexpression.

Genome editing of human induced pluripotent stem cells (iPSCs) is instrumental for functional genomics, disease modeling, and regenerative medicine. However, low editing efficiency has hampered the applications of CRISPR-Cas9 technology in creating knockin (KI) or knockout (KO) iPSC lines, which is largely due to massive cell death after electroporation with editing plasmids. Here, we report that the transient delivery of BCL-XL increases iPSC survival by ∼10-fold after plasmid transfection, leading to a 20- to 100-fold increase in homology-directed repair (HDR) KI efficiency and a 5-fold increase in non-homologous end joining (NHEJ) KO efficiency. Treatment with a BCL inhibitor ABT-263 further improves HDR efficiency by 70% and KO efficiency by 40%. The increased genome editing efficiency is attributed to higher expressions of Cas9 and sgRNA in surviving cells after electroporation. HDR or NHEJ efficiency reaches 95% with dual editing followed by selection of cells with HDR insertion of a selective gene. Moreover, KO efficiency of 100% can be achieved in a bulk population of cells with biallelic HDR KO followed by double selection, abrogating the necessity for single cell cloning. Taken together, these simple yet highly efficient editing strategies provide useful tools for applications ranging from manipulating human iPSC genomes to creating gene-modified animal models.

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.

Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage.

BACKGROUND: Precise genome editing via homology-directed repair (HDR) after double-stranded DNA (dsDNA) cleavage facilitates functional genomic research and holds promise for gene therapy. However, HDR efficiency remains low in some cell types, including some of great research and clinical interest, such as human induced pluripotent stem cells (iPSCs). RESULTS: Here, we show that a double cut HDR donor, which is flanked by single guide RNA (sgRNA)-PAM sequences and is released after CRISPR/Cas9 cleavage, increases HDR efficiency by twofold to fivefold relative to circular plasmid donors at one genomic locus in 293 T cells and two distinct genomic loci in iPSCs. We find that a 600 bp homology in both arms leads to high-level genome knockin, with 97-100% of the donor insertion events being mediated by HDR. The combined use of CCND1, a cyclin that functions in G1/S transition, and nocodazole, a G2/M phase synchronizer, doubles HDR efficiency to up to 30% in iPSCs. CONCLUSIONS: Taken together, these findings provide guidance for the design of HDR donor vectors and the selection of HDR-enhancing factors for applications in genome research and precision medicine.

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors.

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies are: 1) the use of a 2A "self-cleavage" peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

A comparison of Staphylococcus aureus biofilm formation on cobalt-chrome and titanium-alloy spinal implants.

The use of cobalt chrome (CoCr) implants in spinal surgery has become increasingly popular. However, there have been no studies specifically comparing biofilm formation on CoCr with that of titanium-alloy spinal implants. The objective of this study was to compare the difference in propensity for biofilm formation between these two materials, as it specifically relates to spinal rods. Staphylococcus aureus subsp. Aureus (ATCC 6538) were incubated with two different types of spinal rods composed of either CoCr or titanium-alloy. The spinal rods were then subject to a trypsin wash to allow for isolation of the colonized organism and associated biofilms. The associated optical density values (OD) from the bacterial isolates were obtained and the bacterial solutions were plated on brain-heart infusion agar plates and the resultant colony-forming units (CFU) were counted. The OD values for the titanium-alloy rods were 1.105±0.096nm (mean±SD) and 1.040±0.026nm at 48hours and 96hours, respectively. In contrast, the OD values for the CoCr rods were 1.332±0.161nm and 1.115±0.207nm at 48 and 96hours, respectively (p<0.05). The CFU values were 1481±417/100mm(2) and 745±159/100mm(2) at 48 and 96hours, respectively for the titanium-alloy group. These values were significantly lower than the CFU values obtained from the CoCr group which were 2721±605/100mm(2) and 928±88/100mm(2) (p<0.001) at both 48 and 96hours respectively. Our findings, evaluating both the OD and CFU values, indicate that implants composed of CoCr had a higher proclivity towards biofilm formation compared to titanium-alloy implants.