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.

Oxidative stress-induced neuronal death has an important role in the pathogenesis of neurodegenerative disorders. The effects and mechanisms of action of the total flavonoids (TFs) from Rosa laevigata Michx fruit against hydrogen peroxide (H₂O₂)-induced oxidative injury in PC12 cells were investigated in this study. The results demonstrated that the TFs protected against cell apoptosis, DNA and mitochondrial damage caused by H₂O₂ based on single cell gel electrophoresis, in situ terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL), flow cytometry and transmission electron microscope (TEM) assays. In addition, the TFs notably decreased cytochrome C release from mitochondria into the cytosol and intracellular Ca²⁺ levels, and diminished intracellular generation of reactive oxygen species (ROS). Furthermore, the TFs inhibited the phosphorylation levels of JNK, ERK and p38 MAPK as well as down-regulated the expressions of IL-1, IL-6, TNF-α, Fas, FasL, CYP2E1, Bak, caspase-3, caspase-9, p53, COX-2, NF-κB, AP-1, and up-regulated the expressions of Bcl-2 and Bcl-xl. In conclusion, these results suggest that the TFs from R. laevigata Michx fruit show good effects against H₂O₂-induced oxidative injury in PC12 cells by adjusting oxidative stress, and suppression of apoptosis and inflammation, and could be developed as a potential candidate to prevent oxidative stress in the future.

Zhenghao Wang, Chunying Liu, Hongshan Yu, Bo Wu, Baoyu Huai, Ziyu Zhuang, Changkai Sun, Longquan Xu, and Fengxie Jin. J. Microbiol. Biotechnol. 2022;32:437-46. https://doi.org/10.4014/jmb.2112.12036

Highly efficient gene knockout (KO) editing of CRISPR–Cas9 has been achieved in iPSCs, whereas homology-directed repair (HDR)-mediated precise gene knock-in (KI) and high-level expression are still bottlenecks for the clinical applications of iPSCs. Here, we developed a novel editing strategy that targets introns. By targeting the intron before the stop codon, this approach tolerates reading frameshift mutations caused by nonhomologous end-joining (NHEJ)-mediated indels, thereby maintaining gene integrity without damaging the non-HDR-edited allele. Furthermore, to increase the flexibility and screen for the best intron-targeting sgRNA, we designed an HDR donor with an artificial intron in place of the endogenous intron. The presence of artificial introns, particularly an intron that carries an enhancer element, significantly increased the reporter expression levels in iPSCs compared to the intron-deleted control. In addition, a combination of the small molecules M3814 and trichostatin A (TSA) significantly improves HDR efficiency by inhibiting NHEJ. These results should find applications in gene therapy and basic research, such as creating reporter cell lines.