Zinc-finger nuclease, transcription activator-like effector nuclease and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) are becoming major tools for genome editing. Importantly, knock-in in several non-rodent species has been finally achieved thanks to these customizable nucleases; yet the rates remain to be further improved. We hypothesize that inhibiting non-homologous end joining (NHEJ) or enhancing homology-directed repair (HDR) will improve the nuclease-mediated knock-in efficiency. Here we show that the in vitro application of an HDR enhancer, RS-1, increases the knock-in efficiency by two- to five-fold at different loci, whereas NHEJ inhibitor SCR7 has minimal effects. We then apply RS-1 for animal production and have achieved multifold improvement on the knock-in rates as well. Our work presents tools to nuclease-mediated knock-in animal production, and sheds light on improving gene-targeting efficiencies on pluripotent stem cells.

Laboratory animal models play an important role in the study of human diseases. Using appropriate animals is critical not only for basic research but also for the development of therapeutics and diagnostic tools. Rabbits are widely used for the study of human atherosclerosis. Because rabbits have a unique feature of lipoprotein metabolism (like humans but unlike rodents) and are sensitive to a cholesterol diet, rabbit models have not only provided many insights into the pathogenesis and development of human atherosclerosis but also made a great contribution to translational research. In fact, rabbit was the first animal model used for studying human atherosclerosis, more than a century ago. Currently, three types of rabbit model are commonly used for the study of human atherosclerosis and lipid metabolism: (1) cholesterol-fed rabbits, (2) Watanabe heritable hyperlipidemic rabbits, analogous to human familial hypercholesterolemia due to genetic deficiency of LDL receptors, and (3) genetically modified (transgenic and knock-out) rabbits. Despite their importance, compared with the mouse, the most widely used laboratory animal model nowadays, the use of rabbit models is still limited. In this review, we focus on the features of rabbit lipoprotein metabolism and pathology of atherosclerotic lesions that make it the optimal model for human atherosclerotic disease, especially for the translational medicine. For the sake of clarity, the review is not an attempt to be completely inclusive, but instead attempts to summarize substantial information concisely and provide a guideline for experiments using rabbits.

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Summary Phytohormone, particularly jasmonate (JA) and salicylate (SA) signaling, plays a central role in plant responses to herbivore and pathogen attack. Generally, SA mediates resistance responses against biotrophic pathogens and phloem‐feeding insects, while JA mediates responses against necrotrophic pathogens and chewing insects. The phytohormonal responses mediating rice resistance to a piercing‐sucking herbivore, the brown planthopper (BPH), remains unknown. Here, we combined transcriptome analysis, hormone measurements, genetic analysis and a field study to address this issue. Infestation by BPH adult females resulted in significant transcriptional reprograming. The upregulated genes were enriched in the JA signaling pathway. Consistently, the concentrations of JAs, but not SA, were dramatically increased in response to BPH attack. Two JA‐deficient lines (AOC and MYC2 knockout) and two SA‐deficient lines (nahG overexpression and NPR1 knockout) were constructed. BPH performed better on JA‐deficient lines than on wild‐type (WT) plants, but similarly on SA‐deficient and WT plants. During BPH attack, the accumulation of defensive secondary metabolites was attenuated in JA‐deficient lines compared with WT plants. Moreover, MYC2 mutants were more susceptible to planthoppers than WT plants in nature. This study reveals that JA signaling functions in rice defense against BPH.

CRISPR/Cas9 efficiently generates gene knock-out via nonhomologous end joining (NHEJ), but the efficiency of precise homology-directed repair (HDR) is substantially lower, especially in the hard-to-transfect human stem cells and primary cells. Herein we report a tube electroporation method that can effectively transfect human stem cells and primary cells with minimal cytotoxicity. When applied to genome editing using CRISPR/Cas9 along with single stranded DNA oligonucleotide (ssODN) template in human induced pluripotent stem cells (iPSCs), up to 42.1% HDR rate was achieved, drastically higher than many reported before. We demonstrated that the high HDR efficiency can be utilized to increase the gene ablation rate in cells relevant to clinical applications, by knocking-out β2-microglobulin (B2M) in primary human mesenchymal stem cells (MSCs, 37.3% to 80.2%), and programmed death-1 (PD-1) in primary human T cells (42.6% to 58.6%). Given the generality and efficiency, we expect that the method will have immediate impacts in cell research as well as immuno- and transplantation therapies.

Regulated activity of SLC6A3, which encodes the human dopamine transporter (DAT), contributes to diseases such as substance abuse disorders (SUDs); however, the exact transcription mechanism remains poorly understood. Here, we used a common genetic variant of the gene, intron 1 DNP1B sequence, as bait to screen and clone a new transcriptional activity, 3'UTR, for SLC6A3.3'UTR is a 3' untranslated region (3'UTR) of the human 5-Azacytidine Induced 2 gene (AZI2) but appeared to be transcribed independently of AZI2. Found to be present in both human cell nuclei and dopamine neurons, this RNA was shown to downregulate promoter activity through a variant-dependent mechanism in vitro. Both reduced RNA density ratio of 3'UTR/AZI2 and increased DAT mRNA levels were found in ethanol-naive alcohol-preferring rats. Secondary analysis of dbGaP GWAS datasets (Genome-Wide Association Studies based on the database of Genotypes and Phenotypes) revealed significant interactions between regions upstream of3'UTR and SLC6A3 in SUDs. Jointly, our data suggest that 3'UTR confers variant-dependent transcriptional regulation of SLC6A3, a potential risk factor for SUDs.

Cystic fibrosis (CF) is a lethal autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Nuclease-mediated precise gene editing (PGE) represents a promising therapy for CF, for which an efficient strategy that is free of viral vector, drug selection, and reporter enrichment (VDR free) is desirable. Here we compared different transfection methods (lipofectamine versus electroporation) and formats (plasmid DNA versus ribonucleoprotein) in delivering the CRISPR/Cas9 elements along with single-stranded oligodeoxynucleotides (ssODNs) to clinically relevant cells targeting major CFTR mutation loci. We demonstrate that, among different combinations, electroporation of CRISPR/Cas9 and guide RNA (gRNA) ribonucleoprotein (Cas9 RNP) is the most effective one. By using this VDR-free method, 4.8% to 27.2% efficiencies were achieved in creating dF508, G542X, and G551D mutations in a wild-type induced pluripotent stem cell (iPSC) line. When it is applied to a patient-derived iPSC line carrying the dF508 mutation, a greater than 20% precise correction rate was achieved. As expected, genetic correction leads to the restoration of CFTR function in iPSC-derived proximal lung organoids, as well as in a patient-derived adenocarcinoma cell line CFPAC-1. The present work demonstrates the feasibility of gene editing-based therapeutics toward monogenic diseases such as CF.

Plant seed size is tightly regulated by the development of seed coat, embryo, and endosperm; however, currently, its underlying mechanism remains unclear. In this study, we revealed a regulatory role of an R2R3 MYB transcription factor MYB56 in controlling seed size specifically in Arabidopsis thaliana L. Loss-of-function or knock-down of MYB56 yielded smaller seeds as compared with the wild type. Conversely, overexpression of MYB56 produced larger seeds. Further observation using semi-thin sections showed that myb56 developed smaller contracted endothelial cells and reduced cell number in the outer integument layer of the seed coat during the seed development; by contrast, MYB56 overexpressing lines had expanded endothelial cells and increased cell number in the outer integument layer of the seed coat, suggesting the essential role of MYB56 in regulating seed development. In addition, reciprocal cross-analysis showed that MYB56 affected the seed development maternally. MYB56 was shown to be dominantly expressed in developing seeds, consistently with its function in seed development. Moreover, quantitative reverse transcription polymerase chain reaction analysis revealed that MYB56 regulates the expression of genes involved in cell wall metabolism such as cell division and expansion. Altogether, our results demonstrated that MYB56 represents an unknown pathway for positively controlling the seed size.