A Single Administration of CRISPR/Cas9 Lipid Nanoparticles Achieves Robust and Persistent In Vivo Genome Editing

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Cloning and Drought Resistance Analysis of Soybean <i>GmHsps_p23-like</i> Gene

Soybean (Glycine max (L.) Merr.) is an important cultivated crop, which requires much water during its growth, and drought seriously affects soybean yields. Studies have shown that the expression of small heat shock proteins can enhance drought resistance, cold resistance and salt resistance of plants. In this experiment, soybean GmHsps_p23-like gene was successfully cloned by RT-PCR, the protein encoded by the GmHsps_p23-like gene was subjected to bioinformatics analysis, and the pCAMBIA3301-GmHsps_p23-like overexpression vector and pCBSG015-GmHsps_p23-like gene editing vector were constructed. Agrobacterium-mediated method was used to transform soybeans to obtain positive plants. RT-PCR detection, rehydration experiment and drought resistance physiological and biochemical index detection were performed on the T₂ generation positive transgenic soybean plants identified by PCR and Southern hybridization. The results showed that the overexpression vector plant GmHsps_p23-like gene expression increased. After rehydration, the transgenic overexpression plants returned to normal growth, and the damage to the plants was low. After drought stress, the SOD and POD activities and the PRO content of the transgenic overexpression plants increased, while the MDA content decreased. The reverse was true for soybean plants with genetically modified editing vectors. The drought resistance of the overexpressed soybeans under drought stress was higher than that of the control group, and had a stronger drought resistance. It showed that the expression of soybean GmHsps_p23-like gene can improve the drought resistance of soybean. The cloning and functional verification of soybean GmHsps_p23-like gene had not been reported yet. This is the first time that PCR technology has been used to amplify the soybean GmHsps_p23-like gene and construct an expression vector for this gene. This research has laid the foundation for transgenic technology to improve plant drought resistance and cultivate new drought-resistant transgenic soybean varieties.     

CRISPR/Cas9技术编辑MPK7和MPK14基因创制抗穗发芽水稻新种质

穗发芽对籼稻生产危害严重.编辑休眠调控基因,创制高休眠性新种质,进而提高穗发芽抗性,是探索改良穗发芽抗性的新途径.泰丰B(TB)是籼型优质杂交稻保持系,主要缺点是种子休眠性偏低,易受穗发芽危害.MPK7/14具有抑制粳稻种子休眠作用,尚不清楚是否适用于籼稻品种.本研究利用CRISPR/Cas9技术编辑泰丰B(TB)的M...     

谷氨酸棒杆菌中基于CRISPR/Cas9的多位点碱基编辑系统的优化

作为新型的基因组编辑工具,碱基编辑技术结合了CRISPR/Cas系统的定位功能和碱基脱氨酶的编辑功能,可实现特定位点的碱基突变,具有不产生双链DNA断裂,无需外源模板且不依赖染色体DNA同源重组的优势.目前,研究者们已在重要的工业生产菌株谷氨酸棒杆菌(Corynebacterium glutamicum)中开发了多种碱...     

Heritable Custom Genomic Modifications in Caenorhabditis elegans via a CRISPR–Cas9 System

We adapted the CRISPR–Cas9 system for template-mediated repair of targeted double-strand breaks via homologous recombination in Caenorhabditis elegans, enabling customized and efficient genome editing. This system can be used to create specific insertions, deletions, and base pair changes in the germline of C. elegans.     

基于CRISPR/Cas系统的单碱基编辑技术研究进展*

基于CRISPR/Cas系统出现的单碱基编辑技术可以实现高效且简便的单个碱基的替换编辑,其原理是将胞嘧啶脱氨酶(cytosine deaminase)或腺苷脱氨酶(adenosine deaminase)与Cas9n(D10A)形成融合蛋白,通过CRISPR/Cas精准识别和定位DNA上的靶位点后,利用胞嘧啶脱氨酶或腺苷脱氨酶将靶点距离sgRNA位点基序(protospacer adjacent motif,PAM)序列端的4~7位的单个碱基发生单碱基转换或颠换。对基于CRISPR/Cas系统的单碱基编辑技术发现的历史、组成和分类、工作原理进行了概述,并总结了该系统最新进展及应用。     

CRISPR/Cas9基因编辑技术在致瘤病毒感染研究中的应用

成簇的规律间隔的短回文重复序列及其相关蛋白9〔clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9),CRISPR/Cas9〕基因编辑技术的发现源于真细菌和古细菌中CRISPR/Cas系统介导的适应性免疫机制研究。该技术利用特异性向导RNA识别靶点基因,引导核酸内切酶Cas9对其切割,并通过同源重组或非同源末端连接完成对目的DNA的编辑。某些病毒感染机体后,可将其基因组整合到宿主细胞基因组中或潜伏于组织中而无法被彻底清除,从而引起持续性感染。本文参考2013年以来CRISPR/Cas9基因组编辑技术的最新相关研究报道,重点综述其在人类免疫缺陷病毒1型(human immunodeficiency virus type 1,HIV-1)、人乳头瘤病毒(human papillomavirus,HPV )、乙型肝炎病毒(hepatitis B virus, HBV)、 Epstein-Barr病毒(Epstein-Barr virus,EBV)等致瘤病毒感染相关疾病研究中的应用,并概括其作用于这些病毒的有效靶点。     

A palmitoyltransferase Approximated gene Bm-app regulates wing development in Bombyx mori

The silkworm Bombyx mori is an important lepidopteran model insect in which many kinds of natural mutants have been identified.However,molecular mechanisms of most of these mutants remain to be explored.Here we report the identification of a gene Bm-app is responsible for the silkworm minute wing(mw)mutation which exhibits exceedingly small wings during pupal and adult stages.Compared with the wild type silkworm,relative messenger RNA expression of Bm-app is significantly decreased in the ul 1 mutant strain which shows mw phenotype.A 10 bp insertion in the putative promoter region of the Bm-app gene in mw mutant strain was identified and the dual luciferase assay revealed that this insertion decreased Bm-app promoter activity.Furthermore,clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases-mediated depletion of the Bm-app induced similar wing defects which appeared in the mw mutant,demonstrating that Bm-app controls wing development in B.mori.Bm-app encodes a palmitoyltransferase and is responsible for the palmitoylation of selected cytoplasmic proteins,indicating that it is required for cell mitosis and growth during wing development.We also discuss the possibility that Bm-app regulates wing development through the Hippo signaling pathway in B.mori.