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61.
Monogenic diseases are often severe, life-threatening disorders for which lifelong palliative treatment is the only option. Over the last two decades, a number of strategies have been devised with the aim to treat these diseases with a genetic approach. Gene therapy has been under development for many years, yet suffers from the lack of an effective and safe vector for the delivery of genetic material into cells. More recently, gene targeting by homologous recombination has been proposed as a safer treatment, by specifically correcting disease-causing mutations. However, low efficiency is a major drawback. The emergence of two technologies could overcome some of these obstacles. Terminally differentiated somatic cells can be reprogrammed, using defined factors, to become induced pluripotent stem cells (iPSCs), which can undergo efficient gene mutation correction with the aid of fusion proteins known as zinc finger nucleases (ZFNs). The amalgamation of these two technologies has the potential to break through the current bottleneck in gene therapy and gene targeting. 相似文献
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Simon Schiml Friedrich Fauser Holger Puchta 《The Plant journal : for cell and molecular biology》2014,80(6):1139-1150
The CRISPR/Cas nuclease is becoming a major tool for targeted mutagenesis in eukaryotes by inducing double‐strand breaks (DSBs) at pre‐selected genomic sites that are repaired by non‐homologous end joining (NHEJ) in an error‐prone way. In plants, it could be demonstrated that the Cas9 nuclease is able to induce heritable mutations in Arabidopsis thaliana and rice. Gene targeting (GT) by homologous recombination (HR) can also be induced by DSBs. Using a natural nuclease and marker genes, we previously developed an in planta GT strategy in which both a targeting vector and targeting locus are activated simultaneously via DSB induction during plant development. Here, we demonstrate that this strategy can be used for natural genes by CRISPR/Cas‐mediated DSB induction. We were able to integrate a resistance cassette into the ADH1 locus of A. thaliana via HR. Heritable events were identified using a PCR‐based genotyping approach, characterised by Southern blotting and confirmed on the sequence level. A major concern is the specificity of the CRISPR/Cas nucleases. Off‐target effects might be avoided using two adjacent sgRNA target sequences to guide the Cas9 nickase to each of the two DNA strands, resulting in the formation of a DSB. By amplicon deep sequencing, we demonstrate that this Cas9 paired nickase strategy has a mutagenic potential comparable with that of the nuclease, while the resulting mutations are mostly deletions. We also demonstrate the stable inheritance of such mutations in A. thaliana. 相似文献
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基因组靶向修饰技术对基因功能研究、基因治疗以及转基因育种研究都具有重要的意义和价值。近年来发展起来的人工核酸酶如ZFNs、TALENs和CRISPR/Cas9等的应用大大提高了基因组靶向修饰的效率。但是由于核酸酶表达载体转染效率、核酸酶表达效率及活性以及基因组被打靶后的修复效率等因素在一定程度上制约着基因组靶向修饰阳性细胞的获得。因此富集和筛选基因组靶向修饰阳性细胞是一个亟待解决的问题。报告载体系统可以间接地反映核酸酶的工作效率并有效富集核酸酶修饰的阳性细胞,进而提高基因组靶向修饰阳性细胞的富集和筛选效率。本文主要针对由非同源末端连接(Non-homologous end joining,NHEJ)和单链退火(Single-strand annealing,SSA)两种修复机制分别介导的报告载体系统的原理和应用进行了详细的介绍,以期为以后的相关研究提供借鉴和参考。 相似文献
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敲除猪肌肉生长抑制素(Myostatin, MSTN)基因可能提高猪瘦肉率, MSTN基因敲除猪也可作为相关疾病的动物模型。文章利用锌指核酸酶(Zinc-finger nucleases, ZFNs)技术敲除五指山小型猪胎儿成纤维细胞MSTN基因, 为制备MSTN基因敲除猪奠定基础。ZFNs质粒或编码ZFNs的mRNA均能高效敲除MSTN基因, 使用ZFNs mRNA能直接得到MSTN+/-和MSTN-/-两种基因型的细胞克隆。DNA序列测定与分析发现, 细胞克隆的突变类型多为ZFNs作用靶位点处不大于10 bp的碱基插入或缺失(92.18 %); 氨基酸预测发现, 突变型MSTN基因的终止密码子常常提前出现。将MSTN基因敲除的细胞进行体细胞核移植(Somatic cell nuclear transfer, SCNT)发现, 胚胎体外早期发育潜力与野生型无显著差异, 表明这些细胞可用于后续MSTN基因敲除猪的制备。 相似文献
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N. I. Aleksandrushkina E. M. Kof A. V. Seredina A. A. Borzov B. F. Vanyushin 《Russian Journal of Plant Physiology》2008,55(1):23-32
Senescence and wilting of the leaves of pea (Pisum sativum L.) of normal (AfAf) and aphyllous (afaf) genotypes were accompanied by DNA degradation. In young (12th–9th) subapical leaves of AfAf plants, total DNA was high-polymeric; in the 6th leaf, DNA degradation was appreciable; and in the 4th and 3rd leaves, hydrolysis of DNA was pronounced. Similar degradation of DNA was also observed in senescing leaves of aphyllous plants, but there it started later than in the plants of normal type. The extent of DNA degradation was closely related to the elevation of total nuclease activity in pea leaves associated with the age. The leaves of plants of different genotypes distinctly differed in the activity of acid and alkaline nucleases. Senescence of the leaves was accompanied by the induction of Ca2+-and Mg2+-dependent nucleases with mol wts of 42, 37, 34, 26, and 21 kD. In different stages of leaf senescence, different sets of nucleases were detected. 相似文献