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近年来,可编程核酸酶介导的基因编辑技术迅猛发展。CRISPR/Cas9技术源于细菌和古生菌的适应性免疫系统,主要由Cas9内切酶和向导RNA(guide RNA,gRNA)组成。Cas9内切酶在gRNA的指导下造成DNA的双链断裂,从而使研究人员能够精准高效地操纵特定基因组位点。同时,该系统可以揭示基因在疾病进程中所扮演的未知角色,在临床治疗中有应用潜能。现总结了CRISPR/Cas9技术在疾病模型构建与基因修复领域应用的研究进展。 相似文献
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我国是家猪养殖和消费大国,提高母猪的繁殖力对于促进我国生猪产业的发展具有重要的作用。排卵率和产仔数是影响家畜繁殖力的关键因素,其中BMP15 (bone morphogenetic protein 15)基因已被鉴定是控制绵羊排卵数和多胎性状的一个主效基因,然而目前在家猪BMP15基因中尚未发现类似绵羊多胎品系的天然突变。基于高等哺乳动物基因功能的保守性和CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)等基因组编辑技术对动物基因组定点修饰的高效性,应用CRISPR/Cas9技术对家猪BMP15基因进行精确的遗传修饰,使家猪获得类似多胎绵羊的天然突变,对于研究该基因对家猪繁殖力的影响以及培育高繁殖力家猪新品系具有重要的意义。本研究通过CRISPR/Cas9对长白猪胎儿成纤维(porcine embryonic fibroblasts, PEF)细胞中BMP15基因进行打靶,T7E1分析显示打靶效率仅有5%。随后通过共转染RGS双荧光替代性报告载体(RFP-GFP surrogate reporter),并应用流式细胞术分选出双荧光细胞,富集到基因组被CRISPR/Cas9修饰的细胞,使基因打靶效率提高至18%。本研究结果表明,应用RGS双荧光替代性报告载体可以有效提高CRISPR/Cas9在PEF细胞中对BMP15基因的打靶效率,为今后通过体细胞核移植技术培育BMP15基因编辑猪进行了有效的探索。 相似文献
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《遗传学报》2016,(5)
Tissue-specific knockout technology enables the analysis of the gene function in specific tissues in adult mammals.However,conventional strategy for producing tissue-specific knockout mice is a time- and labor-consuming process,restricting rapid study of the gene function in vivo.CRISPR-Cas9 system from bacteria is a simple and efficient gene-editing technique,which has enabled rapid generation of gene knockout lines in mouse by direct injection of CRISPR-Cas9 into zygotes.Here,we demonstrate CRISPR-Cas9-mediated spermatogenic cell-specific disruption of Scp3 gene in testes in one step.We first generated transgenic mice by pronuclear injection of a plasmid containing Hspa2 promoter driving Cas9 expression and showed Cas9 specific expression in spermatogenic cells.We then produced transgenic mice carrying Hspa2 promoter driven Cas9 and constitutive expressed sgRNA targeting Scp3 gene.Male founders were infertile due to developmental arrest of spermatogenic cells while female founders could produce progeny normally.Consistently,male progeny from female founders were infertile and females could transmit the transgenes to the next generation.Our study establishes a CRISPR-Cas9-based one-step strategy to analyze the gene function in adult tissues by a temporal-spatial pattern. 相似文献
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Alexander J. Weitzel Hannah A. Grunwald Ceri Weber Rimma Levina Valentino M. Gantz Stephen M. Hedrick Ethan Bier Kimberly L. Cooper 《PLoS biology》2021,19(12)
Highly efficient gene conversion systems have the potential to facilitate the study of complex genetic traits using laboratory mice and, if implemented as a “gene drive,” to limit loss of biodiversity and disease transmission caused by wild rodent populations. We previously showed that such a system of gene conversion from heterozygous to homozygous after a sequence targeted CRISPR/Cas9 double-strand DNA break (DSB) is feasible in the female mouse germline. In the male germline, however, all DSBs were instead repaired by end joining (EJ) mechanisms to form an “insertion/deletion” (indel) mutation. These observations suggested that timing Cas9 expression to coincide with meiosis I is critical to favor conditions when homologous chromosomes are aligned and interchromosomal homology-directed repair (HDR) mechanisms predominate. Here, using a Cas9 knock-in allele at the Spo11 locus, we show that meiotic expression of Cas9 does indeed mediate gene conversion in the male as well as in the female germline. However, the low frequency of both HDR and indel mutation in both male and female germlines suggests that Cas9 may be expressed from the Spo11 locus at levels too low for efficient DSB formation. We suggest that more robust Cas9 expression initiated during early meiosis I may improve the efficiency of gene conversion and further increase the rate of “super-mendelian” inheritance from both male and female mice.This study shows that while Cas9 expression during meiosis I promotes genotype conversion - the mechanism underlying CRISPR ’gene drive’ - in both male and female mice, timing and high levels of Cas9 protein are critical to achieve robust efficiency. 相似文献
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CRISPR/Cas9‐induced disruption of gene expression in mouse embryonic brain and single neural stem cells in vivo
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Stefania Tavano Fong Kuan Wong Dana Suchold Sylke Winkler Wieland B Huttner Mihail Sarov 《EMBO reports》2016,17(3):338-348
We have applied the CRISPR/Cas9 system in vivo to disrupt gene expression in neural stem cells in the developing mammalian brain. Two days after in utero electroporation of a single plasmid encoding Cas9 and an appropriate guide RNA (gRNA) into the embryonic neocortex of Tis21::GFP knock‐in mice, expression of GFP, which occurs specifically in neural stem cells committed to neurogenesis, was found to be nearly completely (≈90%) abolished in the progeny of the targeted cells. Importantly, upon in utero electroporation directly of recombinant Cas9/gRNA complex, near‐maximal efficiency of disruption of GFP expression was achieved already after 24 h. Furthermore, by using microinjection of the Cas9 protein/gRNA complex into neural stem cells in organotypic slice culture, we obtained disruption of GFP expression within a single cell cycle. Finally, we used either Cas9 plasmid in utero electroporation or Cas9 protein complex microinjection to disrupt the expression of Eomes/Tbr2, a gene fundamental for neocortical neurogenesis. This resulted in a reduction in basal progenitors and an increase in neuronal differentiation. Thus, the present in vivo application of the CRISPR/Cas9 system in neural stem cells provides a rapid, efficient and enduring disruption of expression of specific genes to dissect their role in mammalian brain development. 相似文献
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Verónica Pazmiño-Ibarra Adrià Mengual-Martí Alexandra Marisa Targovnik Salvador Herrero 《Biotechnology and bioengineering》2019,116(11):2823-2833
The clustered regularly interspaced short palindromic repeats (CRISPR) system–associated Cas9 endonuclease is a molecular tool that enables specific sequence editing with high efficiency. In this study, we have explored the use of CRISPR/Cas9 system for the engineering of baculovirus. We have shown that the delivering of Cas9-single guide RNA ribonucleoprotein (RNP) complex with or without DNA repair template into Sf21 insect cells through lipofection might be efficient to produce knockouts as well as knock-ins into the baculovirus. To evaluate potential application of our CRISPR/Cas9 method to improve baculovirus as protein expression vector and as biopesticide, we attempted to knockout several genes from a recombinant AcMNPV form used in the baculovirus expression system as well as in a natural occurring viral isolate from the same virus. We have additionally confirmed the adaptation of this methodology for the generation of viral knock-ins in specific regions of the viral genome. Analysis of the generated mutants revealed that the editing efficiency and the type of changes was variable but relatively high. Depending on the targeted gene, the editing rate ranged from 10% to 40%. This study established the first report revealing the potential of CRISPR/Cas9 for genome editing in baculovirus, contributing to the engineering of baculovirus as a protein expression vector as well as a biological control agent. 相似文献
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CRISPR/Cas9 system is a natural immune system in prokaryotes protecting them from infectious viral or plasmid DNA invading the cells. This RNA-guided system can act as powerful tool for introducing genomic alterations in eukaryotic cells with high efficiency. In the present study, Rig-Igene is taken as model gene to study the efficiency of CRISPR/Cas9 system induced gene deletion in primary fibroblast cell culture. Rig-I(retinoic acid-inducible gene-1) is involved in regulating immune response in mammals. In this study, we optimized the CRISPR/Cas9 method for knocking out Rig-Igene in Goat primary fibroblasts by using a NHEJ pathway. Cells were screened for inactivation of the Rig-Igene and two positive clones were found out of thirty colonies screened. Thus, cells containing Rig-Igene inactivation could be achieved by CRISPR/Cas9 in goat fibroblast cells. 相似文献
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Alexis Eschstruth Sylvie Schneider‐Maunoury Franois Giudicelli 《Genesis (New York, N.Y. : 2000)》2020,58(1)
CRISPR/Cas9‐based strategies are widely used for genome editing in many organisms, including zebrafish. Although most applications consist in introducing double strand break (DSB)‐induced mutations, it is also possible to use CRISPR/Cas9 to enhance homology directed repair (HDR) at a chosen genomic location to create knock‐ins with optimally controlled precision. Here, we describe the use of CRISPR/Cas9‐targeted DSB followed by HDR to generate zebrafish transgenic lines where exogenous coding sequences are added in the nefma gene, in frame with the endogenous coding sequence. The resulting knock‐in embryos express the added gene (fluorescent reporter or KalTA4 transactivator) specifically in the populations of neurons that express nefma, making them convenient tools for research on these populations. 相似文献
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Linlin Zhang Jiankui Zhou Jinxiong Han Bian Hu Ningning Hou Yun Shi Xingxu Huang Xin Lou 《PloS one》2016,11(4)
The CRISPR/Cas9 system has been developed as an easy-handle and multiplexable approach for engineering eukaryotic genomes by zygote microinjection of Cas9 and sgRNA, while preparing Cas9 for microinjection is laborious and introducing inconsistency into the experiment. Here, we describe a modified strategy for gene targeting through using oocyte-specific Cas9 transgenic mouse. With this mouse line, we successfully achieve precise gene targeting by injection of sgRNAs only into one-cell-stage embryos. Through comprehensive analysis, we also show allele complexity and off-target mutagenesis induced by this strategy is obviously lower than Cas9 mRNA/sgRNA injection. Thus, injection of sgRNAs into oocyte-specific Cas9 transgenic mouse embryo provides a convenient, efficient and reliable approach for mouse genome editing. 相似文献
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CRISPR/Cas9的发现为多种生物的基因编辑提供了强有力的工具。然而,该系统在提供靶向性基因修饰的同时,会产生一些不需要的突变,即脱靶现象。为提高CRISPR/Cas9的特异性,我们将野生型FokI核酸内切酶的功能结构域与催化功能区失活的Cas9蛋白(dCas9)进行融合,形成融合蛋白用于降低脱靶效应。FokⅠ是一种依赖于二聚化才能行使内切酶活性的核酸酶,在本研究中,通过将FokⅠ功能结构融合到dCas9的N端,构建表达质粒pST1374-dCas9-FokⅠ。我们前期研究中,发现一个sgRNA在介导Cas9编辑Dnmt1基因建立条件敲除大鼠时,存在显著的脱靶现象。以此为基础,我们利用dCas9-FokⅠ/sgRNA系统编辑大鼠Dnmt1基因,研究该系统是否能够进行基因编辑以及是否能够提高基因编辑特异性。将转录好的dCas9-FokⅠ mRNA和sgRNA显微注射到SD大鼠的受精卵中,用于产生基因编辑大鼠。通过显微注射以及胚胎移植,最终获得43只F0代大鼠,其中两只在靶点位置包含突变,突变效率达4.5%。对脱靶情况进行分析,结果显示,无脱靶现象存在。综上,表明dCas9-FokⅠ/sgRNA可以应用于编辑大鼠基因,并能显著提高特异性。尽管dCas9-FokⅠ/sgRNA系统相比于Cas9/sgRNA系统,基因编辑效率有所下降,但是该技术的发展为基因治疗提供了可供选择的潜在工具。 相似文献
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Dong Zhanqi Qin Qi Hu Zhigang Chen Peng Huang Liang Zhang Xinling Tian Ting Lu Cheng Pan Minhui 《中国病毒学》2019,34(4):444-453
Recently the developed single guide(sg)RNA-guided clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease(CRISPR/Cas9) technology has opened a new avenue for antiviral therapy. The CRISPR/Cas9 system uniquely allows targeting of multiple genome sites simultaneously. However, there are relatively few applications of CRISPR/Cas9 multigene editing to target insect viruses. To address the need for sustained delivery of a multiplex CRISPR/Cas9-based genome-editing vehicle against insect viruses, we developed a one-vector(pSL1180-Cas9-U6-sgRNA) system that expresses multiple sgRNA and Cas9 protein to excise Bombyx mori nucleopolyhedrovirus(BmNPV) in insect cells.We screened the immediate-early-1 gene(ie-1), the major envelope glycoprotein gene(gp64), and the late expression factor gene(lef-11), and identified multiple sgRNA editing sites through flow cytometry and viral DNA replication analysis. In addition, we constructed a multiplex editing vector(PSL1180-Cas9-sgIE1-sgLEF11-sgGP64, sgMultiple) to efficiently regulate multiplex gene-editing and inhibit BmNPV replication after viral infection. This is the first report of the application of a multiplex CRISPR/Cas9 system to inhibit insect virus replication. This multiplex system can significantly enhance the potential of CRISPR/Cas9-based multiplex genome engineering in insect virus. 相似文献
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Genome Editing—Principles and Applications for Functional Genomics Research and Crop Improvement 总被引:1,自引:0,他引:1
Genome editing technologies are powerful tools for studying gene function and for crop improvement. The technologies rely on engineered endonucleases to generate double stranded breaks (DSBs) at target loci. The DSBs are repaired through the error-prone non-homologous end joining (NHEJ) and homology-directed repair (HDR) pathways in cells, resulting in mutations and sequence replacement, respectively. In the widely used CRISPR/Cas9 system, the endonuclease Cas9 is targeted by a CRISPR small RNA to DNA sequence of interest. In this review, we describe the four available types of genome editing tools, ZFN, TALEN, CRISPR/Cas9 and CRISPR/Cpf1, and show their applications in functional genomics research and precision molecular breeding of crops. 相似文献