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Rimsha Farooq, Khadim Hussain, Shahid Nazir, Muhammad Rizwan Javed, Nazish Masood
CRISPR/Cas9 is a technology evolved from modified type II immune system of bacteria and archaea. Exploitation of this bacterial immune system in all eukaryotes including plants may lead to site-specific targeted genome engineering. Genome engineering is objectively utilized to express/silence a trait harbouring gene in the plant genome. In this review, different genetic engineering techniques including classical breeding, RNAi and genetic transformation and synthetic sequence-specific nucleases (zinc finger nucleases; ZFNs and transcription activator-like effector nuclease; TALENs) techniques have been described and compared with advanced genome editing technique CRISPR/Cas9, on the basis of their merits and drawbacks...
November 30, 2018: Cellular and Molecular Biology
Naoaki Mizuno, Eiji Mizutani, Hideyuki Sato, Mariko Kasai, Aki Ogawa, Fabian Suchy, Tomoyuki Yamaguchi, Hiromitsu Nakauchi
Intra-embryo genome editing by CRISPR/Cas9 enables easy generation of gene-modified animals by non-homologous end joining (NHEJ)-mediated frameshift mutations or homology-directed repair (HDR)-mediated point mutations. However, large modifications, such as gene replacement or gene fusions, are still difficult to introduce in embryos without costly micromanipulators. Moreover, micromanipulation techniques for intra-embryo genome editing have been established in only a small set of animals. To overcome these issues, we developed a method of large-fragment DNA knockin without micromanipulation...
November 2, 2018: iScience
Justin S Antony, Ngadhnjim Latifi, A K M Ashiqul Haque, Andrés Lamsfus-Calle, Alberto Daniel-Moreno, Sebastian Graeter, Praveen Baskaran, Petra Weinmann, Markus Mezger, Rupert Handgretinger, Michael S D Kormann
BACKGROUND: β-Thalassemia is an inherited hematological disorder caused by mutations in the human hemoglobin beta (HBB) gene that reduce or abrogate β-globin expression. Although lentiviral-mediated expression of β-globin and autologous transplantation is a promising therapeutic approach, the risk of insertional mutagenesis or low transgene expression is apparent. However, targeted gene correction of HBB mutations with programmable nucleases such as CRISPR/Cas9, TALENs, and ZFNs with non-viral repair templates ensures a higher safety profile and endogenous expression control...
November 14, 2018: Molecular and Cellular Pediatrics
Khadijeh Zare, Milad Shademan, Mohammad M Ghahramani Seno, Hesam Dehghani
Background: With the increasing discovery of long noncoding RNAs (lncRNAs), the application of functional techniques that could have very specific, efficient, and robust effects and readouts is necessary. Here, we have applied and analyzed three gene knockout (KO) strategies to ablate the CCAT1 gene in different colorectal adenocarcinoma cell lines. We refer to these strategies as "CRISPR excision", "CRISPR HDR", and "CRISPR du-HITI". Results: In order to obstruct the transcription of lncRNA or to alter its structure, in these strategies either a significant segment of the gene is removed, or a transcription termination signal is inserted in the target gene...
2018: Biological Procedures Online
C J Pickett, Robert W Zeller
Eliminating or silencing a gene's level of activity is one of the classic approaches developmental biologists employ to determine a gene's function. A recently developed method of gene perturbation called CRISPR-Cas, which was derived from a prokaryotic adaptive immune system, has been adapted for use in eukaryotic cells. This technology has been established in several model organisms as a powerful and efficient tool for knocking out or knocking down the function of a gene of interest. It has been recently shown that CRISPR-Cas functions with fidelity and efficiency in Ciona robusta...
October 30, 2018: Genesis: the Journal of Genetics and Development
Anastasia Lomova, Danielle N Clark, Beatriz Campo-Fernandez, Carmen Flores-Bjurström, Michael L Kaufman, Sorel Fitz-Gibbon, Xiaoyan Wang, Eric Y Miyahira, Devin Brown, Mark A DeWitt, Jacob E Corn, Roger P Hollis, Zulema Romero, Donald B Kohn
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated system (Cas9)-mediated gene editing of human hematopoietic stem cells (hHSCs) is a promising strategy for the treatment of genetic blood diseases through site-specific correction of identified causal mutations. However, clinical translation is hindered by low ratio of precise gene modification using the corrective donor template (homology-directed repair, HDR) to gene disruption (nonhomologous end joining, NHEJ) in hHSCs. By using a modified version of Cas9 with reduced nuclease activity in G1 phase of cell cycle when HDR cannot occur, and transiently increasing the proportion of cells in HDR-preferred phases (S/G2), we achieved a four-fold improvement in HDR/NHEJ ratio over the control condition in vitro, and a significant improvement after xenotransplantation of edited hHSCs into immunodeficient mice...
October 29, 2018: Stem Cells
Annekatrien Boel, Hanna De Saffel, Wouter Steyaert, Bert Callewaert, Anne De Paepe, Paul J Coucke, Andy Willaert
Targeted genome editing by CRISPR/Cas9 is extremely well fitted to generate gene disruptions, although precise sequence replacement by CRISPR/Cas9-mediated homology-directed repair (HDR) suffers from low efficiency, impeding its use for high-throughput knock-in disease modeling. In this study, we used next-generation sequencing (NGS) analysis to determine the efficiency and reliability of CRISPR/Cas9-mediated HDR using several types of single-stranded oligodeoxynucleotide (ssODN) repair templates for the introduction of disease-relevant point mutations in the zebrafish genome...
October 18, 2018: Disease Models & Mechanisms
Nan Hu, Sami N Malek
The emergence of the clustered, regularly interspaced, short palindromic repeat (CRISPR) technology provides tools for researchers to modify genomes in a specific and efficient manner. The Type II CRISPR-Cas9 system enables gene editing by directed DNA cleavage followed by either non-homologous end joining (NHEJ) or homology-directed repair (HDR). Here, we described the use of the Type II CRISPR-Cas9 system in detail from designing the guides to analyzing the desired gene disruption events.
2019: Methods in Molecular Biology
Nitasha Sehgal, M Eileen Sylves, Ansuman Sahoo, Jacky Chow, Sarah E Walker, Paul J Cullen, James O Berry
Clustered regularly interspaced short palindromic repeats (CRISPR) are a revolutionary tool based on a bacterial acquired immune response system. CRISPR has gained widespread use for gene editing in a variety of organisms and is an increasingly valuable tool for basic genetic research, with far-reaching implications for medicine, agriculture, and industry. This lab is based on the premise that upper division undergraduate students enrolled in a Life Sciences curriculum must become familiar with cutting edge advances in biotechnology that have significant impact on society...
November 2018: Biochemistry and Molecular Biology Education
Alewo Idoko-Akoh, Lorna Taylor, Helen M Sang, Michael J McGrew
Primordial germ cells (PGCs), the embryonic precursors of the sperm and egg, are used for the introduction of genetic modifications into avian genome. Introduction of small defined sequences using genome editing has not been demonstrated in bird species. Here, we compared oligonucleotide-mediated HDR using wild type SpCas9 (SpCas9-WT) and high fidelity SpCas9-HF1 in PGCs and show that many loci in chicken PGCs can be precise edited using donors containing CRISPR/Cas9-blocking mutations positioned in the protospacer adjacent motif (PAM)...
October 11, 2018: Scientific Reports
Yanchi Wang, Junya Zhao, Nannan Duan, Wei Liu, Yuxuan Zhang, Miaojin Zhou, Zhiqing Hu, Mai Feng, Xionghao Liu, Lingqian Wu, Zhuo Li, Desheng Liang
Hemophilia B (HB) is an X-linked recessive bleeding disorder, caused by F9 gene deficiency. Gene therapy combined with the CRISPR/Cas9 technology offers a potential cure for hemophilia B. Now the Cas9 nickase (Cas9n) shows a great advantage in reducing off-target effect compared with wild-type Cas9. In this study, we found that in the multicopy ribosomal DNA (rDNA) locus, the homology directed recombination (HDR) efficiency induced by sgRNA-Cas9n was much higher than sgRNA-Cas9, meanwhile without off-target in six predicted sites...
October 5, 2018: International Journal of Molecular Sciences
Lukas Villiger, Hiu Man Grisch-Chan, Helen Lindsay, Femke Ringnalda, Chiara B Pogliano, Gabriella Allegri, Ralph Fingerhut, Johannes Häberle, Joao Matos, Mark D Robinson, Beat Thöny, Gerald Schwank
CRISPR-Cas-based genome editing holds great promise for targeting genetic disorders, including inborn errors of hepatocyte metabolism. Precise correction of disease-causing mutations in adult tissues in vivo, however, is challenging. It requires repair of Cas9-induced double-stranded DNA (dsDNA) breaks by homology-directed mechanisms, which are highly inefficient in nondividing cells. Here we corrected the disease phenotype of adult phenylalanine hydroxylase (Pah)enu2 mice, a model for the human autosomal recessive liver disease phenylketonuria (PKU)1 , using recently developed CRISPR-Cas-associated base editors2-4 ...
October 2018: Nature Medicine
Lijuan Du, Amy Zhou, Alex Sohr, Sougata Roy
Binary transcription systems are powerful genetic tools widely used for visualizing and manipulating cell fate and gene expression in specific groups of cells or tissues in model organisms. These systems contain two components as separate transgenic lines. A driver line expresses a transcriptional activator under the control of tissue-specific promoters/enhancers, and a reporter/effector line harbors a target gene placed downstream to the binding site of the transcription activator. Animals harboring both components induce tissue-specific transactivation of a target gene expression...
September 19, 2018: Journal of Visualized Experiments: JoVE
Yuning Song, Yuxin Zhang, Mao Chen, Jichao Deng, Tingting Sui, Liangxue Lai, Zhanjun Li
BACKGROUND: Oculocutaneous albinism (OCA) is a group of autosomal recessive disorders characterized by reduced melanin that are caused by mutations in the gene encoding tyrosinase (TYR), which is the rate-limiting enzyme in the production of the pigment melanin. Many studies or meta-analyses have suggested an association between the TYR T373K SNP and OCA1, but there is limited biochemical and genetic evidence to support this association. METHODS: We overexpressed TYR-WT and TYR-T373K mutants on HK293T cells and tested the changes of melanin production and tyrosinase activity...
October 2018: EBioMedicine
Eric J Aird, Klaus N Lovendahl, Amber St Martin, Reuben S Harris, Wendy R Gordon
The CRISPR-Cas9 system is a powerful genome-editing tool in which a guide RNA targets Cas9 to a site in the genome, where the Cas9 nuclease then induces a double-stranded break (DSB). The potential of CRISPR-Cas9 to deliver precise genome editing is hindered by the low efficiency of homology-directed repair (HDR), which is required to incorporate a donor DNA template encoding desired genome edits near the DSB. We present a strategy to enhance HDR efficiency by covalently tethering a single-stranded oligodeoxynucleotide (ssODN) to the Cas9-guide RNA ribonucleoprotein (RNP) complex via a fused HUH endonuclease, thus spatially and temporally co-localizing the DSB machinery and donor DNA...
2018: Communications biology
Cristina Parola, Derek M Mason, Andreas Zingg, Daniel Neumeier, Sai T Reddy
From the perspective of academic and small research laboratories, the most common and practical strategy for recombinant expression of full-length monoclonal antibodies is to perform transient plasmid transfection of mammalian cells, resulting in small-scale and limited protein production. The generation of stable antibody producing mammalian cell lines enables larger-scale and consistent expression, however this approach is rarely pursued due to the time-consuming and expensive process of single colony screening and characterization...
2018: Methods in Molecular Biology
Xiao-Lan Li, Guo-Hua Li, Juan Fu, Ya-Wen Fu, Lu Zhang, Wanqiu Chen, Cameron Arakaki, Jian-Ping Zhang, Wei Wen, Mei Zhao, Weisheng V Chen, Gary D Botimer, David Baylink, Leslie Aranda, Hannah Choi, Rachel Bechar, Prue Talbot, Chang-Kai Sun, Tao Cheng, Xiao-Bing Zhang
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...
November 2, 2018: Nucleic Acids Research
Nicholas D Bonawitz, W Michael Ainley, Asuka Itaya, Sivarama R Chennareddy, Tobias Cicak, Katherine Effinger, Ke Jiang, Tejinder Kumar Mall, Pradeep Reddy Marri, J Pon Samuel, Nagesh Sardesai, Matthew Simpson, Otto Folkerts, Rodrigo Sarria, Steven R Webb, Delkin O Gonzalez, Daina H Simmonds, Dayakar R Pareddy
Emerging genome editing technologies hold great promise for the improvement of agricultural crops. Several related genome editing methods currently in development utilize engineered, sequence-specific endonucleases to generate DNA double strand breaks (DSBs) at user-specified genomic loci. These DSBs subsequently result in small insertions/deletions (indels), base substitutions or incorporation of exogenous donor sequences at the target site, depending on the application. Targeted mutagenesis in soybean (Glycine max) via non-homologous end joining (NHEJ)-mediated repair of such DSBs has been previously demonstrated with multiple nucleases, as has homology-directed repair (HDR)-mediated integration of a single transgene into target endogenous soybean loci using CRISPR/Cas9...
September 16, 2018: Plant Biotechnology Journal
Amanda Haupt, Tanya Grancharova, Joy Arakaki, Margaret A Fuqua, Brock Roberts, Ruwanthi N Gunawardane
A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame N- or C-terminal fluorescent tags. The prokaryotic CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) may be used to introduce large exogenous sequences into genomic loci via homology directed repair (HDR). To achieve the desired knock-in, this protocol employs the ribonucleoprotein (RNP)-based approach where wild type Streptococcus pyogenes Cas9 protein, synthetic 2-part guide RNA (gRNA), and a donor template plasmid are delivered to the cells via electroporation...
August 25, 2018: Journal of Visualized Experiments: JoVE
Huibin Tian, Jun Luo, Zhifei Zhang, Jiao Wu, Tianying Zhang, Sebastiano Busato, Lian Huang, Ning Song, Massimo Bionaz
Stearoyl-CoA desaturase 1 (SCD1) is a fatty acid desaturase catalyzing cis-double-bond formation in the Δ9 position to produce monounsaturated fatty acids essential for the synthesis of milk fat. Previous studies using RNAi methods have provided support for a role of SCD1 in goat mammary epithelial cells (GMEC); however, RNAi presents several limitations that might preclude a truthful understanding of the biological function of SCD1. To explore the function of SCD1 on fatty acid metabolism in GMEC, we used CRISPR-Cas9-mediated SCD1 knockout through non-homologous end-joining (NHEJ) and homology-directed repair (HDR) pathways in GMEC...
September 26, 2018: Journal of Agricultural and Food Chemistry
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