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Aki Hayashi, Katsunori Tanaka
The CRISPR/Cas9 system enables the editing of genomes of numerous organisms through the induction of the double-strand breaks (DSB) at specific chromosomal targets. We improved the CRISPR/Cas9 system to ease the direct introduction of a point mutation or a tagging sequence into the chromosome by combining it with the noncanonical homology-directed DNA repair (HDR) based genome editing in fission yeast. We constructed convenient cloning vectors, which possessed a guide RNA (gRNA) expression module, or the humanized Streptococcus pyogenes Cas9 gene that is expressed under the control of an inducible promoter to avoid the needless expression, or both a gRNA and Cas9 gene...
February 12, 2019: G3: Genes—Genomes—Genetics
(no author information available yet)
Quality control (QC) analysis is an integral part of nucleic acid and CRISPR applications. Utilizing QC checkpoints at critical junctures in the CRISPR process eliminates unsuitable samples, saves time and money, and maximizes resources. This in turn increases confidence in results and builds consistency in the workflow. Advanced Analytical Technologies, Inc. provides reliable and consistent quality analysis with parallel capillary electrophoresis (CE) instruments. CE instruments provide extreme sensitivity and resolution that make them ideal for QC in CRISPR experiments, from detection of minimal degradation in sgRNA to detection of dsDNA starting product when generating ssDNA end-product for HDR, to determining the length of polyadenylated Cas9 mRNA...
June 2018: BioTechniques
Jian Gao, Thorsten Bergmann, Wenli Zhang, Maren Schiwon, Eric Ehrke-Schulz, Anja Ehrhardt
Gene therapy represents an attractive alternative to treat hemophilia B. Here we established three hepatocyte-derived cell lines based on Huh7, PLC/PRF/5, and Hep3B cells stably carrying a mutated canine FIX (cFIXmut) transgene containing a single point mutation in the catalytic domain. Based on these in vitro models resembling a commonly used canine large animal model, the tetracycline-controlled transcriptional activator (Tet-on)-inducible CRISPR/Cas9 system and an optimized donor were used to correct mutated cFIX gene through homology-directed repair (HDR)...
December 20, 2018: Molecular Therapy. Nucleic Acids
Mohamed Darwish, Hirofumi Nishizono, Hideki Uosaki, Hitomi Sawada, Taketaro Sadahiro, Masaki Ieda, Keizo Takao
BACKGROUND: The CRISPR/Cas9 technique has undergone many modifications to decrease the effort and shorten the time needed for efficient production of mutant mice. The use of fresh embryos consumes time and effort during oocytes preparation and fertilization before every experiment, and freeze-thawed embryos overcome this limitation. However, cryopreservation of 1-cell embryos is challenging. NEW METHOD: We introduce a protocol that combines a modified method for cryopreserving 1-cell C57BL/6 J embryos with optimized electroporation conditions to deliver CRISPR reagents into embryos, 1 hour after thawing...
January 23, 2019: Journal of Neuroscience Methods
Nataša Savić, Femke Cas Ringnalda, Christian Berk, Katja Bargsten, Jonathan Hall, Martin Jinek, Gerald Schwank
The CRISPR-Cas9 system is a powerful genome-editing tool that promises application for gene editing therapies. The Cas9 nuclease is directed to the DNA by a programmable single guide (sg)RNA, and introduces a site-specific double-stranded break (DSB). In mammalian cells, DSBs are either repaired by non-homologous end joining (NHEJ), generating small insertion/deletion (indel) mutations, or by homology-directed repair (HDR). If ectopic donor templates are provided, the latter mechanism allows editing with single-nucleotide precision...
January 5, 2019: Bio-protocol
Wasu Supharattanasitthi, Emil Carlsson, Umar Sharif, Luminita Paraoan
CRISPR/Cas9 causes double-stranded DNA breaks that can undergo DNA repair either via non-homologous end joining (NHEJ) or, in the presence of a template, homology-directed repair (HDR). HDR is typically used to insert a specific genetic modification into the genome but has low efficiency compared to NHEJ, which is lowered even further when trying to create a homozygous change. In this study we devised a novel approach for homozygous single base editing based on utilising simultaneously two donor DNA templates cloned in plasmids with different antibiotic resistant genes...
January 17, 2019: Scientific Reports
Jianwei Li, Alfred M Handler
CRISPR/Cas9-mediated gene-editing, using injected Cas9 protein, was achieved in the Caribbean fruit fly, Anastrepha suspensa, by initially targeting an exogenous transgene, polyubiquitin-regulated EGFP (PUb-EGFP), for heritable non-homologous end-joining (NHEJ) knock-outs using an individual sgRNA. Multiple deletion mutations, ranging from two to five nts proximal to the target site, were identified phenotypically by the loss of green fluorescence in transgenic flies that were also marked with PUb-DsRed. This represented a relatively high efficiency rate of 29% for germ-line mutations...
January 3, 2019: Gene
Eirini M Kallimasioti-Pazi, Keerthi Thelakkad Chathoth, Gillian C Taylor, Alison Meynert, Tracy Ballinger, Martijn J E Kelder, Sébastien Lalevée, Ildem Sanli, Robert Feil, Andrew J Wood
Genome editing occurs in the context of chromatin, which is heterogeneous in structure and function across the genome. Chromatin heterogeneity is thought to affect genome editing efficiency, but this has been challenging to quantify due to the presence of confounding variables. Here, we develop a method that exploits the allele-specific chromatin status of imprinted genes in order to address this problem in cycling mouse embryonic stem cells (mESCs). Because maternal and paternal alleles of imprinted genes have identical DNA sequence and are situated in the same nucleus, allele-specific differences in the frequency and spectrum of mutations induced by CRISPR-Cas9 can be unequivocally attributed to epigenetic mechanisms...
December 12, 2018: PLoS Biology
Benoit Danilo, Laura Perrot, Emmanuel Botton, Fabien Nogué, Marianne Mazier
Targeted insertion of transgenes in plants is still challenging and requires further technical innovation. In the present study, we chose the tomato DFR gene involved in anthocyanin biosynthesis as a landing pad for targeted transgene insertion using CRISPR-Cas9 in a two-step strategy. First, a 1013 bp was deleted in the endogenous DFR gene. Hypocotyls and callus of in vitro regenerated plantlets homozygous for the deletion were green instead of the usual anthocyanin produced purple colour. Next, standard Agrobacterium-mediated transformation was used to target transgene insertion at the DFR landing pad in the dfr deletion line...
2018: PloS One
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
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