Search

Your search keyword '"genetics [Endonucleases]"' showing total 3 results
Start Over Descriptor "genetics [Endonucleases]"
3 results on '"genetics [Endonucleases]"'

1. Gene editing in mouse zygotes using the CRISPR/Cas9 system

Author

Sanum Bashir, Wolfgang Wurst, Jana Rossius, Benedikt Wefers, and Ralf Kühn

Subjects
metabolism [RNA, Guide], 0301 basic medicine, DNA End-Joining Repair, RNA, Untranslated, Zygote, Mutant, Gene Knockout Techniques, Mice, Genome editing, CRISPR-Associated Protein 9, RNA, Guide, CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats, DNA Breaks, Double-Stranded, Gene Knock-In Techniques, genetics [RNA, Untranslated], Cas9, Crispr, Gene Editing, Mouse, Zygotes, Genetics, Genome, Cas9 endonuclease Streptococcus pyogenes, Electroporation, Gene Transfer Techniques, methods [Electroporation], ddc:540, methods [Gene Targeting], Gene Targeting, genetics [Endonucleases], genetics [Bacterial Proteins], RNA, Guide, Kinetoplastida, methods [Gene Editing], Microinjections, metabolism [Bacterial Proteins], metabolism [Endonucleases], Mice, Transgenic, Computational biology, Biology, genetics [RNA, Guide, Kinetoplastida], General Biochemistry, Genetics and Molecular Biology, metabolism [RNA, Untranslated], 03 medical and health sciences, Bacterial Proteins, genetics [RNA, Guide], metabolism [RNA, Guide, Kinetoplastida], Gt(ROSA)26Sor non-coding RNA, mouse, Animals, Molecular Biology, Genotyping, Microinjection, genetics [DNA], Recombinational DNA Repair, DNA, Endonucleases, cytology [Zygote], metabolism [Zygote], 030104 developmental biology, Animals, Newborn, metabolism [DNA], CRISPR-Cas Systems
Abstract

The generation of targeted mouse mutants is a key technology for biomedical research. Using the CRISPR/Cas9 system for induction of targeted double-strand breaks, gene editing can be performed in a single step directly in mouse zygotes. This article covers the design of knockout and knockin alleles, preparation of reagents, microinjection or electroporation of zygotes and the genotyping of pups derived from gene editing projects. In addition we include a section for the control of experimental settings by targeting the Rosa26 locus and PCR based genotyping of blastocysts.

Published
2016

2. Genome Editing in Mice Using TALE Nucleases

Author

Wefers, Benedikt, Brandl, Christina, Ortiz, Oskar, Wurst, Wolfgang, Kühn, Ralf, Kuehn, R., Wurst, W., and Wefers, B.

Subjects
Cancer Research, Genome, Microinjections, methods [Gene Knockout Techniques], genetics [RNA Editing], Endonucleases, genetics [Animals, Genetically Modified], Animals, Genetically Modified, genetics [RNA, Messenger], Gene Knockout Techniques, Mice, ddc:570, Mutation, Animals, genetics [Endonucleases], DNA Breaks, Double-Stranded, RNA Editing, RNA, Messenger, Technology Platforms
Abstract

Gene engineering for generating targeted mouse mutants is a key technology for biomedical research. Using TALENs as sequence-specific nucleases to induce targeted double-strand breaks, the mouse genome can be directly modified in zygotes in a single step without the need for embryonic stem cells. By embryo microinjection of TALEN mRNAs and targeting vectors, knockout and knock-in alleles can be generated fast and efficiently. In this chapter we provide protocols for the application of TALENs in mouse zygotes.

Published
2015

3. Efficient CRISPR/Cas9 genome editing with low off-target effects in zebrafish

Author

Alexandra Rechenberg, Peter Krawitz, Jochen Hecht, Alexander Hruscha, Verena Heinrich, Christian Haass, and Bettina Schmid

Subjects
methods [Gene Editing], CRISPR-Associated Proteins, Mutagenesis (molecular biology technique), Biology, Genome, Genome engineering, 03 medical and health sciences, 0302 clinical medicine, Genome editing, Bacterial Proteins, ddc:570, CRISPR-Associated Protein 9, CRISPR, Animals, Gene Knock-In Techniques, Molecular Biology, Gene, Zebrafish, 030304 developmental biology, genetics [Zebrafish], Genetics, Gene Editing, genetics [CRISPR-Associated Proteins], 0303 health sciences, CRISPR interference, Base Sequence, Cas9 endonuclease Streptococcus pyogenes, Cas9, genetics [Oligonucleotides, Antisense], Sequence Analysis, DNA, Oligonucleotides, Antisense, Endonucleases, Mutagenesis, genetics [Endonucleases], CRISPR-Cas Systems, 030217 neurology & neurosurgery, genetics [Bacterial Proteins], Developmental Biology
Abstract

Gene modifications in animal models have been greatly facilitated through the application of targeted genome editing tools. The prokaryotic CRISPR/Cas9 type II genome editing system has recently been applied in cell lines and vertebrates. However, we still have very limited information about the efficiency of mutagenesis, germline transmission rates and off-target effects in genomes of model organisms. We now demonstrate that CRISPR/Cas9 mutagenesis in zebrafish is highly efficient, reaching up to 86.0%, and is heritable. The efficiency of the CRISPR/Cas9 system further facilitated the targeted knock-in of a protein tag provided by a donor oligonucleotide with knock-in efficiencies of 3.5-15.6%. Mutation rates at potential off-target sites are only 1.1-2.5%, demonstrating the specificity of the CRISPR/Cas9 system. The ease and efficiency of the CRISPR/Cas9 system with limited off-target effects make it a powerful genome engineering tool for in vivo studies.

Published
2013

Catalog

Books, media, physical & digital resources
See catalog results