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      OutKnocker: a web tool for rapid and simple genotyping of designer nuclease edited cell lines

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          Abstract

          The application of designer nucleases allows the induction of DNA double-strand breaks (DSBs) at user-defined genomic loci. Due to imperfect DNA repair mechanisms, DSBs can lead to alterations in the genomic architecture, such as the disruption of the reading frame of a critical exon. This can be exploited to generate somatic knockout cell lines. While high genome editing activities can be achieved in various cellular systems, obtaining cell clones that contain all-allelic frameshift mutations at the target locus of interest remains a laborious task. To this end, we have developed an easy-to-follow deep sequencing workflow and the evaluation tool OutKnocker ( www.OutKnocker.org), which allows convenient, reliable, and cost-effective identification of knockout cell lines.

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          A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.

          Martin Jinek, Krzysztof Chylinski, Ines Fonfara … (2012)
          Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.
          Article 0 comments Cited 3526 times – based on 0 reviews     Review now
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            Multiplex genome engineering using CRISPR/Cas systems.

            Le Cong, F. Ran, David T. Cox … (2013)
            Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
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              RNA-guided human genome engineering via Cas9.

              P. Mali, L. YANG, K. M. Esvelt … (2013)
              Bacteria and archaea have evolved adaptive immune defenses, termed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems, that use short RNA to direct degradation of foreign nucleic acids. Here, we engineer the type II bacterial CRISPR system to function with custom guide RNA (gRNA) in human cells. For the endogenous AAVS1 locus, we obtained targeting rates of 10 to 25% in 293T cells, 13 to 8% in K562 cells, and 2 to 4% in induced pluripotent stem cells. We show that this process relies on CRISPR components; is sequence-specific; and, upon simultaneous introduction of multiple gRNAs, can effect multiplex editing of target loci. We also compute a genome-wide resource of ~190 K unique gRNAs targeting ~40.5% of human exons. Our results establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.
              Article 0 comments Cited 1616 times – based on 0 reviews     Review now
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                Author and article information

                Journal
                Journal ID (nlm-ta): Genome Res
                Journal ID (iso-abbrev): Genome Res
                Journal ID (hwp): genome
                Journal ID (pmc): genome
                Journal ID (publisher-id): GENOME
                Title: Genome Research
                Publisher: Cold Spring Harbor Laboratory Press
                ISSN (Print): 1088-9051
                ISSN (Electronic): 1549-5469
                Publication date (Print): October 2014
                Volume: 24
                Issue: 10
                Pages: 1719-1723
                Affiliations
                [1 ]Institute of Molecular Medicine, University Hospital, University of Bonn, 53127 Bonn, Germany;
                [2 ]Institute of Innate Immunity, University Hospital, University of Bonn, 53127 Bonn, Germany
                Author notes
                [3]

                These authors contributed equally to this work.

                Corresponding author: veit.hornung@ 123456uni-bonn.de
                Article
                Medline ID: 9518021
                DOI: 10.1101/gr.176701.114
                PMC ID: 4199374
                PubMed ID: 25186908
                SO-VID: 5a0b452b-4a5f-4aac-888a-7d7247c5bb2e
                Copyright © © 2014 Schmid-Burgk et al.; Published by Cold Spring Harbor Laboratory Press
                License:

                This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

                History
                Date received : 2 April 2014
                Date accepted : 14 July 2014
                Page count
                Pages: 5
                Categories
                Subject: Resource

                Data availability:

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