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. 2014 Oct;24(10):1719-23.
doi: 10.1101/gr.176701.114. Epub 2014 Sep 3.

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

Jonathan L Schmid-Burgk  1 , Tobias Schmidt  1 , Moritz M Gaidt  1 , Karin Pelka  2 , Eicke Latz  2 , Thomas S Ebert  1 , Veit Hornung  3
Affiliations

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

Jonathan L Schmid-Burgk et al. Genome Res. 2014 Oct.

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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Figures

Figure 1.
Figure 1.
The workflow to generate and identify knockout cell clones using OutKnocker. (A) Schematic view of the developed workflow to obtain gene-targeted cell clones for subsequent deep sequencing analysis. Forty-eight hours after transfection of a designer nuclease, cells are seeded under limiting dilution conditions and cultured for 2 wk. Grown single-cell clones are picked and duplicated. One duplicate is lysed to perform a locus-specific PCR and a subsequent second PCR is performed to attach barcodes and sequencing adapters. Obtained PCR products are then pooled and subjected to deep sequencing. (B) Schematic view of the alignment and indel calling algorithm used by OutKnocker (see text for details).
Figure 2.
Figure 2.
Application of the OutKnocker analysis tool to generate TLR2 knockout THP-1 cells. (A) The genomic locus of the human TLR2 gene is depicted. Small, black square represents noncoding exons, whereas the large, gray square represents coding exons. The red arrow highlights the target site of the CRISPR that is magnified below. (B) Shown is the analysis performed by OutKnocker of 96 THP-1 clones that were treated with a CRISPR targeting human TLR2. Every pie chart represents a clone, whereas the size of each chart corresponds to the number of reads that were analyzed to evaluate the clone (see legend in the top right). Colors of the individual pie areas indicate in-frame mutations (blue), out-of-frame mutations (red), or no indel calls (gray) (see legend in the bottom right). (C) The identified indel mutations of two knockout clones (5 and 40) are depicted. Orange letters highlight the PAM sequence and red letters indicate the CRISPR target site. (D) Two knockout clones (clones 5 and 40) as well as two unmodified clones (clones 6 and 8) were stimulated with increasing amounts of either the TLR4 ligand LPS (serial dilutions 1:10 from 1 µg/mL to 0.1 ng/mL) or with the TLR2 ligand Pam3CSK4 (serial dilutions 1:10 from 1 µg/mL to 0.1 ng/mL). A representative result out of two independent experiments is depicted as mean value + SEM of biological duplicates.
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References

    1. Ablasser A, Schmid-Burgk JL, Hemmerling I, Horvath GL, Schmidt T, Latz E, Hornung V. 2013. Cell intrinsic immunity spreads to bystander cells via the intercellular transfer of cGAMP. Nature 503: 530–534 - PMC - PubMed
    1. Ablasser A, Hemmerling I, Schmid-Burgk JL, Behrendt R, Roers A, Hornung V. 2014. TREX1 deficiency triggers cell-autonomous immunity in a cGAS-dependent manner. J Immunol 192: 5993–5997 - PubMed
    1. Aliprantis AO, Yang RB, Mark MR, Suggett S, Devaux B, Radolf JD, Klimpel GR, Godowski P, Zychlinsky A. 1999. Cell activation and apoptosis by bacterial lipoproteins through toll-like receptor-2. Science 285: 736–739 - PubMed
    1. Bedell VM, Wang Y, Campbell JM, Poshusta TL, Starker CG, Krug RG 2nd, Tan W, Penheiter SG, Ma AC, Leung AY, et al. . 2012. In vivo genome editing using a high-efficiency TALEN system. Nature 491: 114–118 - PMC - PubMed
    1. Carroll D. 2011. Genome engineering with zinc-finger nucleases. Genetics 188: 773–782 - PMC - PubMed

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