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

Schmid-Burgk, Jonathan L.; Schmidt, Tobias; Gaidt, Moritz M.; Pelka, Karin; Latz, Eicke; Ebert, Thomas S.; Hornung, Veit

doi:10.1101/gr.176701.114

Cited by 132 publications

(143 citation statements)

References 15 publications

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“…To validate the role of Nek7 in inflammasome signaling and to explore its epistatic role in Nlrp3 activation, we generated single cell clones of Nlrp3-Cas9 macrophages that had been transduced with a gRNA targeting Nek7 (15). Following this targeting approach, we obtained several cell clones bearing allallelic frameshift mutations within the Nek7 target region.…”

Section: Resultsmentioning

confidence: 99%

“…From each PCR reaction, 1 l was transferred to a second PCR reaction using the same cycling conditions, but individual combinations of barcode primers described in Ref. 15. PCR products were pooled, gel-purified, precipitated as described (15) and sequenced using the MiSeq deep sequencing platform.…”

Section: Methodsmentioning

confidence: 99%

“…15. PCR products were pooled, gel-purified, precipitated as described (15) and sequenced using the MiSeq deep sequencing platform. Raw data were evaluated using custom-written software that counts the number of primer barcode combinations with which each individual library gRNA sequence was sequenced, which corresponds to the absolute number of initially sorted cells.…”

Section: Methodsmentioning

confidence: 99%

“…Subsequently, growing cell clones were picked and duplicated. One of the duplicates was lysed for deep sequencing-based genotyping (15,16). Two all-allelic Nek7 knock-out cell clones as well as two clones generated with a control gRNA transduction were expanded for functional analysis.…”

Section: Methodsmentioning

confidence: 99%

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A Genome-wide CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Screen Identifies NEK7 as an Essential Component of NLRP3 Inflammasome Activation

Schmid-Burgk

Chauhan

Schmidt

et al. 2016

Journal of Biological Chemistry

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382

276

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Inflammasomes are high molecular weight protein complexes that assemble in the cytosol upon pathogen encounter. This results in caspase-1-dependent pro-inflammatory cytokine maturation, as well as a special type of cell death, known as pyroptosis. The Nlrp3 inflammasome plays a pivotal role in pathogen defense, but at the same time, its activity has also been implicated in many common sterile inflammatory conditions. To this effect, several studies have identified Nlrp3 inflammasome engagement in a number of common human diseases such as atherosclerosis, type 2 diabetes, Alzheimer disease, or gout. Although it has been shown that known Nlrp3 stimuli converge on potassium ion efflux upstream of Nlrp3 activation, the exact molecular mechanism of Nlrp3 activation remains elusive. Here, we describe a genome-wide CRISPR/Cas9 screen in immortalized mouse macrophages aiming at the unbiased identification of gene products involved in Nlrp3 inflammasome activation. We employed a FACS-based screen for Nlrp3-dependent cell death, using the ionophoric compound nigericin as a potassium efflux-inducing stimulus. Using a genome-wide guide RNA (gRNA) library, we found that targeting Nek7 rescued macrophages from nigericininduced lethality. Subsequent studies revealed that murine macrophages deficient in Nek7 displayed a largely blunted Nlrp3 inflammasome response, whereas Aim2-mediated inflammasome activation proved to be fully intact. Although the mechanism of Nek7 functioning upstream of Nlrp3 yet remains elusive, these studies provide a first genetic handle of a component that specifically functions upstream of Nlrp3.Employing an evolutionary conserved set of pattern recognition receptors (PRRs), 2 the innate immune system senses the presence of microbial pathogens (1). Although PRRs can directly detect microbe-associated molecular patterns, some PRRs also respond to endogenous, host-derived signals that are formed or released upon perturbation or damage that is caused by microbial infections. These signals, which are commonly referred to as damage-associated molecular patterns, can also trigger PRR activation in the context of sterile inflammatory conditions (2). At the cell-autonomous level, PRR engagement and associated signaling cascades can result in a diverse set of responses, such as the induction of pro-inflammatory gene expression, the control of cytoskeletal rearrangement (e.g. in the context of phagocytosis), as well as the activation of proteolytic cascades, such as the inflammasome pathway. The inflammasome is a cytosolic multiprotein complex that regulates the activation and processing of caspase-1 (3). Inflammasome sensor proteins employ the adapter protein ASC to recruit caspase-1, which in turn results in the proximity-induced autoprocessing and activation of caspase-1. Active caspase-1 cleaves and thereby matures pro-inflammatory cytokines such as IL-1␤ and IL-18, and at the same time, it triggers an inflammatory type of cell death known as pyroptosis (3). The exact mechanisms of this caspase-1-dependent ce...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

A Genome-wide CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Screen Identifies NEK7 as an Essential Component of NLRP3 Inflammasome Activation

Schmid-Burgk

Chauhan

Schmidt

et al. 2016

Journal of Biological Chemistry

Self Cite

382

276

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“…Human UNC93B1-knockout THP-1 monocytes generated by CRISPR/ Cas9-based gene editing were retrovirally transduced with UNC93B1-mCitrine WT or nonfunctional H412R mutant and have been described previously (24,25). THP-1 cells were differentiated overnight with 100 nM PMA in RPMI 1640 supplemented with 10% FCS prior to stimulation.…”

Section: Cell Linesmentioning

confidence: 99%

TLR8 Senses Bacterial RNA in Human Monocytes and Plays a Nonredundant Role for Recognition of Streptococcus pyogenes

Eigenbrod

Pelka

Latz

et al. 2015

The Journal of Immunology

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Microbial nucleic acids constitute an important group of pathogen-associated molecular patterns (PAMPs) that efficiently trigger innate immune activation. In mice, TLR13 has recently been identified to sense a highly conserved region within bacterial 23S rRNA. However, TLR13 is not expressed in humans, and the identity of its human homolog remains elusive. Moreover, the contribution of bacterial RNA to the induction of innate immune responses against entire bacteria is still insufficiently defined. In the current study, we show that human monocytes respond to bacterial RNA with secretion of IL-6, TNF, and IFN-β, which is critically dependent on lysosomal maturation. Using small interfering RNA and overexpression, we unambiguously identify TLR8 as receptor for bacterial RNA in primary human monocyte-derived macrophages. We further demonstrate that the sequence motif sensed by TLR8 is clearly distinct from that recognized by TLR13. Moreover, TLR8-dependent detection of bacterial RNA was critical for triggering monocyte activation in response to infection with Streptococcus pyogenes. Bacterial RNA within streptococci was also a dominant stimulus for murine immune cells, highlighting the physiological relevance of RNA sensing in defense of infections.

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Trapping all ERBB ligands decreases pancreatic lesions in a murine model of pancreatic ductal adenocarcinoma

Hedegger¹,

Blutke²,

Hommel

et al. 2023

Molecular Oncology

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Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest of cancers. Attempts to develop targeted therapies still need to be established. Some oncogenic mechanisms in PDAC carcinogenesis harness the EGFR/ERBB receptor family. To explore the effects on pancreatic lesions, we attempted simultaneous blockade of all ERBB ligands in a PDAC mouse model. To this end, we engineered a molecular decoy, TRAP‐FC, comprising the ligand‐binding domains of both EGFR and ERBB4 and able to trap all ERBB ligands. Next, we generated a transgenic mouse model (CBATRAP/0) expressing TRAP‐FC ubiquitously under the control of the chicken‐beta‐actin promoter and crossed these mice with KRASG12D/+ mice (Kras) to generate Trap/Kras mice. The resulting mice displayed decreased emergence of spontaneous pancreatic lesion areas and exhibited reduced RAS activity and decreased activities of ERBBs, with the exception of ERBB4, which showed increased activity. To identify the involved receptor(s), we employed CRISPR/Cas9 DNA editing to singly delete each ERBB receptor in the human pancreatic carcinoma cell line Panc‐1. Ablation of each ERBB family member, especially the loss of EGFR or ERBB2/HER2, altered signaling downstream of the other three ERBB receptors and decreased cell proliferation, migration, and tumor growth. We conclude that simultaneously blocking the entire ERBB receptor family is therapeutically more effective than individually inhibiting only one receptor or ligand in terms of reducing pancreatic tumor burden. In summary, trapping all ERBB ligands can reduce pancreatic lesion area and RAS activity in a murine model of pancreatic adenocarcinoma; hence, it might represent a promising approach to treat PDAC in patients.

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

Cited by 132 publications

References 15 publications

A Genome-wide CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Screen Identifies NEK7 as an Essential Component of NLRP3 Inflammasome Activation

A Genome-wide CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Screen Identifies NEK7 as an Essential Component of NLRP3 Inflammasome Activation

TLR8 Senses Bacterial RNA in Human Monocytes and Plays a Nonredundant Role for Recognition of Streptococcus pyogenes

Trapping all ERBB ligands decreases pancreatic lesions in a murine model of pancreatic ductal adenocarcinoma

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