Determining antigen specificity of a monoclonal antibody using genome-scale CRISPR-Cas9 knockout library

Zotova, Anastasia; Zotov, I. A.; Filatov, Alexander; Mazurov, Dmitriy

doi:10.1016/j.jim.2016.09.006

Cited by 10 publications

(8 citation statements)

References 22 publications

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“…CRISPR-KO screening identifies receptors and a role for heparan sulfate in low-affinity interactions While high-affinity mAbs are useful research tools and a few studies have previously shown the utility of CRISPR screens to identify receptor-related cellular pathways (Parnas et al 2015;Zotova et al 2016;Burr et al 2017), we next sought to determine if this approach could be used to identify low-affinity receptors for cell signaling ligands. As a model system, we selected the interaction between Plasmodium falciparum RH5 and its host receptor basigin (BSG) because it is a low-affinity interaction (K D ∼ 1 µM), is biochemically and structurally well characterized and because BSG was highly expressed on our Cas9-expressing HEK293 cell line (Crosnier et al 2011;Wright et al 2014).…”

Section: Genetic Determinants Of Mab Cell Surface Epitope Display By mentioning

confidence: 99%

Genome-scale identification of cellular pathways required for cell surface recognition

et al. 2018

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Interactions mediated by cell surface receptors initiate important instructive signaling cues but can be difficult to detect in biochemical assays because they are often highly transient and membrane-embedded receptors are difficult to solubilize in their native conformation. Here, we address these biochemical challenges by using a genome-scale, cell-based genetic screening approach using CRISPR gene knockout technology to identify cellular pathways required for specific cell surface recognition events. By using high-affinity monoclonal antibodies and low-affinity ligands, we determined the necessary screening parameters, including the importance of establishing binding contributions from the glycocalyx, that permitted the unequivocal identification of genes encoding directly interacting membrane-embedded receptors with high statistical confidence. Importantly, we show that this genome-wide screening approach additionally identified receptor-specific pathways that are required for functional display of receptors on the cell surface that included chaperones, enzymes that add post-translational modifications, trafficking proteins, and transcription factors. Finally, we demonstrate the utility of the approach by identifying IGF2R (insulin like growth factor 2 receptor) as a binding partner for the R2 subunit of GABA receptors. We show that this interaction is direct and is critically dependent on mannose-6-phosphate, providing a mechanism for the internalization and regulation of GABA receptor signaling. We conclude that this single approach can reveal both the molecular nature and the genetic pathways required for functional cell surface display of receptors recognized by antibodies, secreted proteins, and membrane-embedded ligands without the need to make any prior assumptions regarding their biochemical properties.

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Section: Genetic Determinants Of Mab Cell Surface Epitope Display By mentioning

confidence: 99%

Genome-scale identification of cellular pathways required for cell surface recognition

et al. 2018

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“…Additionally, genes involved in vemurafenib resistance were found in a melanoma model, including previously identified genes NF1 and MED12, and the newly identified NF2, CUL3, TADA2B, and TADA1. At present, genome-wide CRISPR-Cas9 screening libraries have been adopted to screen for drug resistance, virulence, and tumor suppressor genes in multiple cell lineages and primary cells [24][25][26][27][28][29][30][31][32][33].…”

Section: Introductionmentioning

confidence: 99%

Screening Genes Promoting Exit from Naive Pluripotency Based on Genome-Scale CRISPR-Cas9 Knockout

Yang

Kuang

et al. 2020

Stem Cells International

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Two of the main problems of stem cell and regenerative medicine are the exit of pluripotency and differentiation to functional cells or tissues. The answer to these two problems holds great value in the clinical translation of stem cell as well as regenerative medicine research. Although piling researches have revealed the truth about pluripotency maintenance, the mechanisms underlying pluripotent cell self-renewal, proliferation, and differentiation into specific cell lineages or tissues are yet to be defined. To this end, we took full advantage of a novel technology, namely, the genome-scale CRISPR-Cas9 knockout (GeCKO). As an effective way of introducing targeted loss-of-function mutations at specific sites in the genome, GeCKO is able to screen in an unbiased manner for key genes that promote exit from pluripotency in mouse embryonic stem cells (mESCs) for the first time. In this study, we successfully established a model based on GeCKO to screen the key genes in pluripotency withdrawal. Our strategies included lentiviral package and infection technology, lenti-Cas9 gene knockout technology, shRNA gene knockdown technology, next-generation sequencing, model-based analysis of genome-scale CRISPR-Cas9 knockout (MAGeCK analysis), GO analysis, and other methods. Our findings provide a novel approach for large-scale screening of genes involved in pluripotency exit and offer an entry point for cell fate regulation research.

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“…There are not many cell line models suitable for screening or subsequent validation of antibodies for subsequent use in brain, as such validation assays need to be performed in brain tissue or in primary cultures of brain cells. This limits the use of knockdown/knockout approaches, especially those employing powerful CRISPR/Cas9 approaches (Zotova, Zotov, Filatov, & Mazurov, 2016), that have gained widespread use in antibody validation for use in other cell types. However, one can advantageously employ the neuroanatomical complexity of the brain when screening and validating antibodies, whereby knowledge of the specific subcellular compartment (e.g., axon, dendrite, synapse, node of Ranvier), cell type, and circuit in which a target protein is (and is not) likely to be expressed can be effectively used to evaluate immunolabeling in brain sections and in primary cultures of brain cells (Gong et al., 2016; Rhodes & Trimmer, 2006).…”

Section: Characterization and Validation Of Recombinant Antibodies Fomentioning

confidence: 99%

Recombinant Antibodies in Basic Neuroscience Research

Trimmer

2020

CP Neuroscience

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Basic neuroscience research employs antibodies as key reagents to label, capture, and modulate the function of proteins of interest. Antibodies are immunoglobulin proteins. Recombinant antibodies are immunoglobulin proteins whose nucleic acid coding regions, or fragments thereof, have been cloned into expression plasmids that allow for unlimited production. Recombinant antibodies offer many advantages over conventional antibodies including their unambiguous identification and digital archiving via DNA sequencing, reliable expression, ease and reliable distribution as DNA sequences and as plasmids, and the opportunity for numerous forms of engineering to enhance their utility. Recombinant antibodies exist in many different forms, each of which offers potential advantages and disadvantages for neuroscience research applications. I provide an overview of recombinant antibodies and their development. Examples of their emerging use as valuable reagents in basic neuroscience research are also discussed. Many of these examples employ recombinant antibodies in innovative experimental approaches that cannot be pursued with conventional antibodies.

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Determining antigen specificity of a monoclonal antibody using genome-scale CRISPR-Cas9 knockout library

Cited by 10 publications

References 22 publications

Genome-scale identification of cellular pathways required for cell surface recognition

Genome-scale identification of cellular pathways required for cell surface recognition

Screening Genes Promoting Exit from Naive Pluripotency Based on Genome-Scale CRISPR-Cas9 Knockout

Recombinant Antibodies in Basic Neuroscience Research

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