Name: Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens (Video) | JoVE Journal
Uploaded: 2020-06-06T13:30:00.000+00:00
Duration: 8 min 49 s
Description: Learn genome-scale CRISPR/Cas9 screening to identify cell surface receptor-ligand interactions without prior biological assumptions. This genetic method enables unbiased discovery for therapeutic development.

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5 minutes

This work was supported by the Wellcome Trust grant number 206194 awarded to GJW. We thank the Cytometry Core facility: Bee Ling Ng, Jennifer Graham, Sam Thompson, and Christopher Hall for help with FACS.

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The authors have nothing to disclose.

A CRISPR-based screening strategy to identify genes encoding cellular components involved in cellular recognition is described. A similar approach using CRISPR activation also provides a genetic alternative to identify directly interacting receptors of recombinant proteins without the need to generate large protein libraries26. However, one major advantage of this approach is that it is applicable to interactions mediated by surface molecules natively displayed on the cell and does not depend on the overexpression of receptors, which can influence the binding avidity of the receptor. Unlike other methods, therefore, this technique makes no assumptions regarding the biochemical nature or cell biology of the receptors and provides an opportunity to study interactions mediated by proteins that are normally difficult to study using biochemical approaches, such as very large proteins, or those that traverse the membrane multiple times or form complexes with other proteins, and molecules other than proteins such as glycans, glycolipids, and phospholipids. Given the genome-scale nature of the method, this approach also has the advantage of not only identifying the receptor but also additional cellular components that are required for the binding event, thereby providing insights into the cell biology of the receptor.
One of the major limitations of this method when using it to identify the receptor of an orphan protein is the initial requirement to first identify a cell line that binds to the protein. This is not always easy and identifying a cell line that displays a binding phenotype that is also permissive to genetic screens can be the time-limiting step for deploying this assay. Some cell lines tend to bind to more proteins than others. This is especially relevant for proteins that bind HS, because these proteins tend to bind to any cell line that displays HS side chains, irrespective of the native binding context. Additionally, we have observed that upregulation of syndecans (i.e., proteoglycans that contain HS) in cell lines leads to increased binding of HS-binding proteins26. This could be a factor to take into consideration when selecting the cell line for screening. However, also important to note is that the additive binding of HS does not interfere with the binding to a specific receptor. This means that if binding is observed, it is possible that it is mediated solely by HS because the binding mediated by HS in this assay is additive rather than codependent19. In such a scenario, the heparin blocking approach described can identify such behaviors without having the need to perform a full genetic screen.
A useful resource for choosing cell lines is Cell Model Passport, which contains genomics, transcriptomics, and culture condition information for ~1,000 cancer cell lines27. Depending on the biological context, cells can be chosen based on their expression profiles. To aid the selection of cell lines, we clustered ~1,000 cell lines in Cell Model Passport according to the expression of ~1,500 preannotated human cell surface glycoproteins28 (Supplementary Figure 2; cluster information for each cell line together with growth conditions are provided in Supplementary Table 2). When testing the binding of a protein with unknown function, it is useful to select a panel of representative cell lines from each cluster to increase the chance of covering a wide range of receptors. Given a choice, it is recommended to choose cell lines that are easy to culture and easy to transduce. As these cell lines will be used in genome-scale screening, it is preferable that they can be grown easily in large quantities and are permissive to lentiviral transduction, because it is the most commonly available method for delivery of sgRNA for CRISPR-based genetic screening in the later steps.
Generally, the phenotype selections are carried out in a single sort. However, this is determined by the brightness of the stained cell population compared to the control. Iterative rounds of selections could be adopted for scenarios in which the signal-to-noise ratio of the desired phenotype is low, or when the aim of the screen is to identify mutants that have strong phenotypes. When using an iterative selection approach for FACS-based screens, it is important to consider that the sorting process can cause cell death, mainly due to the sheer force of the sorter. Thus, not all collected cells will be represented in the next round of sorting.
Library complexity is a very important factor in performing successful genetic screens, especially for negative selection screens because the extent of depletion in these can only be determined by comparing results to what was present in the starting library. For negative selection screens, it is common to maintain libraries of 500-1,000 x complexity. Positive selection screens, however, are more robust to library sizes, because in such screens only a small number of mutants are expected to be selected for a particular phenotype. Therefore, in the positive selection screen described here, the library size can be decreased to 50-100x complexity without compromising the quality of the screen. In addition, in these screens it is also possible to use a control library for a given cell line on a given day as a &#34;general control&#34; for all samples sorted on the day for that given cell line. This will reduce the number of control libraries that need to be produced and sequenced.
Another important consideration for using this approach is the limitations of loss-of-function screens in identifying genes that are essential for in vitro cell growth. The timing of the screens is crucial in this regard, as the longer the mutant cells are kept in culture, the higher the likelihood that cells with mutations in essential genes become nonviable and are no longer represented in the mutant library. The recent genetic screens performed as a part of the Project Score initiative in over 300 cell lines show that multiple genes in the KEGG-annotated protein secretion and N-glycosylation pathway are often identified as being essential for a number of cell lines (Supplementary Figure 3)29. This can be taken into consideration when designing screens if the effect of genes required for proliferation and viability is to be investigated in the context of cellular recognition process. In this case, carrying out screens at an early timepoint (e.g., day 9 posttransduction) would be generally appropriate. However, if the approach is used to identify a few targets with strong size effects rather than general cellular pathways, it might be appropriate to perform screens at a later time point (e.g., day 15-16 posttransduction).
The results from the screening are very robust; in eight recombinant protein binding screens performed in the past, the cell surface receptor was the top hit in every case19. When using this approach to identify the interaction partner, one should therefore expect the receptor and the factors contributing to its presentation on the surface to be identified with a high statistical confidence. Once the screen is performed and a hit is validated using a single gRNA knockout, further follow-ups can be performed using existing biochemical methods such as AVEXIS4 and direct saturable binding of purified proteins using surface plasmon resonance. The approach described here is applicable for all proteins for which it is possible to generate a soluble recombinant binding probe.
In summary, this is a genome-scale CRISPR knockout approach to identify interactions mediated by cell surface proteins. This method is generally applicable to identify cellular pathways required for cell surface recognition in a wide range of different biological contexts, including between an organism&#39;s own cells (e.g., neural and immunological recognition), as well as between host cells and pathogen proteins. This method provides a genetic alternative to biochemical approaches designed for receptor identification, and because it does not require any prior assumptions regarding the biochemical nature or cell biology of the receptors it has great potential to make completely unexpected discoveries.

Extracellular interactions by cell surface receptor proteins direct important biological processes such as tissue organization, host-pathogen recognition, and immune regulation. Investigating these interactions is of interest to the wider biomedical community, because membrane receptors are actionable targets of systematically delivered therapeutics such as monoclonal antibodies. Despite their importance, studying these interactions remains technically challenging. This is mainly because membrane-embedded receptors are amphipathic, making them difficult to isolate from biological membranes for biochemical manipulation, and their interactions are typified by the weak interaction affinities (KDs in the &#181;M-mM range)1. Consequently, many commonly used methods are unsuitable to detect this class of protein interactions1,2.
A range of methods has been developed to specifically investigate extracellular receptor-ligand interactions that take their unique biochemical properties into consideration3. A number of these approaches involve expressing the entire ectodomain of a receptor as a soluble recombinant protein in mammalian or insect cell-based systems to ensure that these proteins contain posttranslational modifications that are structurally important, such as glycans and disulfide bonds. To overcome the low-affinity binding, the ectodomains are often oligomerized to increase their binding avidity. Avid protein ectodomains have been successfully used as binding probes to identify interaction partners in direct recombinant protein-protein interaction screens4,5,6,7. While broadly successful, recombinant protein-based methods require that the ectodomain of a membrane receptor be produced as a soluble protein. Therefore, it is only generally applicable to proteins that contain a contiguous extracellular region (e.g., single-pass type I, type II, or GPI-anchored) and is not generally suitable for receptor complexes and membrane proteins that span the membrane multiple times.
Expression cloning techniques in which a library of complementary DNAs (cDNAs) is transfected into cells and tested for a gain-of-binding phenotype have also been used to identify extracellular protein-protein interactions8. The availability of large collections of cloned and sequenced cDNA expression plasmids in recent years has facilitated methods in which cell lines overexpressing cDNAs encoding cell surface receptors are screened for the binding of recombinant proteins to identify interactions9,10. The cDNA overexpression-based approaches, unlike recombinant protein-based methods, afford the possibility to identify interactions in the context of the plasma membrane. However, the success of using cDNA expression constructs depends on the cells&#39; ability to overexpress the protein in the correctly folded form, but this often requires cellular accessory factors such as transporters, chaperones, and correct oligomeric assembly. Transfecting a single cDNA might therefore not be enough to achieve cell surface expression.
Screening techniques using cDNA constructs or recombinant protein probes are resource-intensive and require large collections of cDNA or recombinant protein libraries. Specifically designed mass spectrometry-based methods have been utilized recently to identify extracellular interactions that do not require the assembly of large libraries. However, these techniques require chemical manipulation of the cell surface, which can alter the biochemical nature of the molecules present on the surface of the cells and are currently only applicable for interactions mediated by glycosylated proteins11,12. The majority of the currently available methods also heavily focus on the interactions between proteins while largely ignoring the contribution from the membrane microenvironment, including molecules such as glycans, lipids, and cholesterol.
The recent development of highly efficient bialleleic targeting using CRISPR-based approaches has enabled genome-scale libraries of cells lacking defined genes in a single pool that can be screened in a systematic and unbiased way to identify cellular components involved in different contexts, including dissecting cellular signaling processes, identification of perturbations that confer resistance to drugs, toxins, and pathogens, and determining specificity of antibodies13,14,15,16. Here, we describe a genome-scale CRISPR-based knockout cell screening assay that provides an alternative to the current biochemical approaches to identify extracellular receptor-ligand interactions. This approach of identifying interactions mediated by membrane receptors by genetic screens is particularly suitable for researchers that have a focused interest on individual ligands because it avoids the need to compile large libraries of cDNAs or recombinant proteins.
This assay consists of three major steps: 1) Highly avid recombinant protein binding probes consisting of the extracellular regions of a receptor of interest are produced and used in fluorescence-based flow cytometry-based binding assays; 2) The binding assays are used to identify a cell line that expresses the interaction partner of the recombinant protein probe; 3) A Cas9-expressing version of the cell line that interacts with the protein of interest is produced and a genome-scale CRISPR/Cas9-based knockout screen is performed (Figure 1). In this genetic screen, binding of a recombinant protein to cell lines is used as a measurable phenotype in which cells within the knockout library that have lost the ability to bind the probe are sorted using fluorescence-based activated cell sorting (FACS) and the genes that caused the loss of the binding phenotype identified by sequencing. In principle, the genes encoding the receptor responsible for binding the avid probe and those required for its cell surface display are identified.
The first step of this protocol involves the production of avid recombinant protein probes representing the ectodomain of the membrane-bound receptors. These receptors are known to frequently retain their extracellular binding functions when their ectodomains are expressed as a recombinant soluble protein1. For a protein of interest, soluble recombinant proteins can be produced in any suitable eukaryotic protein expression system in any format provided that it can be oligomerized for increased binding avidity, and it contains tags that can be used in fluorescence-based flow cytometry-based binding assays (e.g., FLAG-tag, biotin-tag). Detailed protocols for the production of soluble ectodomains of membrane receptors using the HEK293 protein expression system, as well as different multimerization techniques and the protein expression constructs for the production of both pentameric proteins and monomeric proteins have been previously described1,17. The protocol here will describe the steps for generating fluorescent avid probes from monomeric biotinylated proteins by conjugating them to streptavidin conjugated to a fluorochrome (e.g., phycoerythrin, or PE), which can be used directly in cell-based binding assays and has the advantage of not requiring a secondary antibody for detection. General protocols for performing genome-scale screens have already been described20,21, thus the protocol mainly focus on the specifics of performing flow cytometry-based recombinant protein binding screens using the CRISPR/Cas9 knockout screening system using the Human V1 (&#34;Yusa&#34;) library18.

<table><tbody><tr><td>Anti-mouse alkaline phosphatase</td><td>Sigma</td><td>A4656</td><td></td></tr><tr><td>Blasticidin</td><td>Chem-Cruz</td><td>SC-204655</td><td></td></tr><tr><td>Blood &amp;amp; Cell Culture DNA Maxi Kit</td><td>Qiagen</td><td>13362</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A9647-100G</td><td></td></tr><tr><td>Diethanolamine</td><td>Sigma</td><td>398179</td><td></td></tr><tr><td>DMEM</td><td>Gibco</td><td>31966-021</td><td></td></tr><tr><td>Dneasy Blood and Tissue kit</td><td>Qiagen</td><td>69504</td><td></td></tr><tr><td>DynaMag-96 Side Magnet</td><td>Invitrogen</td><td>12331D</td><td></td></tr><tr><td>HEK293T packaging cells</td><td>ATCC</td><td>CRL-3216</td><td></td></tr><tr><td>Heparin</td><td>Sigma</td><td>H4784-1G</td><td></td></tr><tr><td>KAPA HiFi HotStart ReadyMix</td><td>Kapa</td><td>KK2602</td><td></td></tr><tr><td>Lipofectamine LTX with PLUS reagent</td><td>Invitrogen</td><td>15338100</td><td></td></tr><tr><td>MoFlo XDP cell sorter</td><td>BD</td><td></td><td></td></tr><tr><td>Ni2+-NTA agarose beads</td><td>Jena Bioscience</td><td>AC-501-25</td><td></td></tr><tr><td>OPTI-MEM</td><td>Life Technologies</td><td>31985-070</td><td></td></tr><tr><td>OX-68 antibody</td><td>AbD Serotec</td><td>MCA1022R</td><td></td></tr><tr><td>p-nitrophenyl phosphate</td><td>Sigma</td><td>1040-506</td><td></td></tr><tr><td>PD-10 desalting columns</td><td>GE healthcare</td><td>17085101</td><td></td></tr><tr><td>Polybrene</td><td>Millipore</td><td>TR-1003-G</td><td></td></tr><tr><td>Polypropylene tubes with 5 mL bed volume</td><td>Qiagen</td><td>34964</td><td></td></tr><tr><td>Proteinase K, recombinant, PCR Grade</td><td>Roche</td><td>3115879001</td><td></td></tr><tr><td>Puromycin</td><td>Gibco</td><td>A11138-03</td><td></td></tr><tr><td>Q5 Hot Start High-Fidelity 2&amp;times; Master Mix</td><td>NEB</td><td>M0494L</td><td></td></tr><tr><td>QIAquick PCR purification kit</td><td>Qiagen</td><td>28104</td><td></td></tr><tr><td>SCFA filter</td><td>Nalgene</td><td>190-2545</td><td></td></tr><tr><td>Sony Cell sorter</td><td>Sony</td><td></td><td></td></tr><tr><td>SPRI beads (Agencourt AMPure XP beads)</td><td>Beckman</td><td>A63881</td><td></td></tr><tr><td>Streptavidin-coated microtitre plates</td><td>Nalgene</td><td>734-1284</td><td></td></tr><tr><td>Streptavidin-PE</td><td>Biolegend</td><td>405204</td><td></td></tr></tbody></table>

cell surface receptor identification using genome-scale crispr/cas9 genetic screens

Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and functioning of multicellular organisms. Detecting these interactions remains technically challenging, however. This manuscript describes a systematic genome-scale CRISPR/Cas9 knockout genetic screening approach that reveals cellular pathways required for specific cell surface recognition events. This assay utilizes recombinant proteins produced in a mammalian protein expression system as avid binding probes to identify interaction partners in a cell-based genetic screen. This method can be used to identify the genes necessary for cell surface interactions detected by recombinant binding probes corresponding to the ectodomains of membrane-embedded receptors. Importantly, given the genome-scale nature of this approach, it also has the advantage of not only identifying the direct receptor but also the cellular components that are required for the presentation of the receptor at the cell surface, thereby providing valuable insights into the biology of the receptor.

This manuscript describes a genome-scale cell-based screening approach to identify extracellular receptor-ligand interactions.

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and ...

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Research

JoVE Journal

Genetics

The genotyping technique described here, which couples fluorescent polymerase chain reaction (PCR) to capillary gel electrophoresis, allows for high-throughput genotyping of nuclease-mediated knockout clones. It circumvents limitations faced by other genotyping techniques and is more cost effective than sequencing methods.

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

This protocol describes the steps needed to design and perform multiplexed targeting of enhancers with the deactivating fusion protein SID4X-dCas9-KRAB, also known as enhancer interference (Enhancer-i). This protocol enables the identification of enhancers that regulate gene expression and facilitates the dissection of relationships between enhancers regulating a common target gene.

Dissection of Enhancer Function Using Multiplex CRISPR-based Enhancer Interference in Cell Lines

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Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution

A CRISPR/sgRNA library has been applied to interrogating protein-coding genes.However, the feasibility of a sgRNA library to uncover the function of a CTCF boundary in gene regulation remains unexplored. Here, we describe a HOX loci specific sgRNA library to elucidate the function of CTCF boundaries in HOX loci.

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

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Pooled CRISPR-Based Genetic Screens in Mammalian Cells

AviTag-specific Location-restricted Illumination-enhanced Biotinylation (ALIBi) is a novel optogenetically activated method developed for labeling and affinity-purifying subcellular populations of ribosomes for downstream sequencing and proteomic analysis.

Isolation of Subcellular Ribosome Subpopulations Based on Recombinant Peptide Tag-Specific Location-Restricted Illumination-Enhanced Biotinylation

A significant barrier to technologies like CRISPR is the off-target events that can disrupt vital genes. 'Circularization for In Vitro Reporting of Cleavage Effects by Sequencing' (CIRCLE-seq) is a technique designed to identify unintended cleavage sites. This method maps the genome-wide activity of CRISPR-Cas9 with high sensitivity and without bias.

CIRCLE-Seq for Interrogation of Off-Target Gene Editing

The article describes a protocol for the application of an in vitro model for exhaustion to complete a genome-wide CRISPR knockout screen in healthy donor chimeric antigen receptor T cells.

Performing an In Vitro Genome-Wide CRISPR Knockout Screen in Chimeric Antigen Receptor T Cells

Here, we present a protocol that integrates CRISPR/Cas9 technology with the Cell Counting Kit-8 (CCK-8) assay to identify candidate genes that are crucial for the abnormal proliferative capacity of hematopoietic stem and progenitor cells.

Evaluation of Abnormal Growth-related Genes of Hematopoietic Stem and Progenitor Cells by Combining CRISPR/Cas9 Technology with Cell Counting

A meticulous and structured approach is given to select resistant and sensitive genes of radiation through the application of a genome-wide CRISPR/Cas9 screen method. This protocol also has the potential to serve as a versatile framework for other research endeavors investigating the mechanisms of resistance to clinically administered chemical drugs.

Genome-Wide CRISPR Screen for Unveiling Radiosensitive and Radioresistant Genes

We developed an optimized CRISPR-Cas9 knockout screening protocol for primary CAR T cells. By reducing gDNA carryover through enzymatic digestion and sgRNA cassette pulldown, this approach minimizes PCR artifacts, ensuring accurate sgRNA detection and robust identification of functional gene targets.

Improving CRISPR-Cas9 Screens in CAR T Cells: A Refined Method for Library Preparation

Learn genome-scale CRISPR/Cas9 screening to identify cell surface receptor-ligand interactions without prior biological assumptions. This genetic method enables unbiased discovery for therapeutic development.

Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens

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