Investigating Target Gene Function in a CD40 Agonistic Antibody-induced Colitis Model using CRISPR/Cas9-based Technologies
Summary
Here, we describe the methodology to knock out a gene of interest in the immune system using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas9)-based technologies and the evaluation of these mice in a cluster of differentiation 40 (CD40) agonistic antibody-induced colitis model.
Abstract
The immune system functions to defend humans against foreign invaders such as bacteria and viruses. However, disorders of the immune system may lead to autoimmunity, inflammatory disease, and cancer. The inflammatory bowel diseases (IBD)-Crohn’s disease (CD) and ulcerative colitis (UC)-are chronic diseases marked by relapsing intestinal inflammation. Although IBD is most prevalent in Western countries (1 in 1,000), incident rates are increasing around the world. Through association studies, researchers have linked hundreds of genes to the pathology of IBD. However, the elaborate pathology behind IBD and the high number of potential genes pose significant challenges in finding the best therapeutic targets. Additionally, the tools needed to functionally characterize each genetic association introduce many rate-limiting factors such as the generation of genetically modified mice for each gene. To investigate the therapeutic potential of target genes, a model system has been developed using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas9)-based technologies and a cluster of differentiation 40 (CD40) agonistic antibody. The present study shows that CRISPR/Cas9-mediated editing in the immune system can be used to investigate the impact of genes in vivo. Limited to the hematopoietic compartment, this approach reliably edits the resulting reconstituted immune system. CRISPR/Cas9-edited mice are generated faster and are far less expensive than traditional genetically modified mice. Furthermore, CRISPR/Cas9 editing of mice has significant scientific advantages compared to generating and breeding genetically modified mice such as the ability to evaluate targets that are embryonic lethal. Using CD40 as a model target in the CD40 agonistic antibody-induced colitis model, this study demonstrates the feasibility of this approach.
Introduction
Autoimmune diseases refer to conditions in which a patient's immune system attacks their own cells and organs, resulting in chronic inflammation and tissue damage. Nearly 100 different types of autoimmune conditions have been described to date, affecting 3-5% of the human population1. Many of the autoimmune conditions, including systemic lupus erythematosus and IBD, lack effective treatments and present significant unmet medical needs. Currently affecting around 1.5 million people in the USA alone, IBD is a devastating disease marked by progressive, persistent, and relapsing intestinal inflammation with no available cure. Unraveling the underlying pathogenesis and pathophysiology is needed to deliver the novel treatment and prevention strategies that IBD patients require2,3.
Over 230 different IBD loci have been identified through genome-wide association analyses (GWAS)4. Although these associations have elucidated new genes that are potentially important players in the key mechanisms and pathways of IBD, only a few genes from these loci have been studied. Some genes have been implicated in specific pathways. For example, the microbe-sensing pathway has been linked to nucleotide-binding oligomerization domain-containing protein 2 (NOD2); the autophagy pathway has been linked to autophagy-related 16 like 1 (ATG16L1), immunity-related GTPase family M (IRGM), and caspase recruitment domain family member 9 (CARD9); and the pro-inflammatory pathway has been linked to interleukin (IL)-23-driven T-cell responses4. Various in vivo mouse models have been used to functionally characterize genes identified through GWAS5,6.
One of the key models used to study IBD pathogenesis7,8 is the CD40 model of colitis, which induces innate immune intestinal inflammation following the injection of a CD40 agonistic antibody into immunodeficient (T and B-cell) mice. Primarily used to examine the contribution of innate immunity to IBD development, mostly macrophages and dendritic cells9, it is unclear if disease can be induced in fully immune-competent wild-type (WT) mice. In addition to animal models, gene-specific tools are also required for the functional characterization of a gene, including chemical compounds and biologics. More importantly, genetically modified animals are essential in revealing the function of a specific gene. However, the strategies typically used to make genetically modified mice-embryo injection and breeding-often take over a year and incur a significant financial cost. This rate-limiting process presents a significant challenge in the quest to elucidate the functions of the IBD-related genes identified by GWAS.
The protocol presented here provides a viable alternative to breeding genetically modified mice. First, as shown in the Figure 1 schematic, lineage-negative, stem cell antigen1-positive, receptor tyrosine kinase Kit-positive (lineage-Sca1+c-Kit+ or LSK) cells are isolated from the bone marrow of Cas9 knockin (KI) mice bearing a specific allele (CD45.2) to allow donor immune cell tracking. Next, these cells are exposed to lentiviruses bearing different guide RNAs (gRNAs) and a fluorescent marker, violet-excited green fluorescent protein (VexGFP), to allow tracking of transduced cells. Two days later, VexGFP+ cells are sorted and injected into lethally irradiated recipient Ly5.1 Pep Boy mice, which are C57Bl/6 mice bearing the CD45.1 allele to allow for recipient immune cell tracking. Twelve weeks later, the immune system is fully reconstituted, and the mice can be enrolled into in vivo models.
In addition to the benefit of cost savings and faster time-to-generation compared to the generation and breeding of genetically modified animals, this methodology is ideal for targets that are embryonic lethal, as it specifically targets the hematopoietic compartment. Furthermore, for targets where there are no tools available, such as an antibody, this system provides a feasible approach. In summary, to address the challenges described thus far, an in vivo CRISPR/Cas9-based genome editing platform was developed to expeditiously generate genetically modified animal models10,11,12,13,14. This study demonstrates that intestinal inflammation in WT C57Bl/6 mice can be induced by a CD40 agonistic antibody. CD40 is a key regulator of disease in this model and was therefore used as a model target to validate the CRISPR/Cas9-based knockout and loss of gene function.
Protocol
Representative Results
Discussion
The results shown here introduce a novel CRISPR/Cas9-based genome editing platform capable of investigating gene function in this CD40 agonistic antibody-induced colitis model. Cell sorting enriched the pool of genetically modified LSK cells, resulting in over 90% reduction in CD40 expression within the reconstituted animals-in just 4 months. Furthermore, the reduced expression of CD40 within the immune system had a profound effect within the CD40 agonistic antibody-induced colitis model, significantly reducing disease e…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Thank you to Ruoqi Peng, Donna McCarthy, Jamie Erikson, Liz O'Connor, Robert Dunstan, Susan Westmoreland, and Tariq Ghayur for your efforts supporting this work. Thank you to Pharmacology leaders including Rajesh Kamath and others for their leadership in establishing the CD40 agonistic antibody-induced colitis model in WT C57Bl/6 mice. Additionally, thank you to all those at AbbVie Bioresearch Center and Cambridge Research Center in the Comparative Medicine East Department supporting in vivo experiments.
We would like to thank the Zhang lab from the Broad Institute and McGovern Institute of Brain Research at the Massachusetts Institute of Technology for providing CRISPR reagents [multiplex Genome Engineering Using CRISPR/Cas Systems. Cong, L, Ran, FA, Cox, D, Lin S, Barretto, R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F Science. 2013 Jan 3].
Materials
| 6-well tissue culture plates | Corning/Costar | #3506 | |
| TransIT-LT1 | Mirus Bio | MIR 2300/5/6 | |
| MACS Buffer (autoMACS Running Buffer) | Miltenyi Biotec | 130-091-221 | |
| 0.45 µm filter unit | Millipore | #SLHV013SL | |
| 0.6 mL microcentrifuge Tube | Axygen | MCT-060-C-S | |
| 1.5 mL Eppendorf Tube | Axygen | MCT-150-C-S | |
| 15mL Conical | VWR | 21008-918 | |
| 23 G Needle | VWR | #305145 | |
| 24 Well Non-TC Plates | Falcon | #351147 | |
| 24-Well TC Plates | Falcon | #353047 | |
| 50 mL Conical tube | VWR | 21008-951 | |
| 5 mL Syringe | BD Biosciences | #309647 | |
| 70 µm Filter | Miltenyi | #130-098-462 | |
| 96-Well Flat Bottom Plates | Corning | #3599 | |
| 96-Well U-Bottom Plates | Corning/Costar | #3365 | |
| Anesthesia Machine | VetEquip – COMPAC5 | #901812 | |
| Anti-CD40 Agonist monoclonal antibody | BioXcell | BE-0016 | |
| Anti-p40 monoclonal antibody | BioXcell | BE-0051 | |
| B220 PE Antibody | BioLegend | #103208 | |
| Bovine serum albumin | Sigma Aldrich | A7906-100G | |
| Cas9 Knock-in Mice | Jackson Labs | #026179 | C57Bl/6 background |
| CD117+ Beads | Miltenyi | #130-091-224 | |
| CD11b PE Antibody | BioLegend | #101208 | |
| CD3 PE Antibody | BD Biosciences | #553240 | |
| Centrifuge | Beckman Coulter | Allegra 6KR Centrifuge | |
| Countertop Centrifuge | Eppendorf | Centrifuge 5424 | |
| DPBS | ThermoFisher | #14190136 | |
| Dulbecco’s Modified Eagle Medium | Mediatech | #10-013-CV | |
| Ethylenediamine tetraacetic acid (EDTA) | Invitrogen | AM9260G | |
| Endoscope | Karl Storz | N/A | Custom Coloview Tower |
| Flow cytometer | BD Biosciences | FACS Aria II | |
| Fms-related tyrosine kinase 3 ligand (Flt-L) | PeproTech | #250-31L | |
| Gr-1 PE Antibody | BD Biosciences | #553128 | |
| Hank's balanced salt solution (HBSS) | ThermoFisher | #14170120 | |
| Heat-Inactivated Fetal Bovine Serum | HyClone | #SH30071.03 | |
| IL-7 | PeproTech | #217-17 | |
| Incubator | Binder | #9040-0116 | |
| Isoflurane | HenrySchein | #6679401710 | |
| LS Column | Miltenyi | #130-042-041 | |
| Ly5.1 Pepboy Mice | Jackson Labs | #002014 | C57Bl/6 background |
| mouse stem cell factor (mSCF) | PeproTech | #250-03 | |
| Sodium chloride (NaCl) | Hospira | #00409488850 | |
| OPTI-MEM serum-free media | Invitrogen | #31985-070 | |
| Penicillin-streptomycin (PenStrep) | ThermoFisher | #15140-122 | |
| Plate Shaker | ThermoFisher | #88880023 | |
| pLentiPuro | Addgene | #52963 | |
| Polybrene (10 µg/µL) | Sigma Aldrich | #TR-1003-G | |
| Red Blood Cell Lysis Buffer | eBioscience | #00-4333 | |
| Retronectin | Takarbio | #T100B | |
| Sca-1 APC Antibody | BioLegend | #108112 | |
| StemSpan | StemCell Technologies | #09600 | |
| Ter119 PE Antibody | eBioscience | #12-5921 | |
| Thrombopoietin (TPO) | PeproTech | #315-14 | |
| X-ray Irradiator | Precision X-Ray | X-Rad 320 |
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