Progress 09/01/20 to 08/31/21
Outputs
Target Audience:There were no presentations for this reporting period due to Covid restrictions. We have shared the project overview andresults with program leaders from other funding bodies. Changes/Problems:With Covid and the previous switch made in cell types for editing,culturing and differentiation; our collaborative team became limited on the number of cell lines that could be processed through the differentiation process (takes several months/cell line). As such, Recombinetics made seven knock-out cell lines and one cell line with a precise change in MARCO1. Although reduced from the original major goal of 20 cell lines, the eight current lines represent a significant resourcefor the study of immune response to M. bovis infection in vitro, if these cells lines can be successfully differentiated and grown out into enough macrophage-like cells for further study. The process of differentiation and infection challenge are still ongoing at Queens University and University College Dublin, respectively. Their funding was extended for another year due to closures that delayed work during the Covid pandemic. What opportunities for training and professional development has the project provided?Technical training was extended to two other Recombientics employeesfor gene editing, characterization, and growth of bovine embryonic stem cells. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Most of our efforts at Recombinetics were focused on Major Goals #2 and #3. Due to the difficulties in generating iPSCDM in previous years at Queens University (Northern Ireland partner), we moved to using bovine embryonic stem cells (ESCs) available to Recombinetics as the target material for gene editing. Witin a progenitor embryonic cell line derived from a Holstein male, seven different "progeny" cell lines were manufactured. The single gene "knock-out" edits for each line were as follows: 1) ARG2, 2) GBP5, 3) IFI16, 4) IFNGR, 5) MYD88, 6) SLC11A1, and 7) TMEM173. An eighth progeny cell line was also made with an exact edit in MARCO1, which converts the typical taurine "fixed" allele to the "fixed" alternative allele found in indicine cattle. Each cell line was then grown up to threshold numbers for export to Northern Ireland, where the Simpson lab at Queens University will take each cell line through a macrophage differentiation process inculture. The SImpson lab has successfully used the founder ESC line to produce macrophage-like cells. These cells were transferred to UC-Dublin for "cell-type" immune response characterization.
Publications
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Progress 09/01/18 to 08/31/21
Outputs
Target Audience:The only formal presentations for this project were at CRWAD 2018 & 2019 conference attended by scientists in the field. We have shared the project overview and preliminary results with program leaders from other funding bodies. Changes/Problems:Before editing, the stem cell team at Queens needed to identify a bovine cell type that could be changed from progenitor cell to macrophage-like cell, in culture. This step was not completed until late 2020, because the initial iPSC cells did not work properly, and the project switched to bovine embryonic stem cells through resources Recombinetics had available through an agreement with UC-Davis. These cells, once identified for potential to produce challenge quality cell types, were then targeted for editing. Due to the limited time left in this project, we changed the targets from 2 to 7 knock out lines and only one line with a specific NSV, since genotyping did not support changing the other 20 candidate NSV. Such a change would offer a more informative outcome on immune response of hub genes and then later we could characterize the lone sub-species difference found in MARCO1. What opportunities for training and professional development has the project provided?Technical training was extended to a total of threeRecombinetics employees for gene editing design and transfection, screening and colony characterization for stem cell biomarkers, and growht maintenance ofbovine embryonic stem cells. How have the results been disseminated to communities of interest?Aside from the mandatory CRWAD conference attendance no other formal reports have been generated. Publishable results depend on the differentiation of the edited ESCs to macrophage like cells and use of these cells in infection challenge experiments with M. bovis. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Recombinetics participated or led research only for activities that contributed to major goals #1-3. Major goals #4-7 are in still progress through extensions given to our Tri-Partite collaborative partners' funding agencies in Ireland and Northern Ireland based on Covid-related delays. In summary, we identified, ranked and prioritized 21 candidate genes and genomic regulatory elements (GREs) with functionally-relevant natural sequence variants (NSVs - both SNPs and small indels) as potential genome editing targets. Ten of these genes were knocked out or altered in bovine fibroblasts, through optimization processes that tested more than 20 different CRISPR/Cas9 targeting complexes. Seven of the knock-out edits were duplicated in a bovine stem cell line that has the potential to diffentiate into macrophage-like cells needed for infection challenge experments. Relatively large quantities of these cells lines were grown out and transferred to our Tri-Partite partners. Additional genotyping for 21 of the NSVs from 8 genes in a panel of 1000 animals (multi-breed and sub-species), identified one gene (MARCO1) as the best candidate for specific gene alterations to change introduce a sub-species change (from tuarine to zebu gene versions). The specific gene edit for MARCO1 was also introduced into the progenitor line of ECSs. In total, we generated 7 fibroblast lines and seven ESCslines with loss-of-function (LOF) edits in seven genes that are key to the mammalian macrophage response to mycobacterial infections. We also generated a fibroblast line and multiple ESC lineswith a specific sub-species alternative allele/NSV editin MARCO1 identified from the scientific literature, a defined macrophage defense process, and computational analysis of extensive macrophagemycobacteria transcriptomics data. The seven knock-out lines are a germplasm resource available for in vitro challenge studies of immune response and pathogen infection.
Publications
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Progress 09/01/19 to 08/31/20
Outputs
Target Audience:The only presentation for this reporting period was at CRWAD 2019 conference attended by scientists in the field. We have shared the project overview and preliminary results with program leaders from other funding bodies. Changes/Problems:All current delays in this project are related to difficulties related to culturing and differentiating bovine stem cells. The switch in late 2019 to embryonic stemcells seems to have solved this technical challenge. What opportunities for training and professional development has the project provided?Technical training was completed for gene editing of bovine embryonic stem cells. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?We now have gene editing methods in pluripotent embryonic stem cells working. The reagent design and editing of 20 different gene targets will be completed in 2021 based upon the prioritized list of 9 target genes involved in immune response to M. bovis.
Impacts
What was accomplished under these goals?
We completed Major Goal #1 in conjunction with UC-Dublin researchers. Our list has 9 core genes which are slated for knock-out mutations and have selected a series of allelic variants for these genes where sequence variations between Indicine and Taurine cattle are differentially fixed. Gene editing Goals 2 and 3 are still in progress as we have successfully switched to using embryonic stem cells for our resource population of cells. However, the editing for 9 gene knock-outs has been optimized in bovine fibroblasts proving our CRISPR/Cas9 tools are functional in vitro. Previous efforts to make bovine iPSCDM had failed due to the difficult challenge of making these cells. Out project partners in Norther Ireland are now focusing on differentiation of enbryonic stem cells into macrophages for testing the pipeline in Goal #4.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Bostrom J, MacHugh D, Simpson D, Carlson D, Vernimmen D, ODoherty A, Hall T, Gordon S, Correia C, Margariti A, Kelaini S, Sonstegard T. Targeted genome editing to understand and enhance genetic resistance to M. bovis infection in domestic cattle. Proceedings of CRWAD 2018 (Chicago). Poster Abstract.
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Progress 09/01/18 to 08/31/19
Outputs
Target Audience:Currently, we have only shown the project overview and early results at the CRWAD meeting in Chicago at the end of 2018, which was only 3 months into the project. The primary audience has been other scientists attending this conference and other potential scientific collaborators. Changes/Problems:At UCD,There was a significant problem with the Rodeo computer server (rodeo.ucd.ie) used for Task 1. On the17th of October 2018, there was a power outage that damaged equipment in the UCD server centre. Rodeowas repaired by the end of January 2019. See Task 1 description for details of this problem and supportingdocumentation. The postdoctoral researcher (ROI-PDOC) Dr Carol Correia was able to accelerate this workthrough a two-week research visit to the group of Professor Hubert Pausch in ETH-Zurich during earlyJanuary 2019. We also have initiated a collaboration with Dr. Pablo Ross to use his stem cells as an alterntive source of iPSCMD; if technical challenges for stem cell derivation at QUB persist. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Only had a poster presentation at the 2018 CRWAD meeting to date. What do you plan to do during the next reporting period to accomplish the goals?Collaboration with Professor Hubert Pausch ondevelopment of pan-genome resources for Bos taurus andBos indicus cattle. These resources are being used toidentify functionally relevant natural sequence variants(NSVs) for the TARGET-TB project. Also, collaboration with Professor Pablo Ross to provide accessto pluripotent embryonic stem cells for genome editingand transformation to a macrophage phenotype. Thiscollaboration will provide cellular materials for theTARGET-TB project.
Impacts
What was accomplished under these goals?
During Year 1, the UCD group has identified more than 20 gene targets for genome editing to study the response to Mycobacterium bovis infection in the bovine cells that first encounter the disease-causing agent in the lungs. During Year 1, the QUB group have developed methods for generating cells suitable for genome editing from bovine embryonic tissues. During Year 1, the REC group have developed the genome editing tools to study nine of the 20 gene targets and have generated bovine cells with these nine genome edits. Through a new collaboration initiated in Year 1 with scientists at the University of California, Davis (UC Davis), the TARGET-TB project will have access to developmentally plastic bovine cells that can also be used to generate cells suitable for genome editing and for testing with laboratory infection studies.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Sonstegard, T. S., Correia, C. N., Margariti, A., Bostrom, J., Carlson, D. F.,
Simpson, D. A., Gordon, S. V., MacHugh, D. E.
Poster: Targeted genome editing to understand and enhance genetic
resistance to M. bovis infection in domestic cattle.
99th Conference of Research Workers in Animal Diseases (CRWAD), Chicago,
Illinois, USA. 2nd-4th, December, 2018.
https://crwad.org/wp-content/uploads/2018/11/CRWAD-2018.FINAL-Author-
Index-and-Abstracts.pdf
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