Recipient Organization
IOWA STATE UNIVERSITY
2229 Lincoln Way
AMES,IA 50011
Performing Department
Veterinary Microbiology and Preventive Medicine
Non Technical Summary
Species of the microorganism Brucella are important pathogens of domestic livestock that also cause disease in humans. Brucellosis causes abortion and infertility in a variety of animal species, which contributes to significant economic losses in many parts of the world where the disease remains endemic. Despite extensive efforts at developing new vaccines, the most efficacious animal vaccines continue to be live bacterial strains with reduced pathogenicity (i.e. attenuated). Improved vaccines are needed that protect against multiple species of Brucella and that are also safer, since current vaccines can still cause disease and can pose a threat to humans who administer them. In particular, B. melitensis is considered the most pathogenic species of Brucella and only a single vaccine strain is currently available. To develop improved attenuated strains for B. melitensis, we propose to use a combination of comparative genomics and CRISPR/Cas technology as a novel approach to rationally design new vaccines. To accomplish this, the project combines the expertise of the investigators in bacterial genetic engineering and Brucella pathobiology to develop improved methods to modify specific genes in B. melitensis. Genes will be selected for editing based on their contribution to the effectiveness of the B. abortus vaccine strain RB51. Development of a robust CRISPR/Cas gene system for B. melitensis should also be applicable to develop improved vaccine strains of other Brucella species and to better understand mechanisms of virulence in these pathogens. This project is consistent with the overall purpose of the AHDR program and the strategic goals of the USDA to protect agricultural health by preventing and mitigating the spread of agricultural pests and disease, which will help provide all Americans access to a safe and secure food supply.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The overall goal of the research described in this USDA Capacity proposal is to construct improved live attenuated Brucella melitensis vaccines using CRISPR/Cas (clustered regularly interspaced short palindromic repeats)/CRISPR associated proteins) technology. Our specific objective is to develop a plasmid-based system to efficiently assemble recombinant vectors to construct new B. melitensis mutants using CRISPR/Cas. The use of CRISPR/Cas technology represents a new approach for Brucella vaccine development and our approach will allow us to more efficiently modify the B. melitensis genome, as compared to currently used genetic methods. As a result, we anticipate our approach will streamline construction of B. melitensis strains that will be tested in future studies as vaccine candidates, as well as to better understand the basic pathobiology of Brucella. To fulfill these objectives, we propose the following single research objective to be conducted in one year:1. Develop new recombinant vectors to express CRISPR/Cas components to construct new attenuated Brucella melitensis mutants. For this aim, we propose to develop a new plasmid-based system to express Cas9 and specific guide RNAs in B. melitensis. To test the robustness of the plasmid-based system, we will construct multiple attenuated mutants using CRISPR/Cas technology that will be tested for their effectiveness as live vaccines.
Project Methods
1. Plasmid-based system to express CRISPR/Cas gene editing components. CRISPR/Cas systems have been developed for creating targeted genetic changes in bacteria. In general, These systems include targeting the Cas9 nuclease to specific DNA sequences by co-expression of a guide-RNA molecule, chromosome cleavage and repair of the lesion by homologous recombination to precisely edit the DNA. To enable precision gene editing in Brucella, we will use our experience in molecular biology and development of CRISPR tools to construct new recombinant plasmids to enable CRISPR/Cas genome engineering in Brucella. Specifically, we will focus on streamlining construction of plasmids to express Cas9, originally from Streptococcus pyogenes (Cas9-Sp), in combination with guide RNA(s) designed to target specific Brucella melitinsis genes. Given the limited number of options for antibiotic selection and recombinant plasmid vectors for genetic manipulation of Brucella, we propose an innovative approach that takes advantage of in vitro and in vivo genetic engineering to improve the overall efficiency of constructing new strains.The system includes a genetic construct currently used in our lab Cas9-Sp is expressed from a tightly repressed transcriptional unit negatively regulated by the TetR repressor. To minimize the number of plasmids and antibiotic resistance markers introduced to Brucella, we will assemble the coding region for specific guide RNAs on a second plasmid that will be joined into a single vector for introduction into B. melitensis cells. This strategy will take advantage of a plasmid that replicates only in specialized strains of E. coli as a platform to assemble guide RNAs. DNA encoding specific single guide RNAs will be introduced to this small (~2-kbp) R6Kori plasmid by inverse PCR, obviating the need to use oligonucleotides, which typically require multiple steps to incorporate into plasmids. The two plasmids will be fused in vivo using an E. coli strain expressing the site-specific recombinase, Flp, which will also serve to eliminate the antibiotic resistance gene on the R6Kori plasmid. The complete CRISPR/Cas9 system will be assembled onto the pBRR1 vector, which is a commonly used cloning vector for Brucella species (29). B. melitensis transformants will be selected using kanamycin resistance, an antibiotic that is not clinically used to treat brucellosis. We propose that this strategy will facilitate more rapid construction of multiple plasmids for empirical testing in B. melitensis than currently used methods.nThe DNA will be introduced to B. melitensis by electroporation, immediately following initiation of chromosome breakage. Following recovery, bacteria will be plated to select for colonies that have undergone recombinational repair. A final feature of the CRISPR/Cas9 plasmid system is that it will be "self-curing" to eliminate the plasmid, and its associated drug resistance, from the engineered strain. This will be accomplished by incorporating a second guide RNA that will target a sequence within the pBRR1 origin of replication, hence inactivating the plasmid by Cas9 cleavage. For this study, all plasmids will be constructed and initially tested in E. coli using guide RNAs that target the lacZ gene, where gene edits can easily be detected by colony color on chromogenic substrates incorporated into the growth media (e.g., X-gal). All future work in Brucella will be done through collaboration with Dr. Steven Olsen at the National Animal Disease Center, Ames, IA.