Progress 10/01/14 to 09/30/17

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported 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? Using methodologies developed for transforming Rickettsia, another bacterial endosymbiont of arthropods, and those for transferring Wolbachia to naïve cell lines, we developed basic protocols for transforming Wolbachia and introducing them back into host cells. Optimization of these methodologies were ongoing. We tested transposon mutagenesis using preformed "transposomes", that is transposons complexed with their cognate transposases, and as well as plasmid systems in which the transposase is expressed from the plasmid borne gene. The generation of antibiotic resistant populations of Wolbachia within the recovered cells and positive PCR screens indicated that transformation using the transposome method was successful. However, we were unable to establish stable populations of cells containing transformed Wolbachia as cells grew and were passaged. One hypothesis for these results is that transposon insertion at a viable site was rare and Wolbachia insertions were lost when cells were diluted upon passage. Because of these results, we decided to focus attention on site-directed mutagenesis and the CRISPR/Cas9 system. Making the same mutation in all the transformed Wolbachia should make it possible to establish cell populations with a common mutated Wolbachia. To this end we are determining which Wolbachia genes to target by comparing the wAlbB genomic sequences with other bacterial species in the literature to identify targets that are likely to be dispensible, as well as investigating wAlbB promoters for use in developing the constructs needed for transformation of Wolbachia with the CRISPR/Cas9 system. We previously, tested the use of transposomes, preassembled transposase/transposon complexes comprised of an enzyme that randomly cuts the Wolbachia DNA and inserts a linear piece of DNA encoding the marker genes. We developed basic protocols for transforming Wolbachia and introducing them back into host cells. The generation of antibiotic resistant populations of Wolbachia within the recovered cells and positive polymerase chain reaction (PCR) screens indicated that transformation using the transposome method was successful. However, we were unable to establish stable populations of cells containing transformed Wolbachia as cells grew and were passaged. Because transposon insert randomly and multiple Wolbachia may enter a single cell, it is likely that cells carrying Wolbachia with transposon insertions were lost when cells were diluted upon passage. We also focused on the use of the recently developed CRISPER/Cas9 system. This system, derived from a bacterial anti-virus defense system, facilitates targeted cutting of genomes, such that a single gene is disabled. Because all of the Wolbachia will have a mutation in the same gene it will be easier to isolate a population of cells carrying the mutated Wolbachia. Because little is known about the functions of most Wolbachia genes one of our first tasks was to identify putative target genes that are likely to be non-essential for Wolbachia survival. Moreover, if possible we wanted to choose genes that could potentially be involved in CI, as this would give us both a functional assay in mosquitoes and could provide insight into the CI mechanism. To do this we relied on a genomic study in Drosophila (Sutton et al. BMC Genomics 2014, 15:928), in which two closely related strains of Wolbachia were compared. They differed in that one strain induced CI and the other strain could not. Several genes that were inactivated or deleted from the non-inducing strain but not the inducing strain were identified. We selected three of these, which were also missing in other non-CI-inducing strains, as potential targets. The genome of the wAlbB strain has not been fully assembled and is available only as a collection of contigs. Of the three selected genes from the Drosophila study we were able to identify a wAlbB homolog for only one of them. To increase the number of test targets two additional genes were selected. These are genes of unknown function containing ankyrin repeat domains. Ankyrin repeat domain proteins are also thought to be involved in CI, and are highly represented in the Wolbachia genome. Guide RNAs for cleaving the wALbB genome and were designed based on these sequences. The rest of our efforts were focused on determining conditions for transforming Wolbachia. In mosquitoes, my colleague's group found that injecting Cas9 protein was more effective at cleaving the mosquito genomic DNA than plasmid expressed Cas9. Moreover, when Wolbachia are isolated from their host cells they remain viable for several days but do not replicate, suggesting they may not be very active metabolically. Therefore we decided to focus on transformation using the Cas9 protein along with guide RNAs rather than expressing the Cas9 and the guide RNAs from plasmids. There is little information in the literature on bacterial transformation with proteins, so we initially tried to determine conditions using a fluorescent protein marker. However this was not sensitive enough for a reliable assay. We worked on developing PCR based assays to evaluate CRISPR/Cas9 cutting. This was somewhat challenging because most prokaryotes do not have the capability to do nonhomologous end joining repairs as do eukaryotes. For rapid detection of cleavage and without the added step of reintroducing the Wolbachia into insect cells, we need to be able to detect a gap rather than a deletion. One possible method is linker ligated PCR, but we sought a more rapid assay. Ultimately, our goal was to introduce a cassette carrying antibiotic and fluorescent protein selection markers, as in the transposome efforts, with end-homology to the Wolbachia genome flanking the cuts. Thus, another strategy would be to include the insertion cassette for homologous recombination. This would facilitate fairly straight-forward PCR analysis but if the results are negative it would be difficult to determine if the problem is genome cleavage or homologous recombination.

Publications

Progress 10/01/15 to 09/30/16

Outputs
Target Audience: Nothing Reported Changes/Problems:Current focus is on CRISPER/Cas9 system and homologous recombination rather than transposon mediated mutagenesis. What opportunities for training and professional development has the project provided? Nothing Reported 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?Our goals for the next reporting system is to continue to work on developing CRISPR/Cas9 for Wolbachia modification with an emphasis on modifying or developing assays to assess cleavage of the Wolbachia genome following electroporation. A reliable assay would facilitate optimization the electroporation protocols. Once these are accomplished we will move on to test for homologous recombination in this system and reintroduction into mosquito cells. There is a paucity of characterized Wolbachia promoters available for use in a Wolbachia genetic system, so as time permits we will characterize additional wAlbB promoters.

Impacts
What was accomplished under these goals? We previously, tested the use of transposomes, preassembled transposase/transposon complexes comprised of an enzyme that randomly cuts the Wolbachia DNA and inserts a linear piece of DNA encoding the marker genes. We developed basic protocols for transforming Wolbachia and introducing them back into host cells. The generation of antibiotic resistant populations of Wolbachia within the recovered cells and positive polymerase chain reaction (PCR) screens indicated that transformation using the transposome method was successful. However, we were unable to establish stable populations of cells containing transformed Wolbachia as cells grew and were passaged. Because transposon insert randomly and multiple Wolbachia may enter a single cell, it is likely that cells carrying Wolbachia with transposon insertions were lost when cells were diluted upon passage. This reporting period we focused on the use of the recently developed CRISPER/Cas9 system. This system, derived from a bacterial anti-virus defense system, facilitates targeted cutting of genomes, such that a single gene is disabled. Because all of the Wolbachia will have a mutation in the same gene it will be easier to isolate a population of cells carrying the mutated Wolbachia. Because little is known about the functions of most Wolbachia genes one of our first tasks was to identify putative target genes that are likely to be non-essential for Wolbachia survival. Moreover, if possible we wanted to choose genes that could potentially be involved in CI, as this would give us both a functional assay in mosquitoes and could provide insight into the CI mechanism. To do this we relied on a genomic study in Drosophila (Sutton et al. BMC Genomics 2014, 15:928), in which two closely related strains of Wolbachia were compared. They differed in that one strain induced CI and the other strain could not. Several genes that were inactivated or deleted from the non-inducing strain but not the inducing strain were identified. We selected three of these, which were also missing in other non-CI-inducing strains, as potential targets. The genome of the wAlbB strain has not been fully assembled and is available only as a collection of contigs. Of the three selected genes from the Drosophila study we were able to identify a wAlbB homolog for only one of them. To increase the number of test targets two additional genes were selected. These are genes of unknown function containing ankyrin repeat domains. Ankyrin repeat domain proteins are also thought to be involved in CI, and are highly represented in the Wolbachia genome. Guide RNAs for cleaving the wALbB genome and were designed based on these sequences. The rest of our efforts have focused on determining conditions for transforming Wolbachia. In mosquitoes, my colleague's group found that injecting Cas9 protein was more effective at cleaving the mosquito genomic DNA than plasmid expressed Cas9. Moreover, when Wolbachia are isolated from their host cells they remain viable for several days but do not replicate, suggesting they may not be very active metabolically. Therefore we decided to focus on transformation using the Cas9 protein along with guide RNAs rather than expressing the Cas9 and the guide RNAs from plasmids. There is little information in the literature on bacterial transformation with proteins, so we initially tried to determine conditions using a fluorescent protein marker. However this was not sensitive enough for a reliable assay. We are now developing PCR based assays to evaluate CRISPR/Cas9 cutting. This is somewhat challenging because most prokaryotes do not have the capability to do non-homologous end joining repairs as do eukaryotes. For rapid detection of cleavage and without the added step of reintroducing the Wolbachia into insect cells, we need to be able to detect a gap rather than a deletion. One possible method is linker ligated PCR, but we are seeking a more rapid assay. Ultimately, our goal is to introduce a cassette carrying antibiotic and fluorescent protein selection markers, as in the transposome efforts, with end-homology to the Wolbachia genome flanking the cuts. Thus, another strategy would be to include the insertion cassette for homologous recombination. This would facilitate fairly straight-forward PCR analysis but if the results are negative it would be difficult to determine if the problem is genome cleavage or homologous recombination. We had planned to identify and characterize wAlbB gene promoters during this reporting period. That work was not done.

Publications

Progress 10/01/14 to 09/30/15

Outputs
Target Audience: Nothing Reported Changes/Problems:Because we were unable to establish stable transformed lines using the random insertion of transposons we plan to focus on site-specific mutagenesis using the CRISPR/Cas9 system. At this time we have few promoters to use in our constructs. We are limited by a paucity of information about potential Wolbachia promoters. Only one promoter from the strain of Wolbachia, wAlbB, we are studying has been characterized. This is the promoter for Wolbachia surface protein, wsp, a highly expressed gene. However, the wAlbB genomic sequence has been determined, although not completely assembled, we use this data to identify prospective promoters for this work. Information on other Wolbachia, as well as other bacteria, from the literature will be used to select prospective promoters for strong constituative expression. We also plan to explore the incorporation of the lambda red recombinase system for enhancing the efficiency of recombination in Wolbachia as reported for transformation of Escherichia coli with CRISPR/Cas9. What opportunities for training and professional development has the project provided? Nothing Reported 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?The major goal for the next reporting system is to characterize additional wAlbB promoters, make the necessary plasmid constructs for CRISPR targeted site-specific mutagenesis of Wolbachia, and work towards establishing CRISPR/Cas9 mutagenesis in Wolbachia.

Impacts
What was accomplished under these goals? Using methodologies developed for transforming Rickettsia, another bacterial endosymbiont of arthropods, and those for transferring Wolbachia to naïve cell lines, we developed basic protocols for transforming Wolbachia and introducing them back into host cells. Optimization of these methodologies are ongoing. We tested transposon mutagenesis using preformed "transposomes", that is transposons complexed with their cognate transposases, and as well as plasmid systems in which the transposase is expressed from the plasmid borne gene. The generation of antibiotic resistant populations of Wolbachia within the recovered cells and positive PCR screens indicated that transformation using the transposome method was successful. However, we were unable to establish stable populations of cells containing transformed Wolbachia as cells grew and were passaged. One hypothesis for these results is that transposon insertion at a viable site was rare and Wolbachia insertions were lost when cells were diluted upon passage. Because of these results, we decided to focus attention on site-directed mutagenesis and the CRISPR/Cas9 system. Making the same mutation in all the transformed Wolbachia should make it possible to establish cell populations with a common mutated Wolbachia. To this end we are determining which Wolbachia genes to target by comparing the wAlbB genomic sequences with other bacterial species in the literature to identify targets that are likely to be dispensible, as well as investigating wAlbB promoters for use in developing the constructs needed for transformation of Wolbachia with the CRISPR/Cas9 system.

Publications