Progress 01/15/18 to 01/14/19
Outputs
Target Audience:Other researchers; our progress has been informally reported by word of mouth to interested researchers and formally at a meetingin Canada. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has given me the opportunity to take advantage of the depth of experience in insect biology, specifically expertise in bee biology availablein the Entomology Department at PSU. Departmental seminares have provided an expansion of entomology undertanding that complements well my molecular expertise.I have also had the opportunity to share this work at conferences, which has provided valuable networking opportunities that will certainly advance my career as a research scientist. 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?This is the last reporting period for this project.
Impacts
What was accomplished under these goals?
Impact: Thusfar, gene-editing has not been accomplished in the bumble bee, Bombus impatiens, preventing the execution of important research that will tell us about bee biology, polinator decline and possible approaches to bolstering the bee populations that support our agriculture. CRISPR/Cas9 and RNAi are both important technologies that can support these goals. These technologies are unavailable for B. impatiens the protein and nucleic acids required for genetic techniques are difficult to deliver to eggs. In effort to improve delivery, I have injected Bumbleb bees with proteins designed to be delivered to the ovaries in bees and preliminary results suggest that the proteins are present in the ovaries, suggested that our method will aid in the delivery of CRISPR/Cas9 and RNAi machinery to the eggs. These methods, when developed and available to pollinator researchers will provide the tools to gain knowledge to support the environment and our agricultural economy. Goal 1: 1.1.This was accomplished and reported on in a previous reporting period. Briefly, GFP is not usable in Bumble bee ovaries because of intense green autofluorescence in this tissue. 1.2 This was also accomplished. Additionally, constructs were designed for expression in E.coli. In parallel, a regions of the B. impatiens vitellogenin that are conserved among other species (herein called BiVg(8))was included in a fusion protein expression construct and expressed in E.coli as BiVg (8)-mCherry 1.3 This BiVg(8)-mCherry was used to treat ovaries in ex vivo culture. Fluorescence imaging suggested uptake, so BiVg(8) was used for downstream construct design and work was discontinued on other fragments of BiVg. Goal 2 2.1 Methods for biotinylation of dsRNA in vitro have been designed and tested and are ready to be used upon validation and optimization of the transport protein 2.2 Monomeric streptavidin wastested for fusion protein design, however, while monomeric streptavidin was initially successfully purified from E.coli preperations as active protien and found to successfully bind biotinylated double-stranded RNA, successful purification was not consistent. New constructs were designed to encode a monomeric streptavidin that is modified in such a way as to avoid sequestration in the pelleted E.coli during purification and so enhance purificaton of active protein as described byKroetsch et al. inAppl Microbiol Biotechnol.2018 Dec;102(23):10079-10089. doi: 10.1007/s00253-018-9377-7. Epub 2018 Sep 24. Fusionprotein has not yet been successfully expressed. 2.3 No additional progress has been made on this objective since the last reporting period, since monomeric streptavidin constructs are being modified to enable consistent purification. From the last reporting period:As part of this objective the following methods for dsRNA biotinylation have been tested. Treatment of IVT-generated dsRNA treated with Photoprobe biotin from vector shield consistently produces biotinylated dsRNA as detectable by western blot and dot blot. Both extension of biotinylated poly-adenosine tail onto single stranded RNA following in vitro transcription (IVT) followed by annealing to produce end-biotinylated dsRNA and integration of biotinylated dATP during IVT failed to produced biotinylated dsRNA detectable by dot blot with a biotin-specific antibody. 2.4 Progress on this objective is precluded by barriers described in previous objectives Goal 3: 3.1 This has been accomplished: BiVg(8)-Cas9 is being used for the next objectives 3.2 and 3.3Activated queens are being utilized instead of worker females, as described in the last reporting period. These queens are being injected and progeny assayed. Successfull knockout of the B. impatiens KMO has yet to be detected.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Transovarial delivery of Cas9 to diverse arthropod species for easy genetic manipulation. Progress was reported on the project in Bombus impatiens that is supported by this USDA funding.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Targeted delivery of CRISPR-Cas9 ribonucleoprotein into arthropod ovaries for heritable germline gene editing
Duverney Chaverra-Rodriguez, Vanessa M. Macias, Grant L. Hughes, Sujit Pujhari, Yasutsugu Suzuki, David R. Peterson, Donghun Kim, Sage McKeand & Jason L. Rasgon
Nature Communicationsvolume 9, Article number: 3008 (2018)
|
Progress 01/15/17 to 01/14/19
Outputs
Target Audience:Other researchers; our progress has been informally reported by word of mouth to interested researchers and formally at a meetingsin Florida, Shanghai andCanada Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has given me the opportunity to take advantage of the depth of experience in insect biology, specifically expertise in bee biology available in the Entomology Department at PSU. Departmental seminares have provided an expansion of entomology undertanding that complements well my molecular expertise. I have also had the opportunity to share this work at conferences, which has provided valuable networking opportunities that will certainly advance my career as a research scientist. How have the results been disseminated to communities of interest?Results have been presented at scientific meetigs in Florida, Shanghai and Vancouver and in discussions in many research collaborative venues. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Impact: Thusfar, gene-editing has not been accomplished in the bumble bee, Bombus impatiens, preventing the execution of important research that will tell us about bee biology, polinator decline and possible approaches to bolstering the bee populations that support our agriculture. CRISPR/Cas9 and RNAi are both important technologies that can support these goals. These technologies are unavailable for B. impatiens because the protein and nucleic acids required for genetic techniques are difficult to deliver to eggs. In effort to improve delivery, I have injectedbees at the adult stage instead of the embryo stage, with proteins designed to be delivered to the ovaries in bees and preliminary results suggest that the proteins are present in the ovaries, suggested that our method will aid in the delivery of CRISPR/Cas9 and RNAi machinery to the eggs. These methods, when developed and available to pollinator researchers will provide the tools to gain knowledge to support the environment and our agricultural economy. Goal 1: 1.1.This was accomplished and reported on in a previous reporting period. Briefly, GFP is not usable in Bumble bee ovaries because of intense green autofluorescence in this tissue. 1.2 This was also accomplished. Additionally, constructs were designed for expression in E.coli. In parallel, a regions of the B. impatiens vitellogenin that are conserved among other species (herein called BiVg(8)) was included in a fusion protein expression construct and expressed in E.coli as BiVg (8)-mCherry 1.3 This BiVg(8)-mCherry was used to treat ovaries in ex vivo culture. Fluorescence imaging suggested uptake, so BiVg(8) was used for downstream construct design and work was discontinued on other fragments of BiVg. Goal 2 2.1 Methods for biotinylation of dsRNA in vitro have been designed and tested and are ready to be used upon validation and optimization of the transport protein 2.2 Monomeric streptavidin was tested for fusion protein design, however, while monomeric streptavidin was initially successfully purified from E.coli preperations as active protien and found to successfully bind biotinylated double-stranded RNA, successful purification was not consistent. New constructs were designed to encode a monomeric streptavidin that is modified in such a way as to avoid sequestration in the pelleted E.coli during purification and so enhance purificaton of active protein as described by Kroetsch et al. in Appl Microbiol Biotechnol. 2018 Dec;102(23):10079-10089. doi: 10.1007/s00253- 018-9377-7. Epub 2018 Sep 24. Fusion protein has not yet been successfully expressed. 2.3 No additional progress has been made on this objective since the last reporting period, since monomeric streptavidin constructs are being modified to enable consistent purification. From the last reporting period: As part of this objective the following methods for dsRNA biotinylation have been tested. Treatment of IVT-generated dsRNA treated with Photoprobe biotin from vector shield consistently produces biotinylated dsRNA as detectable by western blot and dot blot. Both extension of biotinylated poly-adenosine tail onto single stranded RNA following in vitro transcription (IVT) followed by annealing to produce end-biotinylated dsRNA and integration of biotinylated dATP during IVT failed to produced biotinylated dsRNA detectable by dot blot with a biotin-specific antibody. 2.4 Progress on this objective is precluded by barriers described in previous objectives Goal 3: 3.1 This has been accomplished: BiVg(8)-Cas9 is being used for the next objectives 3.2 and 3.3 Activated queens are being utilized instead of worker females, as described in the last reporting period. These queens are being injected and progeny assayed. Successfull knockout of the B. impatiens KMO has yet to be detected.
Publications
|
Progress 01/15/17 to 01/14/18
Outputs
Target Audience:Other researchers;our progress has been informally reported by word of mouth to interested researchers and formally at meetings in Florida and in China. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has given me the opportunity to train in many aspects of Bumble Bee biology, including bumble bee rearing and experimental methods from several skilled faculty and post-docs in the Entomology Department at PSU. 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?Goal1 Objectives 1.1 and1.2All constructs will be redesignedto encodemCherryfusion proteins instead of EGFP and subsequently expressed in E. coli. 1.3 Injections of P2C-mCherry will be repeated to validate uptake of the protein into the ovaries visualy and by western blot. Newly expressed proteins will be used in bothex vivotreatement of bumble ovaries and injected into vitellogenic queens.If further testing confirms ovary translocation by P2C, then I will use P2C forthe remaining objectives, instead of a portion ofBiVg. If not,BiVgfragment-mCherryfusion proteins will be expressed fromE.coliand tested for ovary translocation in explant culture.? Goal 2 Objectives 2.1. dsRNA will be designed and biotylated with the most efficient methods established during the first reporting period 2.2-4 Wll proceed as described in project initiation upon validation of P2C-mediated delivery or identification of BiVgmin Goal 3: All objectives will begin as planned in project initiation upon validation of P2C-mediated deliveryor identification of BiVgmin
Impacts
What was accomplished under these goals?
Impact:Thusfar, gene-editing has not been accomplished in the bumble bee,Bombusimpatiens, preventing the execution of important research that will tell us about bee biology,polinatordecline and possibleapproaches to bolstering the bee populations that support our agriculture. CRISPR/Cas9 and RNAi are both important technologies that can support these goals. These technologies areunavailable for B.impatiens theprotein and nucleicacids required for genetic techniques aredifficult to deliver to eggs. In efforttoimprovedelivery, I have injected Bumbleb bees withproteins designed to be delivered to the ovaries in bees andpreliminary results suggest that the proteins are present in the ovaries, suggested that our method will aid in thedeliveryof CRISPR/Cas9 and RNAi machinery to the eggs. These methods, when developed and available to pollinator researchers will provide the tools to gain knowledge to support the environment and our agricultural economy. Goal 1: Objectives: 1.1.This has been accomplished, however our result (below) suggest that GFP is not a useful marker as many oocytes display very strongautofluorescenceat the same wavelength as GFP. As aresulthese constructs have beenredisignedto includemCherry. Similarly, other reportshave suggested abinding region ofvitellogeninto the vitellogenin recepto;this regions inBiVgspans the second and third exon. Integrating this information into my project, I havedesigned a fusion of this~200 amino acid region, monomeric streptavidin (mSa) andmCherry, which issuccessfullyexpressed in E. coli and upon purification will be injected invitellogenicbumble bees. 1.2.BiVg-GFP fusion protein has been successfully expressed in S2 cells as confirmed by western blot. Analogous work in our laboratory on mosquito suggests that a high concentration of protein is required to achieve Cas9-targeted cleavage, so in anticipation of a similar phenomenon in bees, I have designed the fusion proteins for expression inE.coli.Several constructs have been built for expression of fusion proteins with fragments ofBiVgfused to EGFP, and now redesign of these constructs to bemCherryfusion proteins instead of EGFP is underway. 1.3.Several changes of action have occurred as a result of initial efforts to complete this objective.First, the fluorescent reporter used in these experiments has been changed from EGFP tomCherry.An initial experiment to test thepossibilityof using a fragment called "P2C," which is a peptidevalidated in our lab to mediate ovary translocation in several mosquito specieswasundertaken. P2C-EGFP fusion protein, EGFP or buffer controls solutions were injected intovitellogenicworkers. If P2C is capable of translocation of cargo to the ovaries, I could proceed with construct that have already been built, which would allow me to move forwardseveral steps, since many P2C fusion proteins have been built including P2C-Cas9 and P2C-mSa. However, the results were not conclusive, since during this experiment, Idiscovered that bumble oocytes emit anautofluorescenceat the green wavelengths whichistoostrong to eliminate even with reduced laser intensity. This result is very useful and hasledto a change of action from using fusion protein constructs with an EGFP to anmCherryreporter; the oocytes do not have high levels of redauto-fluorescence. Second, I have switched from using workers to using activated queens.Initially, it was proposed that proteins should be injected intovitellogenicworkers. New offerings from bumble bee vendors allow the purchase of may queens in a single colony. Since queens ovaries havemany more follicles than in workers and can similarly producehapoidprogeny, theyare more suited to this project. Third, prior to injections, proteins will be tested in explant culture. It is technically straightforward to dissect activated ovaries fromvitellogenicqueens and incubate them in culture medium treated with different fusion proteins. Upon recommendation from acollegueworking with mosquito ovaries in similar experiments, this method was tried in the following experiment including previously mentionedchanges of actionfor this objective. A P2C-mCherry fusion construct has been constructed and the resulting proteintested in for ovary translocation in ovaries in explant culture. In this first injection, red fluorescence was detected in several oocytes. This experiment will be repeated and theprescenceofmCherryin the oocytes validated by western blot. If further testing confirms ovary translocation by P2C, then I will use P2C forthe remaining objectives, instead of a portion ofBiVg. If not,BiVgfragment-mCherryfusion proteins will be expressed fromE.coliand tested for ovary translocation in explant culture. Goal 2: Objectives: 2.1.Methods forbiotinylationof dsRNA in vitro have been designed and tested and are ready to be used upon validation and optimization of the transport protein 2.2.BiVgminhas not yet been determined as described above, however, a fusion protein including the portion ofBiVganalogous tovitellogenin in Xenopus leavisthat has been demonstrated to mediate receptor binding and ovary translocation (Li et al. 2003, Rungger et al.2017),mSaandmCherryhas been constructed and will soon be purified and tested in B. impatiens ovary explantculture for translocation to the oocytes. 2.3. As part of this objectivethe following methods for dsRNAbiotinylationhave been tested.Treatment of IVT-generated dsRNA treated withPhotoprobe biotin from vector shield consistently producesbiotinylated dsRNA as detectable by western blot and dot blot.Both extensionof biotinylated poly-adenosine tail onto singlestrandedRNAfollowing in vitro transcription (IVT) followed by annealing to produce end-biotinylateddsRNAand integration of biotinylateddATPduringIVTfailed toproducedbiotinylated dsRNA detectable by dot blot with a biotin-specific antibody. Upon identification of a peptide capable of mediated ovary translocation in bees, biotinylated dsRNA will be boundtoBiVg- or P2C-mSA constructs and tested for ovary specific knockdown activity in the followingobjectin. 2.4.No progress on this objective yet, since all materials are not yet ready. Goal 3:Work on this goal has not yet begun. Objectives: 3.1. Construct and express a BiVgmin-Cas9 fusion protein 3.2. Inject worker females with fusion protein and guide RNAsagainsthe B. impatienskmoeye color gene 3.3. Rear progeny (all haploid males) of injected workers and screen for white eyes.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
International Workshop on Insect Genome Editing
Presentation: Heritable Cas9-mediated transgenesis of Aedes aegypti by adult intrathoracic injection
Progress was reported on the analagous project in Bombus impatiens that is supported by this USDA funding
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Annual meeting of the American Society of Tropical Medicine and Health
Poster: Heritable Cas9-mediated transgenesis of Aedes aegypti by adult intrathoracic injection
Progress was reported on the analagous project in Bombus impatiens that is supported by this USDA funding
|
|