INSECT GENETIC BIOCONTROL: PERFORMANCE OF MOLECULAR COMPONENTS IN FLUCTUATING ENVIRONMENTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: OTHER GRANTS
• Reporting Frequency: Annual
• Accession No.: 1027023
• Grant No.: 2021-33522-35340
• Cumulative Award Amt.: $499,600.00
• Proposal No.: 2021-04205
• Project Start Date: Sep 1, 2021
• Project End Date: Aug 31, 2025
• Grant Year: 2021
• Program Code: [HX]- Biotechnology Risk Assessment

Recipient Organization
UNIV OF MINNESOTA
(N/A)
ST PAUL,MN 55108

Performing Department
Biotechnology Institute

Non Technical Summary
Many recently developed Genetic Control strategies require the use of CRISPR/Cas9 tools. These tools do not perform well at the wide range of temperatures that they would be required to work in for many pest control applications. In this project, we seek to develop new and improved versions of CRISPR/Cas9 tools that work well at moderate temperatures.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
215	3110	2020	100%

Knowledge Area
215 - Biological Control of Pests Affecting Plants;

Subject Of Investigation
3110 - Insects;

Field Of Science
2020 - Engineering;

Keywords

genetic biocontrol

cas9-based tools

insect biotechnology

regulatory

Goals / Objectives
This proposal is concerned with better understanding a new class of technologies relevant to pest control in agricultural settings. Genetic biocontrol is defined in this proposal as genetically engineering a pest organism to become a biocontrol agent. Environmental release of this agent will eradicate local populations of the pest. Many specific strategies for genetic biocontrol utilize Cas9-based tools. Understanding how well these tools will work in the dynamic environmental conditions likely to be encountered in open-air releases is of great concern to technology developers and regulators. We have preliminary evidence to show an impact of temperature on one specific genetic biocontrol approach that was developed by our lab. In this proposal, we aim to understand the mechanistic reasons behind this temperature effect, and expand the utility of Cas9-based tools for low-temperature applications (to the benefit of many genetic biocontrol strategies). We have substantial preliminary data to de-risk the three main objectives, making it highly likely that this project will generate outcomes that will impact the development and regulation of insect genetic biocontrol technologies.

Project Methods
The U6-promoter50,51 driven sgRNAs and synthetic target sequence will be delivered to a naïve D. melanogaster strain by embryo microinjection and attP insertion to characterized landing pads52. These cassettes will be combined with the Cas9 components via mating. Balancer chromosomes will facilitate tracking the homo/heterozygosity of strains, and all characterization of Cas9-tool activity will occur in the first generation offspring of homozygous parents, meaning that they are heterozygous at both the Cas9 locus and the sgRNA/target sequence locus. The genotype of all transgenic fly lines will be verified by Sanger sequencing of relevant loci prior to experimental characterizationHeterozygous flies generated in subtask 1.1 will be reared in different temperatures (13°C, 18°C, 23°C, and 28°C). Animals will be harvested at different life-stages (L3 larva, pupa, virgin adults) and gDNA will be extracted from pools of 50 individuals in the same treatment group. We will use analytical pipelines described briefly in Section 8.1.7.3 above and illustrated in Figure 8 to measure the on- and off-target activity in specially modified reporter strains. Specifically, we will amplify the synthetic target sequence from the extracted gDNA and sequence the ends using GeneWiz NGS Amplicon Sequencing. In-house data processing pipelines already coded on the Mesabi supercomputer at the Minnesota Supercomputing Institute will allow us to detect the editing frequency of Cas9 nucleases and Cas9-ABEs.We will test the variant sgRNA designs in a yeast screening platform. Yeast strains will contain constitutively expressed Cas9 nucleases or nCas9-ABEs, and both the sgRNA expression cassettes and the complementary target sequences will be introduced by transformation on a plasmid backbone. The sgRNA sequence variants that we will test in this subtask are listed in Table 1. For each type of sgRNA design, we will test 2-5 unique sequence variants to make sure that observed effects are due to the GC content or hairpin design, and not artifacts of a specific outlier sequence. We will design target sequences for Cas9 nuclease cleavage or ABE-editing to contain a rare restriction recognition site at the locus being cleaved/modified. Activity of the various sgRNAs will be determined by transforming yeast with the sgRNA / target sequence plasmid, incubating at various temperatures for 8 hours, and performing a PCR amplification and restriction digest of the target sequence. Cas9 or ABE activity will result in a protection against restriction cleavage, and this can be quantified by gel electrophoresis and image analysis.We have designed a high-throughput yeast selection to identify ABEs or nucleases that work well at low temperature. A nonsense antibiotic resistance gene (i.e. with a premature stop codon in the first 10 amino acids) will be present in a plasmid encoding variant nCas9-ABE genes. This plasmid will be introduced to yeast stain stably expressing a sgRNA directing the ABE to the nonsense codon. The yeast will be maintained at low temperature (initially 25°C, but progressively colder with each round of selection) for 8 hours. An Adenine to Guanine transition mutation will create a functional antibiotic resistance gene, but there will be no selection at this point. After the initial low temperature incubation in yeast, plasmids will be isolated from the yeast population and transformed into E. coli and selected on antibiotic. Plasmids conferring resistance will encode an ABE that was active at low temperature, which will seed the next round of mutagenesis and screening (in a fresh plasmid backbone containing the non-sense resistance gene). This two-stage protocol allows us to perform the selection robustly at 37°C while requiring that the ABE was active at low temperature (there will be no sgRNA in the E. coli selection strain). It also takes advantage of the high rate of transformation in yeast and E. coli (Figure 9a,b).To generate sequence diversity in the ABEs, we will begin with error-prone PCR of the region spanning the N-terminal TadA domains and Cas9 nuclease domains. Control experiments will include a positive control (yeast incubated at 37°C with non-mutated Cas9-ABE) and negative controls (plasmid introduced to a yeast strain not expressing the sgRNA) to determine the range of antibiotic resistant E. coli colonies expected from a fully active ABE and from random mutation of the nonsense codon, respectively. We will design separate selection plasmids with stop-codon containing Kanamycin, Chloramphenical, and Ampicillin genes, all with unique sgRNA protospacer sequences. Screens will alternate between the three so that we do not select for active editing of a single protospacer sequence, which has been observed previously during ABE selections48. Plasmids encoding cold-active ABEs will be sequenced to identify the nature of mutations, and we will perform final rounds of selection using combinatorial site saturation mutagenesis to identify possible synergistic interactions. We expect to identify substitution mutations both within and outside of enzyme active sites.

Progress 09/01/23 to 08/31/24

Outputs
Target Audience:The target audience for this project includes researchers and engineers in the area of genetic biocontrol (engineering insect pests to generate new classes of pesticides), as well as scientists involved in assessing regulatory permit applications for these technologies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has primarily supported a single FTE during the past year, but that FTE has changed throughout the year. The first trainee graduated with a master's degree and is currently gainfully employed at a local biotechnology company. The second trainee is currently and advanced-stage doctoral candidate. A third trainee was supported for the last two months of this year (another advanced-stage doctoral candidate) as his research became aligned with the goals of this project (bullet point #4 in the previous section). How have the results been disseminated to communities of interest?Results have been communicated broadly at the following conferences where oral presentations or posters were presented: NSF/UKRI SITS program annual meeting (Alexandria, VA) "Reverse-engineering the soil microbiome", June 2024 (this might seem like a mistake based on the talk title, but I actually discussed the temperature-dependent Cas9-base editing activity here) 23rd International Conference on Aquatic Invasive Species (Halifax, Canada) "Genetic Biocontrol of Common Carp", May 2024 Invited EPSCORE program workshop keynote (Lincoln, NE) "Accelerating the translation of new technologies from the laboratory to the real world", May 2024 BioMADE annual member meeting (Minneapolis, MN) "Unit operations for biomanufacturing" (session chair and moderator), May 2024 ASA/CSSA/SSSA International Annual Meeting (St. Louis, MO) "Progressing Towards Biocontainment in Perennial Ryegrass with Improved Transformation Protocols" October 2023 (Jonathan Cors). In addition, a paper was published in BMC Biology with results from this project. What do you plan to do during the next reporting period to accomplish the goals?We did not make as much progress with the yeast directed evolution aim in the past year and I would like to push harder in that direction in the next year.

Impacts
What was accomplished under these goals? We have made substantial progress along the following three areas: 1. Established a novel protocol for empirically estimating the threshold of an extreme-underdominance based threshold dependent gene drive (TDGD) in a single generation. 2. Used this protocol to determine the threshold of two distinct genotypes of TDGD agents, discovering a unique and unanticipated impact of temperature on the measured threshold of each genotype. 3. Advancing a yeast-based directed evolution platform for increasing the low-temperature activity of Cas9 nucleases and Adenine Base Editors. 4. Discovering a surprising transversion mutation (C to G) at moderate temperatureswhen using C-base editors in non-model species of Streptomyces. If we can decipher the molecular mechanism by which this unanticipated mutation occurs, it could have implications in the design and behavior of genetic biocontrol strategies.

Publications

• Type: Journal Articles Status: Published Year Published: 2024 Citation: Janzen A, Pothula R, Sychla A, Feltman NR, Smanski MJ (2024) Predicting thresholds for population replacement gene drives. BMC Biol 22, 40 (2024). https://doi.org/10.1186/s12915-024-01823-2

Progress 09/01/22 to 08/31/23

Outputs
Target Audience:The target audience for this project includes researchers and engineers in the area of genetic biocontrol (engineering insect pests to generate new classes of pesticides), as well as scientists involved in assessing regulatory permit applications for these technologies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has primarily supported the work of a single graduate student in the past year. She has learned many new skills in experimental design and molecular biology. She was recently offered a great career opportunity and will transition out of the lab shortly. A new student will take over this project in the Fall. How have the results been disseminated to communities of interest?Preliminary results have been disseminated via poster presentations and invited oral talks at conferences and meetings. This includes a project update given to the Minnesota Department of Natural Resources, an update at a stakeholder workshop that included federal regulatory agencies, poster and oral presentions at the Plant Biology conference, and at USDA PD meetings. A new paper containing data generated on this project was published in Nucleic Acids Research. What do you plan to do during the next reporting period to accomplish the goals?We plan to use our optimized protocols for yeast-based measurement of Cas9 activity to select for Cas9 variants that are active at cold temperatures.

Impacts
What was accomplished under these goals? We have made substantial progress along the following three areas: 1. Characterizing the temperature-dependent performance of genetic biocontrol systems in insects that utilize Cas9-derived molecular tools: We have measured the threshold-dependent gene-drive performance of insects with Engineered Genetic Incompatibility (EGI). This advances our previous (preliminary data in the grant proposal) characterization of the hybrid lethality phenotypes at different temperatures. 2. Ruling out temperature-dependent activity of dCas9-based programmable transcription activators (PTAs): We had previously observed a breakdown of the performance of EGI at low temperatures (18*C, 23*C) for certain genotypes. We could not deduce a priori whether this breakdown was due to failure of the dCas9-activators at low temperatures (Cas9 nuclease has been reported to not function well below 25*C), or if the developing insects were just more robust to perturbed expression of developmental morphogens at colder temperatures. To test the low temperature extremes (colder than we could reasonable grow insects), we used a plant PTA expression system grown at 4*C. Even at 4*C, we saw strong overexpression of target loci, providing the first evidence that PTAs can function across the temperature range in which a genetic biocontrol agent would likely be deployed. This suggests that the survival of hybrid wild-type / EGI parents that survived at 18*C did so because the lower temperatures slowed developmental processes and rendered them more robust to pertubations of gene expression. In the future we will obtain direct evidence to address this hypothesis. 3. Create a yeast-based directed evolution platform for increasing the low-temperature activity of Cas9 nuclease and Cas9- based Adenine Base Editors: We generated dozens of plasmids for this yeast evolution system including Cas9-repairable selectable markers and Cas9 or Cas9-ABE expression plasmids. In the past year we have established a measurement system for Cas9 activity in yeast that is compatible with a high-thoughput screening campaign.

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: J Armando Casas-Mollano and others, Efficient gene activation in plants by the MoonTag programmable transcriptional activator, Nucleic Acids Research, Volume 51, Issue 13, 21 July 2023, Pages 70837093, https://doi.org/10.1093/nar/gkad458

Progress 09/01/21 to 08/31/22

Outputs
Target Audience:The target audience for this project includes researchers and engineers in the area of genetic biocontrol (engineering insect pests to generate new classes of pesticides), as well as scientists involved in assessing regulatory permit applications for these technologies. Changes/Problems:We have experienced two major problems/challenges during this reporting period: 1. High experimental variance in the temperature-dependent threshold-dependent gene drive experiment. Determining the threshold for population replacement empirically was fairly straightforward for several EGI genotypes at ideal lab conditions (25*C and controlled humidity), but the experiment at different environmental conditions has suffered from large experimental variance. This has decreased the statistical power of determining the replacement threshold. Initially we increased the N (number of experimental replicates) to try to solve this problem, but have settled on running individual replicates with much larger numbers of flies per replicate (using bottles instead of vials to do the wild-type / EGI matings). This seems to be more reproducible from one experiment to the next, but we have not finished collecting a full dataset yet. 2. We do not observe Cas9-ABE base editing in yeast at levels above the negative control. We have a directed evolution platform build in yeast in which a non-functional antibiotic resistance selectable marker can be corrected by the activity of a Cas9-ABE in yeast. We get low numbers of surviving colonies that do bear the corrected sequence, but the frequency of these colonies is identicle in our negative control experiments lacking the ABE. This means that we are just detecting background levels of spontaneous mutation. On one hand this is promising because it shows that we have a sensitive selection system, but of course it is discouraging that the ABE is not providing higher-than-background levels of rescue. There are several newer iterations of ABEs that we can try in our system to find one with a basal activity that can be improved by directed evolution. First though, we will turn out attention to Cas9 nuclease (also included in our original proposal), because we know that this is active in yeast. A few more genetic constructs need to be made in yeast to test this platform. What opportunities for training and professional development has the project provided?This project provided training and professional development for two doctoral graduate students and one postdoc. All three trainees are still in the lab. One of the students matriculated from a junior doctoral student to an advanced student (i.e. passed her preliminary exams) during this reporting period. Both students have been given opportunities to present this research at regional or international conferences. How have the results been disseminated to communities of interest?Preliminary results have been disseminated via poster presentations and invited oral talks at conferences and meetings. This includes a project update given to the Minnesota Department of Natural Resources, an update at a stakeholder workshop that included federal regulatory agencies, poster and oral presentions at the Synthetic Biology, Evolution, Engineering, and Design (SEED) conference, the Engineering Biology Research Consortia annual meeting, the Gordon Research Conference on Genetic Biocontrol, and the Cold Spring Harbor Laboratory Synthetic Biology Summer Course, What do you plan to do during the next reporting period to accomplish the goals?We plan to interrogate the molecular biology of Cas9-based tools in insects reared different temperatures to provide more direct evidence to support hypotheses that explain the overall system failure at cold temperatures. We also plan to shift our Cold-Cas9 directed evolution project towards Cas9 nuclease (i.e. away from Cas9-ABEs), as the ABEs are notoriously difficult to work with. We still plan on evolving ABEs later in the award period, but would like to demonstrate the directed evolution platform on an enzyme that will be easier to get working first.

Impacts
What was accomplished under these goals? We have made substantial progress along the following three areas: 1. Characterizing the temperature-dependent performance of genetic biocontrol systems in insects that utilize Cas9-derived molecular tools: We have measured the threshold-dependent gene-drive performance of insects with Engineered Genetic Incompatibility (EGI). This advances our previous (preliminary data in the grant proposal) characterization of the hybrid lethality phenotypes at different temperatures. 2. Ruling out temperature-dependent activity of dCas9-based programmable transcription activators (PTAs): We had previously observed a breakdown of the performance of EGI at low temperatures (18*C, 23*C) for certain genotypes. We could not deduce a priori whether this breakdown was due to failure of the dCas9-activators at low temperatures (Cas9 nuclease has been reported to not function well below 25*C), or if the developing insects were just more robust to perturbed expression of developmental morphogens at colder temperatures. To test the low temperature extremes (colder than we could reasonable grow insects), we used a plant PTA expression system grown at 4*C. Even at 4*C, we saw strong overexpression of target loci, providing the first evidence that PTAs can function across the temperature range in which a genetic biocontrol agent would likely be deployed. This suggests that the survival of hybrid wild-type / EGI parents that survived at 18*C did so because the lower temperatures slowed developmental processes and rendered them more robust to pertubations of gene expression. In the future we will obtain direct evidence to address this hypothesis. 3. Create a yeast-based directed evolution platform for increasing the low-temperature activity of Cas9 nuclease and Cas9-based Adenine Base Editors: We generated dozens of plasmids for this yeast evolution system including Cas9-repairable selectable markers and Cas9 or Cas9-ABE expression plasmids. Initial attempts to select for Cas9-ABE activity in yeast were unsuccessful.

Publications