Source: KANSAS STATE UNIV submitted to
ANALYSIS OF CRISPR-BASED GENE DRIVE SYSTEMS IN BUDDING YEAST
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
REVISED
Funding Source
Reporting Frequency
Annual
Accession No.
1013520
Grant No.
(N/A)
Project No.
KS00-0013-HA
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Aug 18, 2017
Project End Date
Jun 30, 2022
Grant Year
(N/A)
Project Director
Finnigan, GR, .
Recipient Organization
KANSAS STATE UNIV
(N/A)
MANHATTAN,KS 66506
Performing Department
Biochemistry and Molecular Biophysics
Non Technical Summary
The entire field of agriculture faces numerous challenges involving biological pests (plants, insects, fungi, and/or pathogens) at all stages of food production, harvet, storage, and transportation. New methods need to be developed to aid in the control of pests. One newly discovered biotechnology that has the power and potential to control living populations with great effectiveness including the "CRISPR/Cas9" gene drive genetic arrangement. This technology allows a programmed bypass and editing of a species (e.g. insect) genetic material (in the form or one or more genes). Destruction of critical genes can cause a reduction in organism breeding, spread, and population size. The CRISPR-based gene drive bypasses traditional genetic actions and "forces/drives" a predetermined genetic element into a population at a very high rate of speed and penetance. This can cause a dramatic decrease in population levels of key insects, pests, or pathogenes in wild populations. However, there are logistical, technical, and ethical considerations for utilizing this very new technology in real applications. Our study aims to (i) develop an artifical gene drive system in a model species (budding yeast) to safely and rapidly test numerous CRISPR-based variants and their effectiveness within a controlled population, (ii) examine modes of regulation for the gene drive mechanism, and (iii) determine how to safely implement this technology with appropraite safeguards. These findings will assess the feasibility, effectiveness, and molecular variants required for development of new, safe, and controllable gene drive elements to be tested in key species.
Animal Health Component
0%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2157410108050%
2157410104050%
Goals / Objectives
1. Construction of artificial "model" Eukaryotic gene drive system in S. cerevisiae (budding yeast) using S. pyogenes Cas9 from the bacterial CRISPR gene editing system.2. Testing and analysis of three distinct components of the CRISPR system required for all gene drives--single guide RNA, target genomic (double-stranded) DNA, and Cas9 nuclease--to determine methods to optimize and regulate (gene) drive function.3. Expression and molecular characterization of new class of proteins termed "anti-CRISPR" peptides and their ability to inhibit Cas9 nuclease(s).
Project Methods
A BRIEF STATEMENT of the PROBLEM and KNOWLEDGE GAP. The ongoing management of pathogens, weeds, and insect pests is an expensive and challenging undertaking in all agricultural systems including production, harvest, and storage of crops. Traditional means to controlling pests have included either physical, chemical, or biological barriers. Development of new, effective, and safe technologies to combat pests of all types is critical to all agricultural industries. Recently, a genome-editing biotechnology (CRISPR/Cas) has emerged with the potential to revolutionize industries including basic research, biomedical application, and uses in agriculture. One arrangement of the CRISPR system has allowed a non-natural spread of genes through a population by "super-Mendelian" inheritance referred to as a "gene drive" as it "forces or drives" a genetic element through a population and bypasses the traditional mechanisms of genetic inheritance. This system is currently being studied in model systems including mosquitos for their role as hosts for pathogens including malaria. The gene drive consists of the Cas9 nuclease, a single-guide RNA, and genomic DNA. Once expressed and activated, the RNA-loaded Cas9 targets the chromosomal DNA and creates a double stranded break (DSB). Cells have evolved repair systems that allow for the two homologous chromosomes to align and provide a source of homology to fix the DSB. The gene drive allows any heterozygous individual within a population to immediately transform into the homozygous state. However, there are three major issues associated with the practical use of gene drive systems in the wild: (i) unintended risk or release of the gene drive, (ii) extinction-level population control of a given species, and (iii) naturally evolved resistance to gene drives.HOW THE PROJECT WILL ADDRESS THE KNOWLEDGE GAP. The proposed research will develop a gene drive system in budding yeast that will allow for examination of the components of the CRISPR system itself and include genetic safety mechanisms to control drives. This study will focus on: the RNA (sgRNA) fragment, Cas9 nuclease, and the genomic DNA. These findings will lead to the development of novel CRISPR-based gene drives that can be directly tested in higher Eukaryotic systems such as flies and mosquitos, fungi, or plants.RESEARCH METHODS/ EXPERIMENTAL PROCEDURES1. An artificial CRISPR gene editing system in yeast. This project involves the creation of several arrangements of Cas9 in yeast for two major assays. The first allows for a rapid test of general Cas9-based editing in a haploid yeast cell using cell viability (yeast cannot tolerate a DSB) as a quantifiable measure of DNA editing in a population. Second, a separate collection of yeast strains will be built using traditional molecular methods (plasmid construction, gene synthesis, and integration into the yeast genome via homologous recombination) to create an active gene drive arrangement (in the diploid yeast state).2. Generation and characterization of sgRNA, Cas9 variants, and target DNA arrangements within a gene drive. We will initially generate a large number (>500) of unique combinations using each component of the CRISPR editing system to carefully assay their individual contribution (if any) to editing in both the haploid genome and in the context of the gene drive system. a. sgRNA mutant analysis. We will examine mutations, changes in RNA length, and identity as it may provide a level of control to editing efficiency within an active gene drive system. b. Cas9 orthologs and fusions. While the majority of CRISPR-related work has used S. pyogenes as the primary nuclease for gene editing, the identification and characterization of additional orthologs from various bacterial species has provided a suite of options for Cas9-based editing applications. We plan on using the yeast system to test the ability of a small number of orthologous Cas9 enzymes (from S. pyogenes, N. meningitidis, and S. thermophilus) to function within a given editing system and directly compare their editing efficiencies. Moreover, one of the powerful uses of Cas9 is that it can tolerate additional protein(s) being fused to its C-terminus. We will examine how each fusion (or various proteins) impacts overall DNA targeting and/or editing as this may be used to titrate gene drive activity.3. Removal and destruction of the Cas9 nuclease following editing. An often-overlooked step of CRISPR-based editing is the removal of the system following the targeting (and subsequent repair) of the desired DNA. It is critical to study removal systems to reduce "off-target" effects at unintended DNA positions and allow subsequent control/editing events at a later date with no cross-reactivity of previously loaded Cas9 enzymes.4. Molecular characterization of anti-CRISPR proteins as inhibitors of S. pyogenes Cas9. a. Two recent discoveries have illustrated the presence of a new class of protein (termed "anti-CRISPR" peptides) for their ability to inhibit the enzymatic action of Cas9. These peptides evolved within the bacteriophage as a response to the CRISPR system. We plan to synthesize both classes (C1, C2, C3, and A2, A4) of anti-CRISPR gene, clone and express them in yeast in combination with our S. pyogenes editing system (Aim 1). We will examine their ability to inhibit Cas9 editing in both haploids as in the context of the developed gene drive. We plan on a molecular characterization of both classes of anti-CRISPR protein to investigate the mode of Cas9 inhibition.b. We plan to develop several in vivo binding assays to examine the association of the Cas9 nuclease with the anti-CRISPR peptide. We will utilize severa independent approaches including "forced recruitment" strategy, a tripartite split GFP system for detection of protein-protein interactions, and co-localization studies using fluorescence microscopy. We plan to utilize these anti-CRISPR peptides to titrate Cas9 editing and gene drive activity.TIMETABLE.Year 1. Design, cloning, and creation of the artificial haploid (and diploid) editing system in budding yeast using synthesized Cas9 targeting sites. Cloning of the following plasmids and strains will also be the major focus: (i) sgRNA mutants and variants, (ii) Cas9 orthologs and variants, (iii) yeast strains (chromosomally integrated) containing all combinations of the artificial site(s) and Cas9/sgRNA components. Pilot experiments to ensure activity of the haploid editing assays as well as testing of the WT gene drive. Design and implementation of drive safety.Year 2. Cloning and molecular characterization of the anti-CRISPR proteins in the context of the developed (Year 1) haploid Cas9 editing assay and gene drive assay will ensure. Cloning and testing of anti-CRISPR deletion mutants, fusions, and targeted mutants will be tested in this context. Development of Cas9 binding/recruitment assays will be performed. Further analysis of sgRNA, Cas9, and target gene site combinations within the gene drive context will be fully assayed (all combinations possible). Development of additional drive assays (cellular, genetic, and biochemical assays) for high-throughput analysis.Year 3. Development and analysis of Cas9 removal system(s) in vivo. Development and testing of high-throughput analysis of gene drive combinations with identified components (sgRNA sequence, Cas9 variant, and DNA target(s)) to identify optimized drive performance in vivo. Testing of tunable anti-CRISPR system in vivo (identified from Year 2).Year 4. Development of an "self-regulated" Cas9-editing system using all the identified components required (Years 1-3). Pilot inhibition/anti-gene drive internal system in vivo.Year 5.Development of an engineered insect target DNA gene drive in budding yeast. Testing of "evolved resistance" against action of gene drives in vivo and appropriate counter-measures.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:The target audience for our research efforts included (i) undergraduate students in the biological sciences (biology, biochemistry, molecular biology, cellular biology, microbiology and related fields) and computer sciences, (ii) graduate students in the aforementioned fields, (iii) post-doctoral researchers, (iv) professors and other professional researchers/scientists. The audience for this reporting period also included minority undergraduate students (STEM fields) at a national conference. Our efforts during the reporting cycle included laboratory instruction (formal and informal),research mentorships, publication of reseasrch findings (open access), public seminars, and presentations at conferences. Changes/Problems:[COVID-19 Impact Statement] Our laboratory has been impacted by the COVID-19 global pandemic. From mid-March 2020 through July 2020, the laboratory was closed. Moreover, Aug/Sept 2020 were focused on "reopening" the laboratory with limited personnel (major cleaning, organization, restocking of appropriate supplies and reagents, and unfreezing and retesting of critical strains). This caused a major disruption in our research productivity that will also extend into the next reporting cycle. However, progress was made during this time through remote work including data analysis, online meetings, literature analysis, and drafting of manuscripts. This has delayed our (in person) laboratory experiments due to the lab closure. Additionally, students have graduated and/or transffered to other research laboratories in the last reporting cycle. The pandemic prevented recruitment and training efforts of (additional/new) undergraduates to replace those that have left. This will have a significant impact on the research productivity for the 2020-2021 reporting cycle. What opportunities for training and professional development has the project provided?Training activities included mentoring of students (undergraduate or graduate level) in the laboratory either in teams or with the mentor (PI). The PI also gave guest lectures within University courses or student clubs (on CRISPR technology) during this reporting cycle. Professional activities included local (graduate student) or invited seminars (PI), conferences attended (oral or poster presentations, undergraduate students or PI), and regular laboratory meeting presentations (in person or remote). How have the results been disseminated to communities of interest?Results have been shared through publication of open-access manuscripts (2 within this reporting cycle), presentations within University courses or public seminars, and presentations at conferences (oral or poster formats). Undergraduate students were able to present research findings at the American Society for Microbiology Annual Biomedical Research Conference for Minority Students (ABRCMS) conference in Anaheim, CA in November 2019. The undergraduate research conference included minority students within STEM fields interested in pursuing advanced degrees and careers in biomedicine, biotechnology, academia, and industry. What do you plan to do during the next reporting period to accomplish the goals?Given the disruption of the COVID-19 pandemic on our research laboratory, we plan to focus on two major areas for the next reporting cycle that address the three primary goals of this project. First, we plan on finalizing the Cas12a-based gene drive manuscript and submitting it for publication (Major Goals 1 & 2). Second, we will revisit our early preliminary results using a "dead" Cas9 variant (dCas9) that allows for transcriptional regulation (Major Goal 2). Our plan includes demonstration of this CRISPR-based technology within our model system along with anti-CRISPR inhibitors. This will serve as a first proof-of-concept for potential future use of (complex) transcriptional modification(s) to be included within a gene drive or to control (or inhibit) gene drive systems. This work also complements the initial gene drive fusion constructs (dCas9-Cas9) published in 2018 by our laboratory. Moreover, it includes use of anti-CRISPR inhibitors (on dCas9, rather than active Cas9) (Major Goal 3). We plan on accomplishing the final data collection in triplicate, data analysis, and preparation of a draft manuscript within the next reporting cycle.

Impacts
What was accomplished under these goals? IMPACT STATEMENT: Control of biological populations is critical for numerous fields including human health, ecological preservation, and agriculture. Entire industries are devoted to detection, mitigation, and/or eradication of human or animal diseases, pests, parasites, or invasive species. Left unchecked, certain creatures (such as insects) can destroy crops, sicken animals (or humans), and perturb ecological balance. Numerous methods to control animal populations have been employed including natural predators, physical barriers, or chemical agents. However, many of these techniques are limited due to cost, difficulty in application, and unintended impacts on other species within the environment. Recently, a gene editing technology called "CRISPR" has provided powerful methods for changing the DNA of living creatures. There is a unique arrangement of this biotechnology (a "gene drive") that can be employed within a range of organisms; this allows a gene of interest to be "driven" through a population. Experiments in laboratories have demonstrated that a gene drive could be used to program a single species to only produce offspring of a single sex, causing populations to "crash" and be greatly reduced in size. Moreover, this type of system appears to be (i) self-contained to a single species, (ii) is self-propagating and does not require additional input, and (iii) is incredibly rapid. This technology could have profound benefits across a range of fields including agriculture. However, there are a number of obvious concerns and challenges facing any future application of this biotechnology. These include (i) impacts (of species removal) on surrounding ecology and other species, (ii) efficacy and penetrance of the technology within a population (issues of "resistance" to the drive), and (iii) control and reversal of gene drives. To date, much of the experimental research has focused on optimizing CRISPR drives. However, recent work has begun to highlight more advanced programming of this system to allow for inhibition, titration of activity, or possible reversal of a population. These are not only critical safeguards but could represent more targeted (localized or temporal) changes to a population rather than a "global" eradication event. Our laboratory uses budding yeast as a model system to study various aspects of CRISPR gene drives. Our work has demonstrated that control over gene drives may be possible through inhibition, titration of activity, or other reversal systems. [COVID-19 Impact Statement] Our laboratory has been impacted by the COVID-19 global pandemic. From mid-March 2020 through July 2020, the laboratory was closed. Moreover, Aug/Sept 2020 were focused on "reopening" the laboratory with limited personnel (major cleaning, organization, restocking of appropriate supplies and reagents, and unfreezing and retesting of critical strains). This caused a major disruption in our research productivity that will also extend into the next reporting cycle. However, progress was made during this time through remote work including data analysis, online meetings, literature analysis, and drafting of manuscripts. The accomplishments(below) will focus on the first five months of this cycle (Oct 2019 through Feb 2020). Major Goal 1: Construction of artificial "model" Eukaryotic gene drive system in S. cerevisiae (budding yeast) using S. pyogenes Cas9 from the bacterial CRISPR gene editing system. The majority of research focused on CRISPR gene drive systems has employed the S. pyogenes type II Cas9 nuclease. It is the most well studied editing system to date, yet in recent years a number of alternative nucleases have been explored for use in genome editing or reprogramming. One of these is the type V nuclease, Cas12a (formally known as Cpf1). Our laboratory has created an artificial drive system using the F. novicida Cas12a modeled after our previous work with S. pyogenes Cas9. This included reprogramming of the unique DNA sites flanking our system to allow for targeting by Cas12a. Moreover, we ensured that the selection marker proximal to the Cas12a gene was a nutritional marker. Finally, we recreated the "target" haploid strain with unique sequences to prevent inappropriate cross-over during recombination. Like our previous gene drives, we were able to assay the efficiency of the Cas12a-based drives using colony growth on selection medium. We quantified the number of separate clonal isolates that had lost the selection marker present within the target allele. Our results demonstrated that Cas12a functioned at nearly 99% efficiency after a 5-hour induction phase. To ensure that the loss of the target allele was due to recombination and copying of the drive to both chromosomes, we performed an analysis of chromosomal DNA from isolates following drive activation using PCR. Similar to previous findings with Cas9, 100% of the tested isolates had maintained their diploid state but had lost the target allele. These data demonstrated that (i) a successful gene drive could utilize the type V Cas12a nuclease and (ii) the system functioned extremely efficiently compared to similar Cas9-based drives. These findings are being drafted into a manuscript for submission during the 2020-2021 cycle. Major Goal 2: Testing and analysis of three distinct components of the CRISPR system required for all gene drives--single guide RNA, target genomic (double-stranded) DNA, and Cas9 nuclease--to determine methods to optimize and regulate (gene) drive function. One of the major areas of interest surrounding CRISPR editing remains the issue of "off-target effects." Briefly, this includes scenarios where the guide RNA strand does not present a completely complementary sequence to a genomic target but is still able to allow targeting by the nuclease to a lower degree (as an example, this might include a guide RNA that matches a genomic sequence in 18 out of 20 bases). This issue is critical in ensuring that editing at the intended site has occurred and not as alternative site(s) throughout the genome (even at a low level). In the case of a gene drive, this issue is also critical in ensuring targeting and editing of the intended gene(s). However, there remains the issue of drive "resistance" where a mutated genome would present a DNA/RNA mismatch to the "WT" guide sequence. It would be critical to understand whether alteration of these (DNA) sequences would provide lower targeting or escape from CRISPR editing as a resistant allele. This effect has been seen in the laboratory and computationally modeled as having a serious impact on drive effectiveness within populations. Therefore, it remains critical to understand the nature of DNA/RNA (and RNA/DNA) mismatches for nucleases used within gene drives. During this reporting cycle, we analyzed the effects of single guide RNA mismatches within the Cas12a system. Briefly, our findings highlighted the minimum length of guide RNA required for editing as well as distinct regions within the RNA that were more or less sensitive to specific substitutions. Our analysis included all three possible mutations for every base at positions 1-17 within the guide RNA. Positions near the 5' PAM were highly sensitive to changes resulting in little to no editing. However, regions near the 3' end distal to the PAM appeared to be sequence dependent as to whether a single substitution caused a strong (or weak) reduction in editing. Our findings will provide additional insight into editing "rules" for Cas12a in other genomes as well as assist in the design of redundant systems to reduce the occurrence of drive resistance. These findings are being drafted into a manuscript for the next reporting cycle. Major Goal 3: Expression and molecular characterization of new class of proteins termed "anti-CRISPR" peptides and their ability to inhibit Cas9 nuclease(s). There is no further data or progress to report at this time.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Yan, Y. and Finnigan, G.C. (2019) Analysis of CRISPR gene drive design in budding yeast. Access Microbiology. doi: 10.1099/acmi.0.000059. PMCID: PMC7472540. (Published on Nov 1, 2019).
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Heffel, M.G., and Finnigan, G.C. (2019) Mathematical modeling of self-contained CRISPR gene drive reversal systems. Scientific Reports 9, 20050 doi:10.1038/s41598-019-54805-8. PMCID: PMC6934693. (Published on Dec 27, 2019).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Conference Poster Presentations (by undergraduate students) entitled "CRISPR gene drive with Cas12a nuclease: only as good as the guide" and "Design of S. pyogenes Cas9 nuclease immune to anti-CRISPR inhibition" at the American Society for Microbiology Annual Biomedical Research Conference for Minority Students (ABRCMS) on Nov 13-16, 2019 in Anaheim, CA.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Conference Poster and Oral Presentations (by undergraduate students) entitled "CRISPR gene drive with Cas12a nuclease: only as good as the guide" and "Design of S. pyogenes Cas9 nuclease immune to anti-CRISPR inhibition" at the Kansas INBRE annual research conference on Jan 17-19, 2020 in Wichita, KS.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Conference Oral Presentation (by PI) entitled "Shifting into second gear: CRISPR gene drive biotechnology and population control" at the American Chemical Society Midwest Regional Meeting on Oct 16-19, 2019 in Wichita, KS.


Progress 10/01/18 to 09/30/19

Outputs
Target Audience:The target audience(s) for this project within the last reporting period have included the scientific community, other researchers, professionals, and students (undergraduate, graduate, postgraduate) within the local, state, and national/international communities. Our project has produced scientific publications that are open access to read online in peer-reviewed journals. Additionally, the PI and students have presented posters and seminar talks in open forums at scientific conferences (state and national). Finally, even the general public has been engaged (small scale) through science outreach (Science Café segment) within the local community (Manhattan, KS) by the PI. As mentioned, our efforts have included formal publications, public seminars, poster presentations, discussions with the general public, and classroom instruction within an upper division biochemistry laboratory (BIOCH 766/767) that teaches advanced molecular cloning and uses the CRISPR/Cas9 editing system. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?TRAINING Students (undergraduate and graduate) receive training (by PI) in the laboratory. This is to learn experimental design, techniques, safety, and collection and analysis of data. The PI, graduate student, or senior undergraduates also help train new students (to a certain degree). Training also includes analysis of relevant literature, practice presenting data at meetings, and collaborative work with other students. PROFESSIONAL DEVELOPMENT This project allows both the PI and students to publish their findings in peer-reviewed journals. Also, presentations (oral or poster) occur at the local (University), state, and national levels at conferences and small symposia. How have the results been disseminated to communities of interest?1) Publication in open access peer-reviewed journals (online access). 2) Presentations at local, state, and national seminars/conferneces/symposia. 3) Teaching within upper division biochemistry laboratory course. 4) Public Outreach (local Science Cafe event) to discuss CRISPR/Cas9 and gene drives. What do you plan to do during the next reporting period to accomplish the goals?FUTURE WORK 1. We anticipate additional experimentation and preparation of a manuscript focusing on Cas12a-based gene drives (work still unpublished and in progress). This focuses on both Goals 1 &2 (guide RNA specificity). This also includes work on the new set of anti-CRISPRs (Goal 3). 2. We anticipate additional experimentation on the dCas9-based reporter methodology and a manuscript on this technique/approach (Goals 1 &2). 3. We anticipate additional experimentation on the design of Cas9 variants that could be immune to anti-CRISPR inhibition (Goal 3).

Impacts
What was accomplished under these goals? IMPACT The collective work of this project in the last reporting period has led to a number of important impacts at both the local and national level. First, through teaching (courses), public presentations (seminars, public outreach, conferences), and publications (open access publishing), we have been able to provide additional awareness of both CRISPR/Cas genome editing and gene drives. While they may only reach a finite number of individuals, providing students, the public, educators, and other researchers information about this application of CRISPR/Cas biotechnology is extremely important. Second, our research findings and methodologies have begun to be adopted by other scientists at other Universities and within other organisms (insects). This was one of the primary goals of this research--use of a simple, inexpensive, and accessible model system to pilot new ideas and technologies to assist other scientists working on CRISPR-based gene drives (such as those in flies or mosquitos). In the last year, we have observed a number of (other) scientific publications that have piloted our findings and ideas in their particular system of choice. The methods of interest include both advanced designs and architectures for gene drives, but also important safety or regulatory features. This type of (rapid) impact within the field is extremely exciting and important to note. MAJOR GOALS 1) Several advances were made under this goal. First, we performed a thorough genetic analysis of our original gene drive system with additional controls (Yan and Finnigan, 2019) for several reasons. First, our original work had not diagnosed our active drives for potential NHEJ-based repair of the target DNA. Thus, this study examined a large number of separate isolates from many independent experimental trials to illustrate an extreme bias for HDR-based repair (in yeast) within our gene drives. We also performed a thorough analysis of "resistant" clones that had been identified in our trials across all our drive systems. This was significant to examine because within insects, the presence of "resistant" alleles that escape action of CRISPR drives is a serious concern and still one technical challenge facing the field. Therefore, we tested a large number of "resistant" clones that appeared to escape action of our drive. However, we discovered that many of these seemingly resistant isolates had not resisted the drive but had likely failed to activate the drive in the first place (subsequent Cas9 induction illustrated they were still drive-competent). This does not rule out the possibility of drive resistance evolving within our microbial system but does provide an explanation for nearly 90% of "resistant" (inactive) drive isolates. This paper was published inAccess Microbiologyin 2019. The second major advanced within the reporting period was to perform an analysis of drive design using a computational approach. A computer science student modified an existing equation for tracking gene drive population dynamicsin silicoto include several novel ideas to allow for gene drive "reversal" to take place--this is the idea of suppressing or completely reversing the effects of a gene drive back to a wild-type population. While several previous systems have been proposed (anti-drives or underdominance drives), those systems require the introduction of additional individuals into a population. We proposed a gene drive design where the original molecular construct would contain the necessary information to alter one of two variables (the effectiveness of the drive or the fitness of the organism). This type of "inducible" drive could be programmed to respond to some external cue (environmental change or small molecule, etc.) such that at a given generation, the success of the drive (or success of the organism) could be altered. Our simulations demonstrated that for some conditions, this could result in a total reversal of a gene drive population back to the original WT population in a reasonable number of generations. Finally, we also tested a theoretical design which would include a self-cleaving drive module--an inducible system in place that could, when activated, cause full excision and removal of the drive itself followed by replacement by the wild-type allele (essentially converting any WT allele into an anti-drive). We found that this type of system was (i) robust against prior activation of the self-cleaving module to a certain degree and (ii) very effective at removing drives within a population. Granted, our findings make a number of assumptions in order to perform the mathematical modeling and there are still many challenges in the actual design and use of said technology, but this type of work may help provide insight into future drive design that could include regulatory switches and a possible mechanism to remove the drive itself, should the need ever arise. This work was under peer review by the end of this reporting period but has recently been accepted (Nov 2019) for publication withinScientificReports; we will provide additional detail in the next reporting period. Third, we have unpublished data that has built a novel gene drive system with the type V Cas12a nuclease and is able to function in budding yeast. These findings will be described in detail in the next reporting period. 2) This goal was aimed at study of components within a drive that could assist in titrating activity or modulating the speed at which a drive moves through a population (effectiveness). Within this reporting period, we published a short report (Goeckel et al., 2019) on a follow-up analysis to a previous study we conducted (previous reporting period). This focused on use of artificial nuclear signals (NLS and NES) to dictate trafficking of the Cas9 enzyme into and out of the yeast nucleus. We and others have found that control of nuclear entry (or exit) can be used as a means to modulate genomic editing since the nuclease must first gain access to the genome of interest. We focused our efforts on a set of artificially generated NLS signals from a previous study as well as two modified NES signals. We found that these small peptides could direct Cas9 trafficking into and out of the nucleus at various rates. The largest effect was use of NES (export) signals to prevent/slow genomic editing. However, our gene drive system utilizes a very strong inducible promoter to activate expression of Cas9 (for technical reasons). We observed that while NLS/NES signals could assist in modifying rates of drive success, these would be limited at higher Cas9 expression levels and provided only a minor or modest degree of titration on longer time scales. However, these findings demonstrate that nuclear export, when paired with other mechanisms of inhibition, could assist in titration of activity. Second, we have unpublished data that has begun using an enzymatically dead (dCas9) variant to alter gene expression levels. We have not yet combined this technology into gene drives but have a number of preliminary experiments using dCas9-mediated reporter assays. 3) We have made progress on this aim involving anti-CRISPRs. However, these data remain unpublished and will be described in detail within the next reporting period. Briefly, we have made several advances. First, we are focusing on the rationale design of a Cas9 variant that might be immune to anti-CRISPR-based inhibition (by AcrIIA4) using mutagenesis and a number of cellular readouts including gene drive activity. Second, we have preliminary data demonstrating that the AcrIIA4 anti-CRISPR is also a potent inhibitor of dCas9 fusions (as mentioned in Goal 2 above). Third, we have begun work on the newly discovered anti-CRISPR family that inhibits type V nucleases, termed AcrVA1 to AcrVA5. We have begun testing each of these anti-CRISPRs with our Cas9 based assays as well as Cas12a-based assays.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Yan, Y. and Finnigan, G.C. (2018) Development of a multi-locus CRISPR gene drive system in budding yeast. 8(1):17277. doi: 10.1038/s41598-018-34909-3.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Goeckel, M.E., Basgall, E.M., Lewis, I.C., Goetting, S.C., Yan, Y., Halloran, M., and Finnigan, G.C. (2019) Modulating CRISPR gene drive activity through nucleocytoplasmic localization of Cas9 in S. cerevisiae. Fungal Biol Biotechnol. 6:2. doi: 10.1186/s40694-019-0065-x.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Yan, Y. and Finnigan, G.C. (2019) Analysis of CRISPR gene drive design in budding yeast. Access Microbiology. doi: 10.1099/acmi.0.000059.
  • Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Heffel, M.G., and Finnigan, G.C. (2019/2020) Mathematical modeling of self-contained CRISPR gene drive reversal systems. Scientific Reports. (Under Review).


Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Our efforts included members (individuals) of the scientific community involved in gene editing. These scientists were from many backgrounds including agriculture, computer science, biology, biochemistry, and genetics. Our efforts in this reporting period resulted in published manuscripts and other data that were presented in poster form (conferences), public seminars at multiple Universities, and teaching lectures (K-State Univ.). The audience included students (undergraduate and graduate level), postdocs, and other faculty from many diciplines. Changes/Problems:There have been no major changes in our goals or aims. We have discovered new methodologies to address these same questions/goals as stated in our future planned work. These include (i) use of computational modeling as a mechanism to inform design of gene drives in vivo and (ii) development of a dCas9 transcriptional regulator to assay in vivo function of nucleases, guideRNAs, and/or anti-CRISPRs. What opportunities for training and professional development has the project provided?Training: I personally train my PhD student as well as the many undergraduate students in the research laboratory on design, experimental technique, and data analysis of our project. Professional Development: One of my undergraduate students has had the opportunity to present her work on this project at local poster sessions (undergrad only) on campus, state-wide Kansas-INBRE conferences (NIH), and even the national N-ISBRE conference in WA, DC on her work on the anti-CRISPR proteins (Aim 3 above). How have the results been disseminated to communities of interest?I have presented seminars at other Kansas Universities (Biology and Chemistry Departments, e.g. Wichita State Univ.), as well as local Kansas State University public seminars to other departments including entomology, plant pathology, biology, microbiology, and computer engineering departments within the last reporting cycle. I have also guest lectured for other undergraduate teaching courses on the topics/findings of the project. What do you plan to do during the next reporting period to accomplish the goals?(Also see "Accomplished" above). 1. Creation of Version 2.0 gene drive and target. We plan to include the idea of biological "fitness" or success to allow for selection for or against the GD component. This will require significiant testing and reprogramming of our initial version 1.0 GD system. Inclusion and testing of the Cas12a nuclease to power gene drives. More advanced forms of regulation and multi-nuclease drive systems will be worked on. 2. We are very interested in discovering and testing additional forms of titration for GD systems. One includes other temporal and spatial means to regulate and titrate Cas9 itself within a cell (using fusions and tethers, and timed expression of other factors). We are also planning on additional computational modeling to understand how best to implement inhibitory systems, or inducible systems to change the effectiveness of gene drives in the wild. 3. We plan to expand our analysis of the Acr proteins (VA1-5, especially) to test how these peptides can inhibit Cas12a in vivo as part of a gene drive countering system. We are also developing a high-throughput system using enzymatically dead Cas9 (dCas9) to rapidly screen mutational changes to parts of the system including the Acr anti-CRISPR proteins.

Impacts
What was accomplished under these goals? 1. Construction of artificial "model" Eukaryotic gene drive system in S. cerevisiae (budding yeast) using S. pyogenes Cas9 from the bacterial CRISPR gene editing system. Our efforts this reporting cycle were successful in generating an artificial model system to study gene drives in vivo. We reported our first arrangement (version 1.0) of this gene drive (GD) in our G3 manuscript published in early 2018. However, given this was our first set of experiments on the topic, we are now working on several "upgrades" to this basic arrangement. Our more recent efforts in the end of 2018 have included altering the system (GD itself) and the artificial "targets" in order to accommodate other nuclease variants and orthologs. Our primary interest is in using Cas12a (formally Cpf1). This is a different type of nuclease with different preferences for PAM sequences, guide lengths, and efficiencies in vivo. Therefore, we are currently collecting data that will be expanded on in the next reporting cycle as a full manuscript using Cas12a in a GD system. Also, we are beginning to examine ways to provide a more complex system of regulation within the GD itself. These will include more potent ON/OFF (genetic) switches for gene drives, and means to incorporate exogenous information (chemical signals) to the nucleus. Finally, we developed the world's most complex gene drive at 3 separate genetic loci within one genome. This work demonstrated that designs of gene drives can be expanded to include multiple positions and target other systems simultaneously at high efficiency. 2. Testing and analysis of three distinct components of the CRISPR system required for all gene drives--single guide RNA, target genomic (double-stranded) DNA, and Cas9 nuclease--to determine methods to optimize and regulate (gene) drive function. We have conducted a thorough analysis of the aforementioned variables (guide RNA, nuclease location and function, fusions, etc) in our manuscript in G3 published in early 2018. We highlight that two of the major effectors of GD activity are (i) Cas9 expression level and (ii) Cas9 localization in the cell (controlled by nuclear import/export signals). While guide sequence specificity and target placement may have effects, these were found to be much more subtle. Therefore, we performed a follow-up analysis on nuclear transit of Cas9 as a means to control editing and GD activity (currently in revision/review). We used artificial/non-native nuclear signals and export signals and demonstrated that a fairly wide spectru of GD activity can be achieved using varying expression patterns along with restrictive nuclear entry. We are currently planning (for next reporting cycle) to extend this further to include temporal and spatial restriction of the nuclease within a cell using other artificial tethers to specific subcellular compartments. Furthermore, we discovered that altering the GD activity/efficiency within our cell system required an additional piece of information to help extend our findings to a larger-scale and potential future applications. Therefore, we also began a project to computationally model population dynamics of gene drives using some of our discovered variables (such as a lower GD activity). These data are being collected into a manuscript for submission in 2019 (next reporting cycle). Our findings thus far have suggested adding several new safety features to gene drive systems, including a means to halt, inhibit, or reverse the system itself while it is still present within a population mixture. These findings will inform how to design the next generation of GD systems. We are currently exploring more advanced means to titrate GD activity, this time with additional control, programmability, and "reversal" systems in mind (that could provide a means to remove from populations if necessary). 3. Expression and molecular characterization of new class of proteins termed "anti-CRISPR" peptides and their ability to inhibit Cas9 nuclease(s). We published our first manuscript on the anti-CRISPR proteins AcrIIA2 and AcrIIA4 in the journal Microbiology in early 2018. We found that both of these small peptides were able to provide potent inhibition of GD systems in vivo. Furthermore, we performed a targeted mutagenesis of each protein (nearly 100 total mutant variants tested) to determine if we could achieve a partial level of inhibition. Several mutant combinations were found to partially disrupt nuclease activity (tied to Aim 2). We demonstrated that the AcrIIA2 variant could not have N- or C-terminal tags of any size without destroying function. We are excited to continue research on the anti-CRISPRs as a means to inhibit and control gene drives. Very recently, several other groups discovered a new class of anti-CRISPR peptide (AcrVA1-5) that inhibit the Cas12a family of nuclease. We plan to continue and extend our research on this aim to test this new class of protein and examine its specificity and utility as a gene drive inhibitor.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Roggenkamp*, E., Giersch*, R.M., Schrock*, M.N., Turnquist, E., Halloran, M., and Finnigan**, G.C. (2018) Tuning CRISPR/Cas9 gene drives in Saccharomyces cerevisiae. G3 (Bethesda) 8: 999-1018. PMCID: PMC5844318.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Basgall*, E.M., Goetting*, S.C., Goeckel*, M.E., Giersch, R.M., Roggenkamp, E., Schrock, M.N., Halloran, M., and Finnigan**, G.C., (2018) Gene drive inhibition by the anti-CRISPR proteins AcrIIA2 and AcrIIA4 in budding yeast. Microbiology 164: 464-474. PMCID: PMC5982135.
  • Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Goeckel*, M.E., Basgall*, E.M., Lewis*, I.C., Goetting*, S.C., Yan, Y., Halloran, M., and Finnigan**, G.C. (2018) Modulating CRISPR gene drive activity through nucleoplasmic localization of Cas9 in S. cerevisiae. Fungal Biology and Biotechnology (In revision).
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Yan, Y. and Finnigan, G.C. (2018) Development of a multi-locus CRISPR gene drive system in budding yeast. Scientific Reports. 8(1):17277. doi: 10.1038/s41598-018-34909-3. PMCID: PMC6250742.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Poster Presentation entitled "Computational Modeling of Tunable CRISPR/Cas9 Gene Drive Population Dynamics" (by undergraduate student) at annual Cold Spring Harbor Laboratory conference Genome Engineering: The CRISPR/Cas Revolution on August 22-25, 2018.


Progress 08/18/17 to 09/30/17

Outputs
Target Audience:The current reporting period includes the dates between August 18, 2017 and September 30, 2018. For these 6 weeks, the target audience includes my laboratory staff and students, as well as the general scientific community as we were able to publish twomethodology papers in this time period that aids in establishing new molecular tools for this NIFA project. Furthermore, a small mini-review that provides a summary of the CRISPR field within our model system.These manuscripts'audience are not only the yeast community, but the scientific community at large as the examples of CRISPR-based cloning can be applicable to many research projects. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?I personally mentored the many undergraduate students that populate my lab, as well as my full-time technician in design, experimentation, and interpretaion of data, analysis, and formation of figures/tables for future publication. 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?Note, also see point 1, accomplishments of the goals (above). Briefly, Goal 1: We intend to continue design of additional model arrangements within yeast to expand our suite of tools available for testing of CRISPR-Cas editing in live cells and within gene drives. These will include use of more than one genomic locus, alterations to target DNA, and multiplexing within our system. Futhermore, we intend to study other Cas9 variants (natural and engineered) aside from only S. pyogenes. Goal 2: We intend to test new alterations to the sgRNA including new mutational analyses, structure, and expression modules. We intend to test additional Cas9 fusions to (new) and mutated cellular signals including nuclear trafficking tags. We intend to test other futures of Cas9 to other Cas9 variants or other proteins of choice. Finally, we intend to re-engineer the genome so as to test new target cleavage sites at various positions across chromosome and epigenetic regions. Goal 3: We will continue experimentation involving the anti-CRISPR proteins, as well as begin design and planning for future testing and combinations with Cas9 and other Cas9 variants. As mentioned abiove, this include both spatial and temporal control of the nuclease and/or the inhibitor, mutational screens to determine molecular function, and genetic bypass for "escape" of Cas9 from its inhibitor. In summary, we have many, many new strains and DNA constructs to build, test, and analyze, and are making excellent progress!

Impacts
What was accomplished under these goals? Goal 1: The majority of the time prior to initiation of this Hatch project, and during the 6-week initial period (August 18, 2017 to September 30, 2017), was focused on design, and molecular construction of the model system in yeast. Several different designs were tested and a final molecular arrangement was selected to proceed with future experiments. Also, a significant amount of time was taken to ensure the biosafety and containment of this artificial system and its ability to act as a "gene drive" in the laboratory setting. Time was invested to develop a unique protocol for testing of haploid editing ("traditional" gene editing) versus diploid editing (gene drive). Future work on this goal includes the testing of additional arrangements (new Cas9 nucleases) and further optimization of the editing system in vivo. Some of our future goals here include developing a system that can allow for rapid "multiplexing" across many loci simultaneously--our current system, for simplicity, only focuses on one DNA position in the genome. Goal 2: Work has begun on testing of various single guide RNAs (sgRNA). However, we chose to focus on varying the length of the crRNA portion, as well as mutational substitutions present within the 5' end of the sgRNA (furthest away from the target PAM sequence). The data associated with this testing was not as fruitful as we wished. However, future experimentation will focus on additional modifications to the guide RNA that are possible including mutations in other positions within the guide sequence, the tracrRNA portion, and structural RNA elements (stem loops), etc. No work was initiated on alterations to the "target" DNA. These will be performed in future iterations of our system and represent achallenging (but exciting) process of engineering the yeast genome itself. For our pilot studies we have restricted our use of "target" DNA to a small number of artificial DNA sites. Future work will focus on including new variations of surrounding sequence, epigentic modification(s), and chromosomal positioning. Work was begun on alterations to the Cas9 nuclease itself, although there are many more experiments to perform in future years. We focused our efforts on fusions to the Cas9 nuclease--attachment of GFP (green fluorescent protein), and combination of cellular localization signals (nuclear import or export signals). We have found that combinations of these intracellular trafficking modules can directly influence CRISPR editing in vivo. Future work will focus heavily on new cellular signals, modifications of exitsting signals, and different molecular arrangements (N-terminal tag, C-terminal tag, etc). Finally, we have begun preliminary work on novel fusions of Cas9 to an identical or enzymatically "dead" second protein of Cas9. These are extremely large proteins and these fusions can also incluence editing and gene drive activity. There are many proposed arrangements and modifications that are possible to the Cas9 nuclease. We intend to focus heavily on new fusions (to other Cas9 nucleases), and fusions of Cas9 to other cellular locations and how these can affect editing in vivo. Goal 3: Preliminary experimental work has also begun on cloning of the "anti-CRISPR" proteins. This class of peptide has been published by others to bind to and inhibit the nuclease activity of Cas9 itself in vitro and in vivo. We are planning to test whether this class of protein can inhibit Cas9 in the context of our gene drive system in vivo. Futhermore, we plan on a mutational screen of these proteins to detemine which portions (domains, residues, motifs) are required for its function and binding to Cas9. There are many future experiments planned including both spatial and temporal control of these inhibitors (and Cas9 itself), new fusions between inhibitor proteins and the nuclease, and mutational screening of Cas9 to develop "bypass" mutants that could escape inhibition. In summary, we have made the most progress with Goal 1, moderate progress with Goal 2, and preliminary progress with Goal 3. Furthermore, while we have developed "simple" molecular versions for our editing system, we intend to begin development of more complex arrangements that can be used to address more challenging questions and exapnd the utility of our system.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Roggenkamp, E., Giersch, R.M., Wedeman, E., Eaton, M., Turnquist, E., Schrock, M.N., Alkotami, L., Jirakittisonthon, T., Schluter-Pascua, S.E., Bayne, G.H., Wasko, C., Halloran, M., and Finnigan, G.C. (2017) CRISPR-UnLOCK: Multipurpose Cas9-Based Strategies for Conversion of Yeast Libraries and Strains. Front Microbiol 8, doi: 10.3389/fmicb.2017.01773. PMCID: PMC5611381.
  • Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Giersch, R.M. and Finnigan, G.C. (2017) Yeast still a Beast: Diverse Application of CRISPR/Cas Editing Technology in S. cerevisiae. Yale J Biol Med 90(4): 643651. PMCID: PMC5733842.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Giersch, R.M. and Finnigan, G.C. (2017) Method for Multiplexing CRISPR/Cas9 in Saccharomyces cerevisiae Using Artificial Target DNA Sequences. Bio-protocol 7(18): e2557. DOI: 10.21769/BioProtoc.2557. PMCID: PMC5659391.
  • Type: Journal Articles Status: Submitted Year Published: 2018 Citation: Roggenkamp, E., Giersch, R.M., Schrock, M.N., Turnquist, E., Halloran, M., and Finnigan, G.C. (2018) Tuning CRISPR/Cas9 gene drives in Saccharomyces cerevisiae. G3 (Bethesda)