Herbicide-resistant weeds result from rare genetic mutations that increase in frequency through selection by herbicides. The ability of scientists to make specific edits in weed genomes including the genes for herbicide resistance is becoming feasible. The value of such work is that studying changes in herbicide response due to specific gene edits would greatly further our understanding of potential solutions to the growing herbicide resistance problem. Gene editing processes could also one day be introduced into weed populations to facilitate increased weed control, including the reversion of resistant weeds back to susceptibility, through systems called gene drives. To develop gene drives that reverse herbicide resistance in weeds, laboratory studies need to first be done using weed tissues that do not have the capacity to escape laboratory containment through the production of seed, pollen, or other propagules. Plants grown in tissue culture as undifferentiated cells do not have such capacity, yet still maintain most of the physiological processes that are targeted by herbicides. We previously developed a tissue culture system in waterhemp, and we propose to target and edit the acetolactate synthase (ALS) gene in these tissue culture cells. The ALS gene is a common herbicide target site, and mutations in this gene are known to confer ALS-inhibiting (Group 2) herbicides. This project seeks to determine the specificity and efficiency of gene editing in the gene for ALS in waterhemp, with the longterm goal of reversing herbicide resistance.

1) Test the specificity of a CRISPR-based gene editing system targeting the waterhemp ALS gene in purified DNA.
2) Test the efficiency of a CRISPR-based gene editing system targeting the ALS gene in waterhemp protoplasts.

1) The ability to produce alterations in the waterhemp ALS herbicide target-site gene without risking unintentional release into the environment. The protocols developed in this project will also facilitate the future study of other herbicide target-site genes in waterhemp.
2) Safe assessment of emerging gene drive technology for reversing herbicide resistance.

Updated December 1, 2021:
Completed work:
Research to evaluate CRISPR-based gene editing of the waterhemp gene for acetolactate synthase (ALS; target of Group 2 herbicides), was initiated. Successful gene editing of waterhemp ALS would allow mutations conferring herbicide resistance to be changed back to susceptibility. In the field, such a system could be utilized in an emerging genetic weed control system called a gene drive, where resistant weed populations could be changed back to susceptibility. CRISPR-based gene editing requires certain sequences, called Protospacer Adjacent Motifs (PAMs), to be present in the target gene in order for most CRISPR gene editing systems to work. The PAM sequences must be adjacent to the portion of the gene that will be edited. To determine the location of ALS gene PAM sequences within currently available waterhemp tissue cultures, DNA was extracted and portions of the ALS gene containing herbicide resistance mutation sites were sequenced. PAM sequences were then detected using CRISPRdirect software (Naito et al. 2015. Bioinformatics 31:1120-1123).

Preliminary results:
Fifty PAM sequences were identified within the sequenced portion of the waterhemp ALS gene. Of these, only one was rejected as unsuitable for CRISPR gene editing due to the presence of nearby sequences known to be problematic. The other 49 PAM sequences will be further evaluated for gene editing potential, including their proximity to herbicide resistance mutation sites.

Work to be completed:
The identified PAM sequences indicate the specific locations within the waterhemp ALS gene that can be targeted for gene editing. Each potential location is currently being evaluated for gene editing potential, including an evaluation of adjacent sequence and the proximity of herbicide resistance sites. CRISPR gene editing components will then be designed to target the desired location within ALS, and tested using purified DNA of the waterhemp ALS gene. Pending successful targeting of the ALS gene, CRISPR components will be introduced into waterhemp protoplasts (cells without cell walls), and the efficiency of gene editing will be evaluated.

Updated August 19, 2022:
Introduction:
The widespread occurrence of herbicide resistance in waterhemp has necessitated research into alternative methods of control, including emerging genetic technologies that may reverse resistance. One emerging technology that might be useful in the fight against herbicide-resistant weeds is called a gene drive, where the ability to reverse resistance back to susceptibility would be self-propagated in weed populations.

Many proposed gene drive systems use CRISPR (clustered regularly interspaced short palindromic repeat) gene editing technology, where the genetic sequence responsible for resistance is targeted, cut by an enzyme called Cas9 (CRISPR-associated protein 9), and repaired using a susceptible gene sequence as a template, thus changing the gene from a resistant to a susceptible form.

To advance this technology in weeds such as waterhemp, we developed a tissue culture system where CRISPR could be tested without producing seed or pollen that could escape the laboratory. The research reported here is an investigation of the CRISPR system’s ability to target the acetolactate synthase (ALS) gene, target-site of group 2 herbicides. This research used Cas9, an enzyme that cuts DNA in the CRISPR system, and sgRNA (single-guide RNA), nucleic acid that directs Cas9 where to cut. Transformation of Cas9 into waterhemp protoplasts was evaluated, along with the ability of Cas9/sgRNA complexes to cut the ALS gene in purified DNA and laboratory-grown waterhemp cells, and the ability of live waterhemp protoplasts to repair the cut DNA.


Materials and Methods:
Objective 1: Test the specificity of a CRISPR-based gene editing system targeting the waterhemp ALS gene in purified DNA.

The waterhemp tissue culture cell line used in the current work was originally derived from the hypotocyl of a germinating waterhemp seedling. DNA was isolated and purified from this culture, and sequences of the waterhemp ALS gene were used to choose four sites in the ALS gene for target-specificity analysis, i.e., testing the ability of Cas9 to recognize and cut DNA at these sites. Single-guide RNAs (sgRNAs) matching these four sites were synthesized and used in the following experiments.

Using the extracted waterhemp cell suspension culture DNA, polymerase chain reaction (PCR) was used to amplify portions of the waterhemp ALS gene spanning the four CRISPR target sites. These PCR products were then purified and incubated with commercial Cas9 and the synthesized sgRNAs, followed by agarose gel electrophoresis to analyze the expected DNA cuts.


Objective 2: Test the efficacy of a CRISPR-based gene editing system targeting the ALS gene in waterhemp protoplasts.

Waterhemp protoplasts (cells without cell walls), were prepared from 4-day-old suspension cell cultures and tested for viability. They were then incubated with each sgRNA along with Cas9 enzyme fused to GFP (green fluorescent protein), so that transformation into protoplasts could be evaluated with a fluorescence microscope.

DNA from each sample was then extracted, purified, and the ALS gene sequenced to detect indels (insertions or deletions) in the gene, resulting from cell repair of the Cas9 cut.


Results and Discussion:
Objective 1: Test the specificity of a CRISPR-based gene editing system targeting the waterhemp ALS gene in purified DNA.

Four sgRNAs targeting the waterhemp ALS gene were tested for their ability to guide Cas9 to the corresponding position in purified DNA, allowing Cas9 to cut the DNA sequence. The sgRNAs were labeled 292, 1053, 1401, and 1644 based on the targeted portion of the waterhemp ALS gene. In four different reactions, the 1053 sgRNA/Cas9 was found to fully cut the gene, indicating good targeting specificity. Partial cutting was observed with the 292 and 1401 sgRNAs, while results were unclear for the 1644 sgRNA.

Overall, results demonstrated the ability of the CRISPR system to target the waterhemp ALS gene, especially with the 1053 sgRNA. Such specific targeting is necessary to mark the ALS gene for repair in live cells, allowing gene editing of herbicide-resistant ALS genes back to susceptibility.


Objective 2: Test the efficacy of a CRISPR-based gene editing system targeting the ALS gene in waterhemp protoplasts.

Polyethylene glycol (PEG) can be used to permeabilize protoplasts, allowing entry of molecules such as protein and/or nucleic acids (RNA or DNA). Our results indicated successful transformation of waterhemp protoplasts with Cas9, ranging from 26 to 39% using 40% PEG, and 42 to 54% using 50% PEG. While this suggests a benefit of 50% PEG over 40% PEG, this will need to be confirmed in future research. In general, addition of sgRNAs decreased transformation efficiency for both 40% and 50% PEG, with transformation ranging from 0 to 20% using 40% PEG, and 8 to 27% using 50% PEG. This reduced efficiency may have been due to the increased size of the Cas9/sgRNA complex compared to Cas9 alone. As sgRNA is necessary to target Cas9 to the waterhemp ALS gene, increased concentrations of Cas9 may be necessary for efficient CRISPR-based gene editing in waterhemp protoplasts. Cas9 alone is also smaller than the Cas9/GFP fusion protein used in these experiments, and it may be possible to increase Cas9/sgRNA transformation by using unfused Cas9.

In DNA sequences from transformed protoplasts, no evidence of Cas9-induced ALS cuts and repair was observed for sgRNAs 292, 1401, and 1644. The target site of sgRNA 1053 could not be amplified from transformed protoplast-derived DNA, preventing sequence analysis.

It is unclear why Cas9/sgRNAs were able to target and cut the waterhemp ALS gene in purified DNA (Objective 1), but such targeting did not result in cleavage/repair when using live protoplasts. It is possible that the protoplast transformation efficiency was not high enough, or that the protoplasts are not repairing the cut ALS gene as expected. We previously determined that our protoplasts, while alive, do not regenerate cell walls and do not divide, i.e., they don’t seem to recover after cell wall digestion. This is a focus of future research. It is possible that ability to repair Cas9-derived cuts is also an activity that is limited in these protoplasts, and that determining the conditions conducive to protoplast recovery and division will also promote DNA repair of Cas9 cuts in the CRISPR system.


Conclusions:
The ALS gene of waterhemp was successfully targeted and cut by Cas9 and at least three different sgRNAs when using purified waterhemp DNA. Cas9 was also successfully transformed into waterhemp protoplasts, although transformation efficiency was reduced when sgRNA, which is necessary for Cas9 function, was included.

When using live protoplasts and targeting the ALS gene for cleavage, expected repair of the gene was not observed. This may have been due to decreased transformation efficiency when sgRNA was included with Cas9, or because DNA repair mechanisms were not active in the waterhemp protoplasts under our experimental conditions. Current research is addressing the conditions necessary for optimum protoplast recovery after cell wall digestion and/or transformation, which may improve DNA repair as well.

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Research Conducted:
CRISPR is a new genetic technique that can produce specified changes in plant DNA. Emerging genetic biocontrol methods may be able to use CRISPR in self-propagating systems that could reverse herbicide resistance in waterhemp populations. This research project evaluated conditions under which CRISPR components could be put into laboratory-grown waterhemp cells, and assessed the ability of CRISPR to target the waterhemp ALS gene, target of group 2 herbicides, in both purified DNA and live cells.

Why the research is important to ND soybean growers:
Herbicide-resistant waterhemp represents an important problem in North Dakota soybean production, and new tools are needed to study emerging methods for controlling this weed. However, when growing herbicide-resistant weeds for research, it is important to minimize the risk of seeds or pollen escaping and spreading into fields. This is especially important when researching new genetic biocontrol methods. The current research advanced the application of CRISPR technology to waterhemp herbicide resistance research using laboratory-grown waterhemp cells incapable of escape into the environment.

Final findings of the research:
The CRISPR system was able to target specified locations in the waterhemp ALS gene, using purified DNA, paving the way for development of genetic biocontrol methods that could reverse herbicide resistance. CRISPR components were also successfully put into live waterhemp cells, but targeting and subsequent repair of the ALS gene was not observed in these cells. This may be due to inactive repair systems in waterhemp cells under our treatment conditions, and is the subject of future research.

Benefits/Recommendations to North Dakota soybean farmers and industry:
This research has introduced new genetic techniques to the study of herbicide resistance in waterhemp. Such techniques will allow the study and potential application of emerging genetic biocontrol methods to a weed control problem that is important in North Dakota soybean production.

Development of new herbicide modes of action has greatly declined in recent decades, while herbicide-resistant weeds are decreasing the effectiveness of existing herbicides for soybean production. Alternative weed control strategies need to be explored, including the potential of emerging genetic technologies for weed control. Gene drives are a genetic technology with potential to reverse herbicide resistance in weed populations and/or directly disrupt the ability of weeds to successfully propagate. While gene drives are gaining worldwide interest, much research needs to be done before they would be available for release. As this research progresses, it is important that: 1) problems experienced by North Dakota soybean farmers are included among the priorities, 2) research is performed in a manner that does not risk negative impacts on North Dakota agriculture through unintentional weed escapes, and 3) efforts are directed toward gene drive systems that are most likely to be successful and accepted by the public. Establishing methods for gene drive research in waterhemp, using laboratory-contained tissue cultures and with a focus on reversing herbicide resistance, upholds these priorities and positions North Dakota soybean growers to benefit from this emerging technology.