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.

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.