Targeted mutagenesis via CRISPR
At Hudson River Biotechnology we employ CRISPR-based genome editing for specific trait development. CRISPR can be used to target highly specific pieces of DNA while leaving the rest of the genome intact, which allows for very fast improvement of already carefully bred organisms. This technology can be used to target a diverse range of traits:
- Disease resistance
- Drought tolerance
- Nutritional content
- Flowering time
- Improved shelf life
Hudson River Biotechnology is a biotech company offering end-to-end genome editing solutions for plants, fungi, and micro-organisms. We have experience with a wide range of crops and can work with vegetables, ornamentals and field crops, including polyploid species, as well as algae and mushrooms. Our main genome-editing tool is CRISPR, which we apply using a proprietary, validated molecular breeding workflow called TiGER, an acronym for Target identification, Guide selection, Entry into the cell and Regeneration. Our CRISPR enzyme of choice is MAD7, due to the low licensing costs. While CRISPR genome editing is straightforward in principle, the functional application is a considerable challenge, especially in the green biotech. Our workflow addresses these challenges head-on, and we are always actively developing specific aspects of the workflow to continuously broaden its range and scope.
Our CRISPR editing approach
The CRISPR system consists of an endonuclease (DNA cutting enzyme) that is complexed with an RNA molecule, the guide RNA (gRNA). The gRNA allows targeting of the endonuclease to any location in the genome where a protospacer adjacent motif (PAM) is present (Figure 1). If the chosen sequence is unique enough in the genome, this location will be the only site targeted. Upon binding of the gRNA to the DNA of the genome, the associated endonuclease will cleave both DNA strands. Next, the cell itself performs the actual genome editing.
DNA breaks occur naturally and, because these are undesirable, the cell has repair systems in place. The most predominant repair system, for natural as well as CRISPR-induced breaks, is the non-homologous end-joining (NHEJ) system. Although this system is fast and effective, often one or more nucleotides are lost as immediately after a break, some nucleotides are removed from the open DNA ends. In most cases loss of a few nucleotides would not affect the integrity of the genome, but when the open reading frame of a gene (the part of a gene that encodes a functional protein) is repaired in such a manner, it can lead to the production of a non-functional protein.
Cells have another, more elegant repair system as well, called the homology directed repair (HDR) pathway, which is only active during specific stages of cell division when genomic breaks occur more frequently. As the name already suggests, it requires pieces of DNA that are homologous to each other (stretches of DNA with a similar sequence) and naturally finds these on the sister chromosome (copy of a chromosome present just before cell division). As mentioned, immediately after the break nucleotides are removed from the open ends of the DNA. The resulting single-stranded stretches of DNA can bind with the complementary strand of DNA on the sister chromosome. Thereafter, the sister chromosome can function as a repair template (Figure 2). Such repairs are ‘flawless’ in the sense that they do not result in omission of nucleotides. As such, this repair system allows for very precise genome editing, because when during genome editing a custom-made piece of DNA, termed ‘donor DNA’, is provided to the cell, this can also serve as the repair template. And while the donor DNA needs to be homologous enough, it does not need to be identical.
HRB employs CRISPR-based genome editing for specific trait development while leaving the rest of the genome intact. This allows for very fast improvement of already carefully bred organisms. Our CRISPR enzyme of choice is MAD7 and HRB has in-house production capabilities for high-grade CRISPR proteins and guide RNAs that enable highly efficient genome editing.
CRISPR guide design and validationStep 2 of our TiGER workflow is CRISPR Guide selection. Guide efficiency is an important factor in overall genome editing efficiency. HRB has proprietary guide design software that allows for the design of efficient guides for multiple different CRISPR enzymes. We can produce all enzymes and their guide RNAs in house, achieving comparable performance to products from commercial suppliers. Prior to mutant organism creation, we validate CRISPR guides via measuring endonuclease efficiency on purified DNA (Figure 4) and genome editing occurrences in live cells (Figure 5). For plants we often use protoplasts for this process allowing in vitro and vivo validation in less than a month.
Moving CRISPR into the cell
Step 3 of our TiGER workflow is enabling Entry of the CRISPR elements into the cells or tissues of interest. For this, we have access to common technologies such as PEG- based transfection and biolistics, which we apply without the need for transgenic selection markers. In addition, HRB has ongoing developments of nanoparticle-based platform technologies for encapsulation and functional delivery of CRISPR protein-guide complexes (RNPs) into different types of cells and (plant) materials in a highly efficient manner. These technologies include delivery to protoplasts, callus and tissues in planta, as well as solutions that circumvent the need for plant regeneration.
Of note, we take a DNA-free approach to CRISPR genome editing, whereas most others rely on the incorporation of transgenic DNA and use of antibiotic selection for the expression of the CRISPR enzyme and the guide RNA inside the plant cell to achieve editing. Our system has substantial benefits as it is faster (no need to outcross transgenes), more efficient (no need to express multiple components in sync in the plant cells), less prone to off-target effects (CRISPR is only active for a short period of time), and more versatile (allows editing of plants where out-crossing of transgenes is not an option because this is biologically impossible or undesirable because of heterogeneous and/or complicated polyploid genetics). An added benefit is that the DNA-free method is likely to reduce the regulatory burden that accompanies the use of transgenes.
Step 4 of our TiGER workflow is Regeneration. In many scenarios we work with in vitro plant tissues or cells. After successful genome editing, these need to be regenerated back into a plant, ready for generation of lines, scale-up and field or greenhouse testing. HRB has validated protocols for regeneration of multiple species. In addition, we are developing advanced materials that substantially shorten the timeline associated with the regeneration process, while improving efficiency.
In some cases, a traditional approach to breeding is desired. HRB can perform EMS mutagenesis to generate a large population of randomly mutated individuals. This population can then be screened, for example for visible traits or specific DNA mutations. This approach can be combined in a powerful way with our TiGER workflow, where we validate a desirable trait using a CRIPSR pre-screen.