About our technology

HRB 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 licencing costs (MAD TiGER).

While CRISPR genome editing is straightforward in principle, the functional application is a considerable challenge, especially in the green biotech. TiGER enables molecular breeding in an end-to-end fashion, providing innovative solutions to the typical barriers encountered in (plant) CRISPR breeding projects. In addition, we are actively further developing specific aspects of the workflow to continuously broaden its range and scope.

Targeted mutagenesis via CRISPR

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.

Figure 1. Elements of a CRISPR genome editing system.
In green the CRISPR enzyme MAD7 with its coupled guide RNA in black. Part of the guide RNA, the crRNA, is bound to a complementary sequence in the genome (the target). Upon binding MAD7 will cleave at a fixed distance from the protospacer adjacent motif (PAM).

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.

Figure 2. Homologous directed repair (HDR).
Either a homologous chromosome or custom- piece of dsDNA can serve as a repair template after a CRISPR-induced double strand break. (Note, the HDR pathway can also include recombination events; not shown).

Identifying unique targets for CRISPR editing

Step 1 of our TiGER workflow is Target identification. A key element to developing new traits is knowing what to edit. Open reading frames of genes are the most common targets for genome editing that we work with. Depending on existing knowledge and tools available (e.g., a sequenced genome), we take a tailored approach to target gene selection. For example, comparing gene expression profiles of different, closely related lines, usually via RNAseq. (If a reference genome is not yet available for your species of interest, we still have several other options available.) In addition to these unbiased approaches, we use literature study and our network in the academic world to gain insights on genetic targets. Combined with promoter and enhancer screening and profiling (see below), this results in a candidate list for mutagenesis that we pursue via targeted and/or random mutagenesis approaches.

Besides genes, HRB focuses on a more unique set of targets: gene regulatory elements (promoters and enhancers). For many genes, completely abolishing their activity by targeting the open reading frame leads to detrimental effects on growth and development, rather than conferring desirable traits. Our strategy is to target the regulatory elements in the genome controlling the level, timing and tissue-specificity of expression, allowing a more nuanced way of genome editing. To identify such regulatory elements, we have multiple solutions available such as the SuRE, STARR-seq and STAP-seq technology.

Figure 3. The SuRE technology workflow.
The SuRE technology was developed at the Netherlands Cancer Institute (NKI) for human applications1. HRB and Gen-X have validated the technique for use in plants. With this technology, a whole genome is assessed for regulatory elements, such as promoters and autonomous enhancers.

Regarding the SuRE technology, HRB has partnered with Gen-X for access to this technology for agricultural applications. HRB has now validated the SuRE technology in plants by demonstrating that it can produce meaningful genome-wide maps of promoters and enhancers in tomato. In more detail; SuRE allows genome-wide screening for regulatory elements (promoters and autonomous enhancers). It assays small fragments of a sheared genome for their ability to autonomously drive transcription (Figure 3). A plasmid library of random, 0.2-2 kb genomic fragments upstream of a 20-bp barcode is constructed and decoded by paired-end sequencing. This library is used to transfect cells, and barcodes in transcribed RNA are quantified by high-throughput sequencing. Over 50-fold genome coverage can be reached, allowing mapping of autonomous promoter activity to a genome, or parts of a sequenced genome. By computational modeling we can further delineate sub-regions within promoters that are relevant for their activity. In short, the technology can be seen as a genome wide truncation experiment with such ample redundancy of the fragments assayed that it can identify single nucleotides as important for promoter/enhancer function.

Additionally, HRB has access to other technologies that screen for gene regulatory elements, such as STARR-seq and STAP-seq2 that follow analogous approaches. 2 Arnold et al. (2013) Science 339(6123), Arnold et al. (2017) Nat. Biotech. 35(2)

There are numerous valuable agricultural applications for promoter and enhancer screening:

  • Unbiased identification of gene regulatory elements can generate targets for CRISPR or TILLING approaches to mutagenesis. Modifying regulatory elements rather than open reading frames allows for partial down-regulation or even up-regulation of gene activity instead of a complete knockout. In addition, elements may be identified that regulate gene expression in a manner that is tissue or time specific, enabling genetic optimizations that are even more specifically targeted and/or circumvent off-target effects.
  • Genomes of closely related lines that differ in traits (e.g., disease resistance) can be compared to pinpoint differing levels of gene expression rather than sequence variations within genes to these traits.
  • Genomic responses to exposure to e.g. (a)biotic stressors can be assayed, to identify genomic elements that mediate the stress response; such elements may function as targets for modification to confer resistance to the stressor.
  • Identification of unique promoter/enhancer regions that can serve as strong, endogenous promoters. These are highly valuable for strong and sustained transgene expression with reduced risk of silencing.

CRISPR guide design and validation

Step 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.
Figure 5. Output of an in vivo CRISPR activity assay.
Alignments of the target region of a wild-type (WT) genome (top sequence) and gene-edited genomes (all other sequences).
Figure 4. In vitro CRISPR activity test.
An image of an agarose gel with DNA molecules separated based on size. In each ‘lane’ a sample was run that included a linear piece of target DNA exposed to a CRISPR enzyme complexed with an unique guide. If the guide allows cleavage activity, the upper target DNA band is cut into two smaller pieces that show up lower on the gel. Varying efficiencies of DNA cleavage can be observed, as the upper band disappears over time while the two lower bands appear.
Figure 5. Output of an in vivo CRISPR activity assay.
Alignments of the target region of a wild-type (WT) genome (top sequence) and gene-edited genomes (all other sequences).

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.

Plant regeneration

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.

Random mutagenesis

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.