About our technology
HRB applies and develops innovative solutions to critical bottlenecks in the application of CRISPR in plant breeding. A typical CRISPR breeding project encounters barriers at several of the steps in the process, including target selection, CRISPR guide design, transfection and plant regeneration. HRB has, or is developing, solutions for each of these bottlenecks.
Targeted mutagenesis via CRISPRIn genetics, clustered regulatory interspaced short palindromic repeats (CRISPRs) are loci that contain multiple short direct repeats, and that provide acquired immunity to bacteria and archaea through sequence-specific silencing of invading foreign DNA. So-called CRISPR-associated systems (CAS) are a family of proteins (including Cas9 and MAD7) that can serve as RNA-guided DNA endonucleases for targeted mutagenesis in plants and other organisms. These endonucleases cleave DNA upon target recognition by their guide RNA (gRNA) sequences. The gRNA connects to the endonuclease and identifies, in a highly specific manner, the correct location in the genome where a cut is to be made. Together, the endonuclease and gRNA form the RNA-protein complex that performs targeted mutagenesis. The cell’s innate DNA damage repair machinery (for example, non-homologous end joining, NHEJ) repairs the break made in the DNA by the endonuclease but introduces errors (mutations) in the process that cause altered function of the targeted gene. As such, CRISPR enables a novel form of targeted mutagenesis, which allows us to modify a specific gene of interest. HRB works with multiple different CRISPR options and has in-house capabilities for CRISPR protein and guide-RNA production.
Identifying unique targets for CRISPR editingGenes are common targets for genome editing. HRB additionally focuses on a unique type of targets: gene regulatory elements (promoters and enhancers). For many genes, completely abolishing their activity 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. One is the proprietary SuRE Platform. Gen-X, a spin-off from the Netherlands Cancer Institute has developed SuRE and validated the platform in mammalian cell culture systems1. HRB has partnered with Gen-X for access to the SuRE technology for agricultural applications, and for validation of this technology in plants. 1Van Arensbergen et al. (2016) Nature Biotechnology
SuRE assays small fragments of a sheared genome for their ability to autonomously drive transcription. A plasmid library of random, 0.2-2kb 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.
Together, HRB and Gen-X have demonstrated the SuRE technology can produce meaningful genome-wide maps of promoters and enhancers in plants.
Additionally, HRB has access to other technologies that screen for gene regulatory elements, such as STARR and STAP2 that follow an analogous approach to SuRE. 2Arnold et al. (2013) Science 339(6123), Arnold et al. (2017) Nat. Biotech. 35(2)
Valuable agricultural applications for promoter and enhancer screening
- Unbiased identification of gene regulatory elements that can serve as targets for CRISPR or TILLING approaches to mutagenesis. Modifying these elements rather than the genes themselves allows for partial down-regulation or even up-regulation of gene activity, rather than complete knockout, and leaves the gene sequence intact. 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 or that circumvent off-target effects.
- Genomes of closely related lines can be compared to pinpoint differences in traits (e.g. disease resistance) that are due to differing levels of gene expression rather than sequence variations within genes.
- 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.
Other Target Selection MethodsIn addition to identifying gene regulatory elements, we also screen for genes as targets for modification. 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, this results in a candidate list for mutagenesis that we pursue via targeted or random mutagenesis approaches.
CRISPR guide design and validationGuide efficiency is an important factor in overall genome editing efficiency. HRB has developed proprietary guide design software that allows to design efficient CRISPR guides for multiple different CRISPR enzymes in a rapid manner. We can produce all enzymes and guide RNAs in house, achieving comparable performance to products from commercial suppliers. Our workflow for validating CRISPR guide efficiency further consists of measuring editing efficiency on purified DNA and on genomic DNA in live protoplasts.
Gel image showing a piece of target DNA (“No guide”) and varying efficiencies of CRISPR-guide RNA complexes targeting that area for gene editing. Here, endonuclease activity results in a break in the DNA that is visualized in the gel. HRB uses this as the first step in the process of guide design and testing.