CARBON Newsletter (19 April) - Your Latest News About CRISPR in AgroBio

Top picks

• Chinese researchers have used CRISPR-Cas9 to switch the leaf colour in ornamental kale (Brassica oleracea) from pink to green. The scientists achieved this by inserting either one or two base pairs in the dihydroflavonol 4-reductase-encoding gene BoDFR1 that drives anthocyanin accumulation. However, knocking out the ortholog BoDFR2 gene did not affect anthocyanin production.
• Rice with superior yield and grain quality has been obtained through CRISPR-Cas9-mediated gene editing of the GS3 gene, a quantitative trait locus (QTL) that regulates rice grain length and weight. Hybrids obtained from gene edited plants had 7.9% longer grains, thousand grain weight increased by 6.7%, and grain yield per plant increased by 14.9%. In addition, other quality traits improved in the gene edited hybrid rice.
• The world's first commercial CRISPR tomato - containing a high level of GABA (ɣ-aminobutyric acid) with well-known human health-promoting effects - was recently launched to the Japanese market. A paper by Japanese researchers now discusses the social-economic impact of its implementation on crop genome editing.

Technical advances

• An efficient multiplex genome editing system for the allotetraploid species Nicotiana benthamiana is presented by researchers in Austria. The method incorporates the DsRed2 fluorescent marker to select transgenic plants and subsequent identification transgene-free T1 lines with homozygous or biallelic mutations. Using preselected gRNAS, the authors achieved plant lines with knockout mutations in all four P4H4 genes simultaneously.
• German researchers have devised a protocol for using CRISPR-Cas9 to create chromosomal rearrangements in the Mb range, such as inversion and translocations, Arabidopsis thaliana. The protocol is based on Agrobacterium-mediated transformation of DNA constructs containing Cas9, which is driven by an egg-cell-specific promoter, and two guide RNAs that have been preselected based on their cutting efficiency.
• CRISPR-Cas9-mediated gene editing is more accurate than base editing using the BE4-Gam system in sheep embryos, Chinese researchers found. They used both methods to introduce the FecG^H mutation that increases prolificacy and achieved equal gene editing efficiencies of 16%. However, no bystander editing events were detected with CRISPR-Cas9, whereas the bystander editing efficiency was as high as 15% in BE4-Gam-edited embryos.
• Gene editing has been used to trigger maternal haploids in the allotetraploid model plant Nicotiana tabacum, a strategy that breeders can use to accelerate the breeding process. Loss-of-function mutations in three homologues of the DMP genes (NtDMP1, 2, and 3) were created by CRISPR-Cas9 with an induction rate of 1.52-1.75% leading to differences in pollen vigour and seed germination rate.
• The gene Wuschel2 (Wus2) is known to be essential for somatic embryo formation and shoot regeneration. This is now utilised by American researchers, who use co-transformation of Wus2 to achieve highly efficient CRISPR-Cas genome editing and rapid de novo shoot regeneration in sorghum.
• Carbon nanotubes (CNTs) loaded with plasmid DNA carrying genes encoding Cas9 and gRNAs are proposed as a new delivery system for gene editing of rice leaves and seeds. CNTs can diffuse through plant cell walls, facilitating the transient expression of foreign genetic elements in plant tissues. Preliminary results indicate that the CNT-delivery of CRISPR reagents can lead to frameshift mutations and premature stop codons in the target gene.

Disease control

• American researchers have used gene editing to obtain resistance in citrus to the bacterial disease citrus canker caused by Xanthomonas citri ssp. citri (Xcc). CRISPR-Cas9 was used to make frameshifting indels in CsDMR6, a repressor of plant immunity that negatively regulates the expression of plant defence genes like NPR1. Compared to wild-type plants, Xcc cell populations were reduced by 99.8% in gene-edited plants at 20 dpi.

Nutritional quality

• Researchers in China propose a new way to reduce residual amounts of the herbicide isoproturon in rice. The scientists used genetic transformation and CRISPR-Cas9 to either overexpress or knock out the uncharacterised cytochrome P450 gene CYP76C6. They found that overexpression of CYP76C6 led to isoproturon metabolism and detoxification.
• American researchers have improved the n-3 fatty acid content in channel catfish (Ictalurus punctatus). This was achieved by CRISPR-Cas9-mediated transgenesis of the masu salmon (Oncorhynchus masou) biosynthetic elovl2 gene. As a result, the content of n-3 fatty acids EPA and DHA increased by approximately 50% in the economically important freshwater aquaculture species that generally have low levels of these polyunsaturated fatty acids.

Agronomic traits

• CRISPR-Cas9 has been used to create a golden version of the economically important fish Nile tilapia (Oreochromis niloticus), a type of cichlid that is both captured and cultured. The Chinese researchers achieved the new colour by knocking out combinations of the two premelanosome protein genes (pmela and pmelb), typically expressed at high levels in the eyes and skin.
• Researchers in Singapore propose another strategy to produce golden tilapias. First, they showed that the knockout of Pmel17 with CRISPR-Cas9 in blackish tilapias resulted in golden colouration. Then, further functional analysis in vitro showed that the insertion in the 3′ UTR of Pmel17 reduced the transcripts of Pmel17.
• Researchers in China have used CRISPR-Cas9 to downregulate gibberellin 3β-hydroxylase (GmGA3ox1) in soybean. Mutants showed lower seed weight but promoted seed yield by increasing seed numbers. The authors suggest that gene editing of GmGA3ox1 might be a clue for future high-yield breeding in soybean and other crops.

Detection

• Researchers in China have developed a CRISPR-Cas12a-driven biosensor to detect the presence of cow milk in goat milk. The ultrasensitive surface-enhanced Raman scattering (SERS) biosensor has a detection limit of 224 aM for target DNA, and a portable Raman spectrometer detects readout.
• Chinese researchers have developed a CRISPR-Cas12a based fluorescence assay to detect organophosphorus pesticides (OPs) in agricultural products. The assay utilises that OPs suppress acetylcholinesterase activity , which is the first step in a cascade required for CRISPR-Cas12a activation and a fluorescence signal. The assay has good analytical performance toward paraoxon, dichlorvos and demeton.

Industry

• UK startup company ALORA, a pioneer in developing ocean agriculture to create a more sustainable future, has announced the development of gene-edited salt-tolerant rice plants. The plants thrive with high yields at salt levels of 12g/L and show an upper tolerance of 16g/L (seawater is 28-32g/L). The company also announced $1.4 million in seed funding from investors, including Toyota Ventures and Mistletoe.

Regulation and opinion

• Testbiotech writes that in a letter written in April 2022, the EU Commission states that CRISPR-Cas applications do not create any new or specific risks due to unintended effects. According to Testbiotech, however, the EU Commission continues to ignore the scientific findings regarding new and specific risks.
• A piece in EatingWell looks at the role of CRISPR in researchers' strategies to modify crops so they fit to the increasing average global temperature. The author points out that we count on fewer than 20 types of crops to feed the world, many of them are at risk, so there is an urgent need to breed climate-hardy crops. Examples for strategies like rediscovering of landraces and orphan crops, gene knockout etc., are presented.

Reviews

• A review by researchers in South Korea gives an overview of the potential applications of CRISPR-Cas9-based methods in developing the cultivation of major agricultural crops. In addition, the limitations and significant challenges of genome editing in grains, vegetables, and fruits are discussed in detail by emphasising its applications in crop refinement strategy.
• Genome editing of genes encoding enzymes, chaperones and transporters involved in catabolic and biosynthesis pathways in rice is a review topic by Japanese researchers. The review details strategies for creating random mutagenesis, targeted mutations, and artificial regulation using dead Cas9 fused to transcriptional activators or repressors.
• German scientists argue that grapevine breeding is lacking behind and that the market stick to well-known varietal wines or blends (e.g. Chardonnay, Cabernet Sauvignon, Riesling). They foresee that climate change with its extreme weather will impose the need for a change in cultivars in many wine growing regions and suggest that CRISPR-Cas can contribute to the rapid creation of new, competitive cultivars.
• Researchers from Pakistan and other countries review CRISPR-Cas9 based abiotic stress tolerance in chickpea (Cicer arietinum). The authors summarise the past achievements with genetic transformation and discuss the potential of CRISPR-Cas9 to develop climate-resilient cultivars and boost the yield of chickpea.