CARBON Newsletter (22 March 2022) - Your Latest News About CRISPR in AgroBio

Top picks

• Chinese scientists have used CRISPR-Cas9 to obtain peanuts (Arachis hypogaea) with low palmitic acid and high oleic acid. Peanuts with reduced content of saturated fatty acids were achieved by two gRNAs targeting the knockout of two homologs of acyl-ACP thioesterase B (FatB). One mutant contained up to 63% oleic acid (46% in WT) and 23% linoleic acid (36% in WT) with little or no effect on plant growth and peanut yield.
• A new approach, named CRISPR-Kill, can be used for tissue engineering in Arabidopsis thaliana by inducing cell death in a controlled way. CRISPR-Kill is based on cell type-specific promoters driving the expression of Cas9 and gRNAs targeting repetitive DNA in tandem repeats. The German researchers demonstrate how the approach can be used to specifically eliminate petals by using the promoter of the floral homeotic gene APETALA1. Moreover, the number and length of lateral roots cold be dramatically reduced by using the xylem pole pericycle (XPP) promoter specific to root pericycle cells.

Technical advances

• Using a single gRNA, American researchers have successfully obtained multiplex knockout of eight alleles of three targeted genes in hybrid poplar (Populus tremula × P. alba). The single gRNA targeted a conserved region in exon two of MYB186, MYB138 and MYB38 - known positive trichome regulators - and led to trichome-less plants with no detectable off-targets.
• An optimised prime editor for gene editing in Arabidopsis thaliana cells is reported by Chinese scientists. The method was optimised via various promoters, reverse transcriptases, and sequence codon-optimisation and achieved an average of 1.15% editing efficiency, which is 16.4-fold higher than previously reported. The authors suggest that the method can provide a blueprint for developing prime editors for genetically engineered high-yield microalgae.
• Japanese researchers demonstrate the effect of timing of electroporation during in vitro maturation (IVM) on CRISPR-Cas9 triple gene editing in porcine embryos. After 44 h of IVM culture, electroporation led to higher blastocyst formation rate, mutation rates, and target numbers than electroporation after 40 or 42 h.
• Researchers from China describe a cytosine base editor toolkit for versatile gene manipulation in plants. The toolkit utilises an engineered human cytosine deaminase, AID10, with varying activity windows at the PAM protospacer. In addition, the toolkit can be used with different Cas9 nickase variants to widen target scopes.
• Chinese researchers have described a ribozyme-mediated CRISPR-Cas9 gene-editing system in hairy roots of pyrethrum (Tanacetum cinerariifolium) using an RNA polymerase II-dependent promoter. The method was used to edit the (E)‐β‐farnesene synthase (EbFS) gene, which catalyses the synthesis of the aphid defence-related compound (E)-β-farnesene in pyrethrum.
• A cationic lipid nanoparticle-mediated CRISPR-Cas9 technique for the production of stable genome-edited citrus plants is described by American researchers. Citrus protoplasts were transfected using Lipofectamine with gRNA targeting the Nonexpressor of Pathogenesis-Related 3 (CsNPR3) gene, a negative regulator of systemic acquired resistance (SAR). Plants from four edited lines had none to little expression of CsNPR3, while expression of CsPR1- a marker for the SAR induction process - was significantly upregulated.
• American researchers present a robust and simplified protocol for protoplast-based ribonucleoprotein-mediated genome editing in the model species Nicotiana benthamiana. A gene-editing efficiency of 30-60% in protoplasts is achieved, and gene-edited protoplasts can be readily regenerated without selection agents to obtain 50-80% gene editing efficiency in regenerated calli and plants.
• A CRISPR-Cas9 platform for targeted genome editing in Hong Kong kumquat (Fortunella hindsii) is described by Chinese scientists. The method employs double gRNAs to achieve 16–673 bp fragment deletions between the two sites with up to 53% efficiency.

Disease control

• Multiplex CRISPR-Cas9-mediated resistance to tomato brown rugose fruit virus (ToBRFV) in tomato (Solanum lycopersicum) is reported by scientists in Japan. The authors targeted four tomato homologs of tobamovirus multiplication 1 (TOM1), an Arabidopsis thaliana gene essential for tobamovirus multiplication. No detectable ToBRFV coat protein accumulation or noticeable growth or fruit production defects were observed in quadruple-mutant plants.
• Researchers from Israel have used CRISPR-Cas9 to obtain resistance in tomato (Solanum lycopersicum) to potyvirus potato virus Y (PVY). The authors targeted two homologs of the eukaryotic translation initiation factor 4E (eIF4E) essential in the potyvirus life cycle, SleIF4E1 and SleIF4E2, and knockout mutants were generated. Accumulation of PVY coat protein was significantly lower in sleIF4E1 mutants - either single or double - and resistance was specific to PVY, with no resistance to either eggplant mild leaf mottle virus, cucumber mosaic virus, pepino mosaic virus or tomato brown rugose fruit virus.

Nutritional quality

• Arabidopsis plants with a two-fold increase in total lipid content in their leaves have been obtained with CRISPR-Cas9 by scientists from the UK. The feat is achieved by using two gRNAs to delete a non-essential intergenic sequence between an upstream strong promoter and the coding region of diacylglycerol acyltransferase 2 (DGAT2) that has a relatively weak promoter. The authors suggest that the approach could be used in forage crops like perennial ryegrass (Lolium perenne) to enhance livestock productivity and reduce enteric methane emissions in pasture-based ruminant farming systems.

Agronomic traits

• Researchers from Iran present a strategy for introducing commercial CRISPR-Cas9-mediated bioengineered poplars with promising cellulose applications. The authors targeted various combinations of four residues of a component in the large transmembrane-localised cellulose synthase (CESA) complexes, CESA4, in Populus alba. As a result, significant alterations in cell wall architecture and improved saccharification efficiency was achieved.
• Researchers in China have identified a novel cadmium (Cd) stress-induced gene, NtNRAMP3, in tobacco and show that CRISPR-Cas9-mediated knockout of this gene leads to enhanced Cd tolerance and accumulation in leaves. The authors suggest that NtNRAMP3 might be a key candidate gene to improve the phytoremediation efficiency of plants via gene editing.

Industry

• The Israelian AgTech company BetterSeeds has signed an agreement with MilliporeSigma (the Life Science division of leading multinational pharmaceutical company Merck KGaA) to accelerate CRISPR in agricultural applications. As part of the agreement, BetterSeeds will have access to MillioporeSigma's technology for plant applications, strengthening BetterSeeds' capabilities to deliver high-quality crops with better traits.

Regulation and opinion

• Kenya will become the second African country after Nigeria to publish guidelines that clarify if and how genome-edited organisms and derived products will be regulated under Kenya's Biosafety Act. The Guidelines will provide an applicant with early consultation to determine the regulatory pathway to adopt and ensure that an early decision will be communicated to the applicant within 30 working days.
• Researchers in India foresee that dual use of the CRISPR-Cas technology will be challenging for law enforcement to combat. In a review, the authors summarise the potential applications of CRISPR in humans and plants and evaluate the laws and regulations imposed by different countries to keep genome editing technology under check.

Reviews

• A review by Chinese researchers looks at modified CRISPR gene-editing systems for various bioengineering tools and crop improvement - including base editors, prime editors, RNA editing, gene editing in mitochondria and chloroplasts, and multiplex gene-editing. The review summarises these modified gene-editing systems' capabilities, deficiencies, and applications and discusses their future developmental direction and challenges.
• Recent advances in genomics research and genetic improvements for insect pest resistance in major field crops is provided in a review by Indian and Australian researchers. The study discusses the scope for gene editing and genomic breeding strategies to develop more durable insect pest-resistant crops. In addition, it provides an overview of the diverse defence mechanisms that plants exert in response to insect pest attacks.
• A review by researchers in China discusses the potential of nanoparticles as an optimal platform to deliver biomolecules to plants for genetic engineering and CRISPR-Cas9 gene-editing. The potential adverse effects of plant nanoparticle technology are discussed, including the risk of transferring nanoparticles through the food chain.
• American and Indian scientists review CRISPR-Cas tools for multiplex genome editing in developing biotic and abiotic stress-resistant crop plants. The review describes the components, mechanisms, and types of CRISPR-Cas techniques and the genes selected for knocking down/out.

Meetings, webinars etc.

• A podcast on "Gene editing with CRISPR and its impact on plant breeding" features Gabino Sanchez from Hudson River Biotechnology. The podcast is an episode of the series "Computomics: Discussions on machine learning algorithms for plant breeding challenges".
• A Plant Physiology webinar entitled "Gene Editing and its Applications" is taking place Thursday, 28 April 2022 at 7:00 am BST London (UTC+1). The webinar is moderated by Yunde Zhao, Plant Physiology Editor-in-Chief, and features speakers Yao-Cheng Lin, Xiaolan Zhang, Ryozo Imai and Heriberto Cerutti.