Breeding exceptionally fragrant soybeans for soy milk with strong aroma

Soybean(Glycine max(L.)Merr.)is a major source of vegetable protein and oil in human diet and animal nutrition.Soybean seeds have been extensively used in various food products and snacks.Taste quality,particularly the aroma,affects cooking and eating,and ultimately influences consumer preference.Soy milk is particularly popular in China and has been gaining popularity in many other countries in the world.

Simple method for transformation and gene editing in medicinal plants

Gene-editing technologies have ushered in a significant advancement in plant genetics research and molecular breeding.However,a critical challenge hindering the widespread adoption of these technologies is the efficient delivery of gene-editing tools.The predominant methods for introducing these tools into plants typically involve Agrobacterium tumefaciens-mediated transformation or particle bombardment(Mao et al.,2019).Unfortunately,these traditional gene delivery methods require delicate and timeconsuming tissue culture procedures and show limited success,especially in medicinal and other less studied plants.A recent breakthrough is the development of the Cut-Dip-Budding(CDB)gene delivery system.The CDB system is highly effective for plants with root-suckering capabilities.It allows the delivery of transgenes and gene-editing tools into plants through hairy root induction followed by shoot regeneration from transformed hairy roots,bypassing the need for tissue culture processes(Cao et al.,2023).

An engineered Cas12i nuclease that is an efficient genome editing tool in animals and plants

The type V-I CRISPR-Cas system is becoming increasingly more attractive for genome editing.However,natural nucleases of this system often exhibit low efficiency,limiting their application.Here,we used structure-guided rational design and protein engineering to optimize an uncharacterized Cas12i nuclease,Cas12i3.As a result,we developed Cas-SF01,a Cas12i3 variant that exhibits significantly improved gene editing activity in mammalian cells.Cas-SF01 shows comparable or superior editing performance compared to SpCas9 and other Cas12 nucleases.Compared to natural Cas12i3,Cas-SF01 has an expanded PAM range and effectively recognizes NTTN and noncanonical NATN and TTVN PAMs.In addition,we identified an amino acid substitution,D876R,that markedly reduced the off-target effect while maintaining high on-target activity,leading to the development of CasSF01^(HiFi)(high-fidelity Cas-SF01).Finally,we show that Cas-SF01 has high gene editing activities in mice and plants.Our results suggest that CasSF01 can serve as a robust gene editing platform with high efficiency and specificity for genome editing applications in various organisms.

Novel Wx alleles generated by base editing for improvement of rice grain quality

Amylose content(AC),which is regulated by the Waxy(Wx)gene,is a major indicator of eating and cooking quality(ECQ)in rice(Oryza sativa).Thus far,only a limited number of mutations in the N-terminal domain of Wx were found to have a major impact on the AC of rice grains and no mutations with such effects were reported for other regions of the Wx protein.Here,nucleotide substitutions in the middle region of Wx were generated by adenine and cytosine base editors.The nucleotide substitutions led to changes in 15 amino acid residues of Wx,and a series of novel Wx alleles with ACs of 0.3%-29.43%(wild type with AC of 19.87%)were obtained.Importantly,the waxy~(abe2)allele showed a"soft rice"AC,improved ECQ,favorable appearance,and no undesirable agronomic traits.The transgenes were removed from the waxy~(abe2)progeny,generating a promising breeding material for improving rice grain quality.

Creation of aromatic maize by CRISPR/Cas

Aroma is an important quality parameter for breeding in rice(Oryza sativa).For example,the aromatic rice varieties basmati and jasmine rice,with a popcorn-like scent,are popular worldwide and routinely command a price premium.2-acetyl-1-pyrroline(2AP)is a key flavor compound among over 200 volatiles identified in fragrant rice.A naturally fragrant germplasm exists in multiple plant species besides rice,which all exhibit lower activity of BETAINE ALDEHYDE DEHYDROGENASE 2(BADH2).However,no equivalent aromatic germplasm has been described in maize(Zea mays).Here,we characterized the two maize BADH2 homologs,ZmBADH2a and ZmBADH2b.We generated zmbadh2a and zmbadh2b single mutants and the zmbadh2a-zmbadh2b double mutant by CRISPR/Cas in four inbred lines.A popcorn-like scent was only noticeable in seeds from the double mutant,but not from either single mutant or in wild type.In agreement,we only detected 2AP in fresh kernels and dried mature seeds from the double mutant,which accumulated between 0.028 and 0.723 mg/kg 2AP.These results suggest that ZmBADH2a and ZmBADH2b redundantly participate in 2AP biosynthesis in maize,and represent the creation of the world's first aromatic maize by simultaneous genome editing of the two BADH2 genes.

Cas12a-based on-site, rapid detection of genetically modified crops

Genetically modified(GM)crops that impart herbicide resistance,improved tolerance to biotic and abiotic stresses,and enhanced yield or nutritional quality have been developed and grown worldwide(ISAAA Brief 55-2019:International Servicefor theAcquisition ofAgri-biotech Applications(ISAAA),2019).In response topublic concern on genetically modified organisms(GMOs),DNA-based and protein-based methods have been developed to detect GMOs infood/feed samples(Fraitureetal.,2015).These methods,however,require expensive equipment and technical expertise and are unsuitable for field diagnosis(DNA-based methods)or cannot detect non-coding sequences such as promoters or terminators(protein-based methods).