Multiplex genome editing targeting soybean with ultra-low anti-nutritive oligosaccharides

Soybean is the primary source of plant protein for humans.Owing to the indigestibility of the raffinose family of oligosaccharides(RFO),raffinose and stachyose are considered anti-nutritive factors in soybean seeds.Low-RFO soybean cultivars are generated by mutagenesis of RFO biosynthesis genes,but the carbohydrate profiles invite further modification to lower RFOs.This study employed a pooled multiplex genome editing approach to target four seed-specifically expressed genes mediating RFO biosynthesis,encoding three raffinose synthases(RS2,RS3,and RS4)and one stachyose synthase.In T1progeny,rs2/rs3 and rs4/sts homozygous double mutants and a rs2/rs3/rs4/sts quadruple mutant(rfo-4m)were characterized.The rs2/rs3 mutant showed reduced raffinose and stachyose contents,but the rs4/sts mutant showed only reduced stachyose in seeds.The RFO contents in the rfo-4m mutant were almost eliminated.Metabolomic analysis showed that the mutation of four RFO biosynthesis genes led to a shift of metabolic profile in the seeds,including the accumulation of several oligosaccharides-related metabolites.These mutants could contribute to precision breeding of soybean cultivars for soy food production.

Doubled haploid technology and synthetic apomixis:Recent advances and applications in future crop breeding

Doubled haploid(DH)technology and synthetic apomixis approaches can considerably shorten breeding cycles and enhance breeding efficiency.Compared with traditional breeding methods,DH technology offers the advantage of rapidly generating inbred lines,while synthetic apomixis can effectively fix hybrid vigor.In this review,we focus on(i)recent advances in identifying and characterizing genes responsible for haploid induction(Hl),(ii)the molecular mechanisms of Hl,(ili)spontaneous haploid genome doubling,and(iv)crop synthetic apomixis.We also discuss the challenges and potential solutions for future crop breeding programs utilizing DH technology and synthetic apomixis.Finally,we provide our perspectives about how to integrate DH and synthetic apomixis for precision breeding and de novo domestication.

Evolutionary mechanisms and practical significance of reproductive success and clonal diversity in unisexual vertebrate polyploids

Unisexual reproduction is generally relevant to polyploidy, and unisexual vertebrates are often considered an evolutionary “dead end” due to the accumulation of deleterious mutations and absence of genetic diversity. However, some unisexual polyploids have developed strategies to avoid genomic decay, and thus provide ideal models to unveil unexplored evolutionary mechanisms, from the reproductive success to clonal diversity creation. This article reviews the evolutionary mechanisms for overcoming meiotic barrier and generating genetic diversity in unisexual vertebrates, and summarizes recent research advancements in the polyploid Carassius complex. Gynogenetic gibel carp(Carassius gibelio) is a unique amphitriploid that has undergone a recurrent autotriploidy and has overcome the bottleneck of triploid sterility via gynogenesis. Recently, an efficient strategy in which ploidy changes, including from amphitriploid to amphitetraploid, then from amphitetraploid to novel amphitriploid, drive unisexual-sexual-unisexual reproduction transition and clonal diversity has been revealed.Based on this new discovery, multigenomic reconstruction biotechnology has been used to breed a novel strain with superior growth and stronger disease resistance. Moreover, a unique reproduction mode that combines both abilities of ameiotic oogenesis and sperm-egg fusion,termed as ameio-fusiongensis, has been discovered, and it provides an efficient approach to synthesize sterile allopolyploids. In order to avoid ecological risks upon escape and protect the sustainable property rights of the aquaculture seed industry, a controllable fertility biotechnology approach for precise breeding is being developed by integrating sterile allopolyploid synthesis and gene-editing techniques.This review provides novel insights into the origin and evolution of unisexual vertebrates and into the attempts being made to exploit new breeding biotechnologies in aquaculture.

Satellite-enabled enviromics to enhance crop improvement

Enviromics refers to the characterization of micro-and macroenvironments based on large-scale environmental datasets.By providing genotypic recommendations with predictive extrapolation at a site-specific level,enviromics could inform plant breeding decisions across varying conditions and anticipate productivity in a changing climate.Enviromics-based integration of statistics,envirotyping(i.e.,determining environmental factors),and remote sensing could help unravel the complex interplay of genetics,environment,and management.To support this goal,exhaustive envirotyping to generate precise environmental profiles would significantly improve predictions of genotype performance and genetic gain in crops.Already,informatics management platforms aggregate diverse environmental datasets obtained using optical,thermal,radar,and light detection and ranging(LiDAR)sensors that capture detailed information about vegetation,surface structure,and terrain.This wealth of information,coupled with freely available climate data,fuels innovative enviromics research.While enviromics holds immense potential for breeding,a few obstacles remain,such as the need for(1)integrative methodologies to systematically collect field data to scale and expand observations across the landscape with satellite data;(2)state-of-the-art AI models for data integration,simulation,and prediction;(3)cyberinfrastructure for processing big data across scales and providing seamless interfaces to deliver forecasts to stakeholders;and(4)collaboration and data sharing among farmers,breeders,physiologists,geoinformatics experts,and programmers across research institutions.Overcoming these challenges is essential for leveraging the full potential of big data captured by satellites to transform 21st century agriculture and crop improvement through enviromics.

CRISPR/Cas: a Nobel Prize award-winning precise genome editing technology for gene therapy and crop improvement

Since it was first recognized in bacteria and archaea as a mechanism for innate viral immunity in the early 2010 s,clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein(Cas)has rapidly been developed into a robust,multifunctional genome editing tool with many uses.Following the discovery of the initial CRISPR/Cas-based system,the technology has been advanced to facilitate a multitude of different functions.These include development as a base editor,prime editor,epigenetic editor,and CRISPR interference(CRISPRi)and CRISPR activator(CRISPRa)gene regulators.It can also be used for chromatin and RNA targeting and imaging.Its applications have proved revolutionary across numerous biological fields,especially in biomedical and agricultural improvement.As a diagnostic tool,CRISPR has been developed to aid the detection and screening of both human and plant diseases,and has even been applied during the current coronavirus disease 2019(COVID-19)pandemic.CRISPR/Cas is also being trialed as a new form of gene therapy for treating various human diseases,including cancers,and has aided drug development.In terms of agricultural breeding,precise targeting of biological pathways via CRISPR/Cas has been key to regulating molecular biosynthesis and allowing modification of proteins,starch,oil,and other functional components for crop improvement.Adding to this,CRISPR/Cas has been shown capable of significantly enhancing both plant tolerance to environmental stresses and overall crop yield via the targeting of various agronomically important gene regulators.Looking to the future,increasing the efficiency and precision of CRISPR/Cas delivery systems and limiting off-target activity are two major challenges for wider application of the technology.This review provides an in-depth overview of current CRISPR development,including the advantages and disadvantages of the technology,recent applications,and future considerations.

Precision Breeding Made Real with CRISPR: Illustration through Genetic Resistance to Pathogens

Since its discovery as a bacterial adaptive immune system and its development for genome editing in eukaryotes,the CRISPR technology has revolutionized plant research and precision crop breeding.The CRISPR toolbox holds great promise in the production of crops with genetic disease resistance to increase agriculture resilience and reduce chemical crop protection with a strong impact on the environment and public health.In this review,we provide an extensive overviewon recent breakthroughs in CRISPR technology,including the newly developed prime editing system that allows precision gene editing in plants.We present how each CRISPR tool can be selected for optimal use in accordance with its specific strengths and limitations,and illustrate how the CRISPR toolbox can foster the development of genetically pathogen-resistant crops for sustainable agriculture.

Causal gene identification and desirable trait recreation in goldfish

Goldfish(Carassius auratus) have long fascinated evolutionary biologists and geneticists because of their diverse morphological and color variations.Recent genome-wide association studies have provided a clue to uncover genomic basis underlying these phenotypic variations,but the causality between phenotypic and genotypic variations have not yet been confirmed.Here,we edited proposed candidate genes to recreate phenotypic traits and developed a rapid biotechnology approach which combines gene editing with high-efficiency breeding,artificial gynogenesis,and temperature-induced sex reversal to establish homozygous mutants within two generations(approximately eight months).We first verified that low-density lipoprotein receptorrelated protein 2B(lrp2a B) is the causal gene for the dragon-eye variation and recreated the dragon-eye phenotype in side-view Pleated-skirt Lion-head goldfish.Subsequently,we demonstrated that the albino phenotype was determined by both homeologs of oculocutaneous albinism type II(oca2),which has subfunctionalized to differentially govern melanogenesis in the goldfish body surface and pupils.Overall,we determined two causal genes for dragon-eye and albino phenotypes,and created four stable homozygous strains and more appealing goldfish with desirable traits.The developed biotechnology approach facilitates precise genetic breeding,which will accelerate re-domestication and recreation of phenotypically desirable goldfish.

Rethinking fish biology and biotechnologies in the challenge era for burgeoning genome resources and strengthening food security

Fish biology has been developed for more than 100 years,but some important breakthroughs have been made in the last decade.Early studies commonly concentrated on morphology,phylogenetics,development,growth,reproduction manipulation,and disease control.Recent studies have mostly focused on genetics,molecular biology,genomics,and genome biotechnologies,which have provided a solid foundation for enhancing aquaculture to ensure food security and improving aquatic environments to sustain ecosystem health.Here,we review research advances in five major areas:(1)biological innovations and genomic evolution of four significant fish lineages including non-teleost ray-finned fishes,northern hemisphere sticklebacks,East African cichlid fishes,and East Asian cyprinid fishes;(2)evolutionary fates and consequences of natural polyploid fishes;(3)biological consequences of fish domestication and selection;(4)development and innovation of fish breeding biotechnologies;and(5)applicable approaches and potential of fish genetic breeding biotechnologies.Moreover,five precision breeding biotechniques are examined and discussed in detail including gene editing for the introgression or removal of beneficial or detrimental alleles,use of sex-specific markers for the production of mono-sex populations,controllable primordial germ cell on-off strategy for producing sterile offspring,surrogate broodstock-based strategies to accelerate breeding,and genome incorporation and sexual reproduction regainbased approach to create synthetic polyploids.Based on these scientific and technological advances,we propose a blueprint for genetic improvement and new breed creation for aquaculture species and analyze the potential of these new breeding strategies for improving aquaculture seed industry and strengthening food security.

Elimination of an unfavorable allele conferring pod shattering in an elite soybean cultivar by CRISPR/Cas9

Pod shattering can lead to devastating yield loss of soybean and has been a negatively selected trait in soybean domestication and breeding.Nevertheless,a significant portion of soybean cultivars are still pod shattering-susceptible,limiting their regional and climatic adaptabilities.Here we performed genetic diagnosis on the shattering-susceptible trait of a national registered cultivar,Huachun6(HC6),and found that HC6 carries the susceptible genotype of a candidate Pod dehiscence 1(PDH1)gene,which exists in a significant portion of soybean cultivars.We next performed genome editing on PDH1 gene by clustered regularly interspaced short palindromic repeats(CRISPR)-CRISPR-associated protein 9(Cas9).In T2 progenies,several transgene-free lines with pdh1 mutations were characterized without affecting major agronomic traits.The pdh1 mutation significantly improved the pod shattering resistance which is associated with aberrant lignin distribution in inner sclerenchyma.Our work demonstrated that precision breeding by genome editing on PDH1 holds great potential for precisely improving pod shattering resistance and adaptability of soybean cultivars.

Genome-edited crops: how to move them from laboratory to market

Recent breakthroughs in CRISPR technology allow specific genome manipulation of almost all crops and have initiated a revolution in precision crop breeding.Rationally-based regulation and widespread public acceptance are needed to propel genome-edited crops from laboratory to market and to translate this innovative technology into agricultural productivity.