CRISPR/Cas9-mediated gene editing to confer turnip mosaic virus (TuMV) resistance in Chinese cabbage (Brassica rapa)

Genome editing approaches,particularly the CRISPR/Cas9 technology,are becoming state-of-the-art for trait development in numerous breeding programs.Significant advances in improving plant traits are enabled by this influential tool,especially for disease resistance,compared to traditional breeding.One of the potyviruses,the turnip mosaic virus(TuMV),is the most widespread and damaging virus that infects Brassica spp.worldwide.We generated the targeted mutation at the eIF(iso)4E gene in the TuMV-susceptible cultivar“Seoul”using CRISPR/Cas9 to develop TuMV-resistant Chinese cabbage.We detected several heritable indel mutations in the edited T0 plants and developed T1 through generational progression.It was indicated in the sequence analysis of the eIF(iso)4E-edited T1 plants that the mutations were transferred to succeeding generations.These edited T1 plants conferred resistance to TuMV.It was shown with ELISA analysis the lack of accumulation of viral particles.Furthermore,we found a strong negative correlation(r=-0.938)between TuMV resistance and the genome editing frequency of eIF(iso)4E.Consequently,it was revealed in this study that CRISPR/Cas9 technique can expedite the breeding process to improve traits in Chinese cabbage plants.

Manipulation of plant height in garden asparagus (Asparagus officinalis L.) through CRISPR/Cas9-mediated aspSPL14 allele editing

Dear Editor,Reduction in plant height has been associated with yield increases and yield stability in a number of important crop species,such as wheat and rice[1].In these plants,dwarfing is mainly attributed to the inability to synthesize or respond to certain phytohormones,predominantly gibberellin(GA)[2].Ideal Plant Architecture 1(IPA1),an miR156 target gene,encodes SPL14 and it is able to bind directly to the promoters of multiple GA biosynthetic,signal,and deactivating genes in rice[3].Moreover,IPA1 loss-of-function mutants exhibit dwarf phenotypes[4].

CRISPR/Cas9-mediated SNAC9 mutants reveal the positive regulation of tomato ripening by SNAC9 and the mechanism of carotenoid metabolism regulation

NAC transcriptional regulators are crucial for tomato ripening.Virus-induced gene silencing(VIGS)of SNAC9(SlNAC19,Gene ID:101248665)affects tomato ripening,and SNAC9 is involved in ethylene and abscisic acid(ABA)metabolic pathways.However,the function of SNAC9 in pigment metabolism in tomatoes remains unclear.This work seeks to discover the mechanism of SNAC9 involvement in pigment metabolism during tomato ripening by establishing a SNAC9 knockout model using CRISPR/Cas9 technology.The results indicated that fruit ripening was delayed in knockout(KO)mutants,and SNAC9 mutation significantly affected carotenoid metabolism.The chlorophyll(Chl)degradation rate,total carotenoid content,and lycopene content decreased significantly in the mutants.The transformation rate of chloroplasts to chromoplasts in mutants was slower,which was related to the carotenoid content.Furthermore,SNAC9 changed the expression of critical genes(PSY1,PDS,CRTISO,Z-ISO,SGR1,DXS2,LCYE,LCYB,and CrtR-b2)involved in pigment metabolism in tomato ripening.SNAC9 knockout also altered the expression levels of critical genes involved in the biosynthesis of ethylene and ABA.Accordingly,SNAC9 regulated carotenoid metabolism by directly regulating PSY1,DXS2,SGR1,and CrtR-b2.This research provides a foundation for developing the tomato ripening network and precise tomato ripening regulation.

CRISPR/Cas9-mediated knockout of SLC15A4 gene involved in the immune response in bovine rumen epithelial cells

The objective of this study was to determine the role of SLC15A4 in the muramyl dipeptide(MDP)-mediated inflammatory response of bovine rumen epithelial cells(BRECs).First,changes in the m RNA expression of proinflammatory factor genes in BRECs following 10μg m L^(–1)MDP treatments were examined.RT-q PCR results showed that the expression of pro-inflammatory factor(IL-1β,IL-6,and TNF-α)m RNAs were significantly increased under MDP stimulation(P<0.001).Moreover,SLC15A4-Knockout(SLC15A4-KO)cells were obtained through lentivirus packaging,transfection,screening,and cell monoclonal culture.In order to gain further insight into the potential function of SLC15A4,we utilized transcriptome data,which revealed a change in the genes between WT-BRECs and SLC15A4-KO.Five down-regulated pro-inflammatory genes and 13 down-regulated chemokine genes related to the inflammatory response were identified.Meanwhile,the down-regulated genes were mostly enriched in the nuclear factorκB(NF-κB)and mitogen-activated protein kinase(MAPK)signaling pathways.The results of RT-q PCR also verified these detected changes.To further determine the mechanism of how WT and SLC15A4-KO BRECs are involved in inflammatory responses,we investigated the inflammatory responses of cells exposed to MDP.WT-BRECs and SLC15A4-KO were treated with a culture medium containing 10μg m L^(–1)MDP,in comparison to a control without MDP.Our results show that SLC15A4-KO BRECs had reduced the expression of genes(IL-6,TNF-α,CXCL2,CXCL3,CXCL9,and CCL2)and proteins(p-p65 and p-p44/42)from the MDP-mediated inflammatory response compared to WT-BRECs(P<0.05).In this experiment,CRISPR-Cas9 was used to KO the di/tripeptide transporter SLC15A4,and its role was confirmed via the MDP-induced inflammatory response in BRECs.This work will provide a theoretical basis for studying the pro-inflammatory mechanism of MDP and its application in the prevention and treatment of subacute rumen acidosis in dairy cows.

Reduction in cadmium accumulation in japonica rice grains by CRISPR/Cas9-mediated editing of OsNRAMP5

Cadmium(Cd) intake is harmful to human health and Cd contamination in rice grains represents a severe threat to those consuming rice as a staple food. Knockout of Cd transporters is a promising strategy to reduce Cd accumulation in rice grains. OsNRAMP5 is the major transporter for Cd and manganese(Mn) uptake in rice. Nevertheless, it is uncertain whether knockout of OsNRAMP5 is applicable to produce low Cd rice without affecting plant growth and grain yield. In this study, we adopted CRISPR/Cas9-based gene editing technology to knock out OsNRAMP5 in two japonica varieties. We generated three independent transgene-free osnramp5 mutants and investigated the effect of osnramp5 mutations on Cd accumulation and plant growth. Hydroponic experiments showed that plant growth and chlorophyll content were significantly reduced in osnramp5 mutants at low Mn conditions, and this defective growth in the mutants could be fully rescued by supply of high levels of Mn. Cd and Mn accumulation in both roots and shoots was markedly reduced in the mutants compared to that in wild-type plants. In paddy field experiments, although Cd in flag leaves and grains was greatly reduced in osnramp5 mutants, some agronomic traits including plant height, seed setting rate, and grain number per panicle were affected in the mutants, which ultimately caused a mild reduction in grain yield. The reduced plant growth in the mutants can be attributed to a marked decrease in Mn accumulation. Our results reveal that the manipulation of OsNRAMP5 should be treated with caution: When assessing the applicability of osnramp5 mutants, soil pH and soil water content in paddy fields need to be taken into consideration, since they might affect the levels of available Mn in the soil and consequently determine the effect of the mutation on grain yield.

Characterization and Evaluation of OsLCT1 and OsN ramp5 Mutants Generated Through CRISPR/Cas9-Mediated Mutagenesis for Breeding Low Cd Rice

To explore how rice(Oryza sativa L.) can be safely produced in Cd-polluted soil, OsLCT1 and OsNramp5 mutant lines were generated by CRISPR/Cas9-mediated mutagenesis. One of OsLCT1 mutant(lct1×1) and two of OsNramp5 mutants(nramp5×7 and nramp5×9) were evaluated for grain Cd accumulation and agronomic performances. In paddy field soil containing approximately 0.9 mg/kg Cd, lct1×1 grains contained approximately 40%(0.17 mg/kg) of the Cd concentration of the wild type parental line, less than the China National Food Safety Standard(0.20 mg/kg). Both OsNramp5 mutants showed low grain Cd accumulation(< 0.06 mg/kg) in the paddy(approximately 0.9 mg/kg Cd) or in pots in soil spiked with 2 mg/kg Cd. However, only nramp5×7 showed normal growth and yield, whereas the growth of nramp5×9 was severely impaired. The study showed that lct1×1 could be used to produce rice grains safe for human consumption in lightly contaminated paddy soils and nramp5×7 used in soils contaminated by much higher levels of Cd.

Creating a novel herbicide-tolerance OsALS allele using CRISPR/Cas9-mediated gene editing

Weeds and weedy rice plague commercial rice fields in many countries. Developingherbicide-tolerance rice is the most efficient strategy to control weed proliferation. CRISPR/Cas9-mediated gene editing, which generates small InDels and nucleotide substitutions atand around target sites using error-prone non-homologous end joining DNA repairing, hasbeen widely adopted for generation of novel crop germplasm with a wide range of geneticvariation in important agronomic traits. We created a novel herbicide-tolerance allele inrice by targeting the acetolactate synthase (OsALS) gene using CRISPR/Cas9-mediated geneediting. The novel allele (G628W) arose from a G-to-T transversion at position 1882 of OsALSand conferred a high level of herbicide tolerance. Transgene-free progeny carryinghomozygous G628W allele were identified and showed agronomic performance similar tothat of wild-type plants, suggesting that the G628W allele is a valuable resource fordeveloping elite rice varieties with strong herbicide tolerance. To promote use of the G628Wallele and to accelerate introgression and/or pyramiding of the G628W allele with other elitealleles, we developed a DNA marker for the G628W allele that accurately and robustlydistinguished homozygous from heterozygous segregants. Our result further demonstratesthe feasibility of CRISPR/Cas9-mediated gene editing in creating novel genetic variation forcrop breeding.

High-Efficiency Reduction of Rice Amylose Content via CRISPR/Cas9-Mediated Base Editing

Rice is a staple food for more than half of the human population.It has been estimated that by 2030,rice production must increase by 40%to meet the growing demand(Khush,2005).In addition,with the improvement of people's living standards,the demand for elite rice with better eating and cooking quality(ECQ)is increasing.ECQ is determined by several factors,including amylose content(AC),gel consistency(GC),gelatinization temperature(GT)and viscosity,where AC is the predominant factor(Juliano,1998).

CRISPR/Cas9-mediated VvPR4b editing decreases downy mildew resistance in grapevine(Vitis vinifera L.)

Downy mildew of grapevine(Vitis vinifera L.),caused by the oomycete pathogen Plasmopara viticola,is one of the most serious concerns for grape production worldwide.It has been widely reported that the pathogenesis-related 4(PR4)protein plays important roles in plant resistance to diseases.However,little is known about the role of PR4 in the defense of grapevine against P.viticola.In this study,we engineered loss-of-function mutations in the VvPR4b gene from the cultivar“Thompson Seedless”using the CRISPR/Cas9 system and evaluated the consequences for downy mildew resistance.Sequencing results showed that deletions were the main type of mutation introduced and that no off-target events occurred.Infection assays using leaf discs showed that,compared to wild-type plants,the VvPR4b knockout lines had increased susceptibility to P.viticola.This was accompanied by reduced accumulation of reactive oxygen species around stomata.Measurement of the relative genomic abundance of P.viticola in VvPR4b knockout lines also demonstrated that the mutants had increased susceptibility to the pathogen.Our results confirm that VvPR4b plays an active role in the defense of grapevine against downy mildew.

Generation of tryptophan hydroxylase 2 gene knockout pigs by CRISPR/Cas9-mediated gene targeting

Unbalanced brain serotonin(5-HT) levels have implications in various behavioral abnormalities and neuropsychiatric disorders. The biosynthesis of neuronal 5-HT is regulated by the rate-limiting enzyme, tryptophan hydroxylase-2(TPH2). In the present study, the clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated(Cas) system was used to target the Tph2 gene in Bama mini pig fetal fibroblasts. It was found that CRISPR/Cas9 targeting efficiency could be as high as 61.5%, and the biallelic mutation efficiency reached at38.5%. The biallelic modified colonies were used as donors for somatic cell nuclear transfer(SCNT) and 10 Tph2 targeted piglets were successfully generated. These Tph2 KO piglets were viable and appeared normal at the birth.However, their central 5-HT levels were dramatically reduced, and their survival and growth rates were impaired before weaning. These Tph2 KO pigs are valuable large-animal models for studies of 5-HT deficiency induced behavior abnomality.

Truncated gRNA reduces CRISPR/Cas9-mediated off-target rate for MSTN gene knockout in bovines

The CRISPR/Cas9 mediates efficient gene editing but has off-target effects inconducive to animal breeding. In this study, the efficacy of CRISPR/Cas9 vectors containing different lengths of g RNA in reduction of the off-target phenomenon in the bovine MSTN gene knockout fibroblast cell lines was assessed, providing insight into improved methods for livestock breeding. A 20-bp g RNA was designed for the second exon of the bovine MSTN gene, and CRISPR/Cas9-B was constructed to guide the Cas9 protein to the AGAACCAGGAGAAGATGGACTGG site. The alternative CRISPR/Cas9-19, CRISPR/Cas9-18, CRISPR/Cas9-17 and CRISPR/Cas9-15 vectors were constructed using g RNAs truncated by 1, 2, 3 and 5 bp, respectively. These vectors were then introduced into bovine fetal fibroblasts by the electroporation method, and single cells were obtained by flow cytometry sorting. PCR was performed for each off-target site. All samples were sequenced and analyzed, and finally the efficiency of each vector in target and off-target sites was compared. The CRISPR/Cas9-B vector successfully knocked out the MSTN gene, but the off-target phenomenon was observed. The efficiencies of CRISPR/Cas-B, CRISPR/Cas9-19, CRISPR/Cas9-18, CRISPR/Cas9-17 and CRISPR/Cas9-15 in triggering gene mutations at MSTN targeting sites were 62.16, 17.39, 7.69, 74.29 and 3.85%, respectively;rates of each at the Off-MSTN-1 locus were 52.86, 0, 0, 8.82 and 0%, respectively;all were 0% at the Off-MSTN-2 locus;rates at the Off-MSTN-3 site were 44.87, 51.72, 86.36, 0 and 50%, respectively. The efficiency of the CRISPR/Cas9-17 plasmid in the MSTN site was higher than that in the CRISPR/Cas9-B plasmid, and the effect at the three off-target sites was significantly lower. This study demonstrated that the CRISPR/Cas9-17 plasmid constructed by truncating 3 bp g RNA can effectively reduce the off-target effect without reducing the efficiency of bovine MSTN gene targeting. This finding will provide more effective gene editing strategy for use of CRISPR/Cas9 technology.

An improved Tet-on system in micro RNA overexpression and CRISPR/Cas9-mediated gene editing

Background: Tetracycline(Tet)-regulated expression system has become a widely applied tool to control gene activity. This study aimed to improve the Tet-on system with superior regulatory characteristics.Results: By comprehensively comparing factors of transactivators, Tet-responsive elements(TREs), orientations of induced expression cassette, and promoters controlling the transactivator, we developed an optimal Tet-on system with enhanced inducible efficiency and lower leakiness. With the system, we successfully performed effective inducible and reversible expression of micro RNA, and presented a more precise and easily reproducible fine-tuning for confirming the target of a mi RNA. Finally, the system was applied in CRISPR/Cas9-mediated knockout of nuclear factor of activated T cells-5(NFAT5), a protective transcription factor in cellular osmoregulation.Conclusions: This study established an improved Tet-on system for powerful and stringent gene regulation in functional genetic studies.

CRISPR/Cas9-mediated targeted mutation reveals a role for AN4 rather than DPL in regulating venation formation in the corolla tube of Petunia hybrida

Venation is a common anthocyanin pattern displayed in flowers that confers important ornamental traits to plants.An anthocyanin-related R2R3-MYB transcription factor,DPL,has been proposed to regulate corolla tube venation in petunia plants.Here,however,we provide evidence redefining the role of DPL in petunia.A CRISPR/Cas9-mediated mutation of DPL resulted in the absence of the vein-associated anthocyanin pattern above the abaxial surface of the flower bud,but not corolla tube venation,thus indicating that DPL did not regulate the formation of corolla tube venation.Alternately,quantitative real-time PCR analysis demonstrated that the spatiotemporal expression pattern of another R2R3-MYB gene,AN4,coincided with the formation of corolla tube venation in petunia.Furthermore,overexpression of AN4 promoted anthocyanin accumulation by increasing the expression of anthocyanin biosynthesis genes.CRISPR/Cas9-mediated mutation of AN4 led to an absence of corolla tube venation,suggesting that this gene in fact determines this key plant trait.Taken together,the results presented here redefine the prime regulator of corolla tube venation,paving the way for further studies on the molecular mechanisms underlying the various venation patterns in petunia.

Egg tanning improves the efficiency of CRISPR/Cas9-mediated mutant locust production by enhancing defense ability after microinjection

The mutant efficiency and hatching ratio are two key factors that significantly affect the construction of genome-modified mutant insects.In the construction of CRISPR/Cas9-mediated dsLmRNase2^(–/–)mutant locusts,we found that the tanned eggs which experienced a 20-min contact with the oocyst exhibited a higher success rate compared to fresh newly-laid eggs that were less tanned.However,the heritable efficiency of the dsLmRNase2 deletion to the next generation G_(1 )progeny was similar between adults derived from the tanned or less tanned engineered eggs.Further,the similar effective mutant ratios in the normally developed eggs and G_(0) adults of tanned and less tanned eggs also indicated that tanning did not reduce the absolute mutation efficiency induced by CRISPR/Cas9.Moreover,we found that the syncytial division period,which was longer than the time for tanning,conferred a window period for microinjection treatment with efficient mutation in both tanned and less tanned eggs.We further found that tanned eggs exhibited a higher hatching rate due to a reduced infection rate following microinjection.Both the anti-pressure and ultrastructure analyses indicated that the tanned eggs contained compressed eggshells to withstand increased external pressure.In summary,tanned eggs possess stronger defense responses and higher efficiency of genome editing,providing an improved model for developing Cas9-mediated gene editing procedures in locusts.

CRISPR/Cas9-mediated mutagenesis of CiBG1 decreased seed size and promoted seed germination in watermelon

Abscisic acid(ABA)is a critical regulator of seed development and germination.β-glucosidases(BGs)have been suggested to be contributors to increased ABA content because they catalyze the hydrolysis of ABA-glucose ester to release free ABA.However,whether BGs are involved in seed development is unclear.In this study,a candidate gene,CiBG1,in watermelon was selected for targeted mutagenesis via the CRISPR/Cas9 system.Seed size and weight were significantly reduced in the Clbg1-mutant watermelon lines,which was mainly attributed to decreased cell number resulting from decreased ABA levels.A transcriptome analysis showed that the expression of 1015 and 1429 unique genes was changed 10 and 18 days after pollination(DAP),respectively.Cytoskeleton-and cell cycle-related genes were enriched in the differentially expressed genes of wild type and Clbg1-mutant lines during seed development.Moreover,the expression of genes in the major signaling pathways of seed size control was also changed.In addition,seed germination was promoted in the Cibg1-mutant lines due to decreased ABA content.These results indicate that ClBG1 may be critical for watermelon seed size regulation and germination mainly through the modulation of ABA content and thereby the transcriptional regulation of cytoskeleton-,cell cycle-and signaling-related genes.Our results lay a foundation for dissecting the molecular mechanisms of controlling watermelon seed size,a key agricultural trait of significant economic importance.

Subculturing cells have no effect on CRISPR/Cas9-mediated cleavage of UL30 gene in pseudorabies virus

CRISPR/Cas9-mediated genome editing can inhibit virus infection by targeting the conserved regions of the viral genomic DNA. Unexpectedly, we found previously that pseudorabies virus(PRV) could escape from CRISPR/Cas9-mediated inhibition.In order to elucidate whether the escape of PRV from Cas9-mediated inhibition was due to cell deficiencies, such as genetic instability of sgRNA or Cas9 protein, the positive cells were passaged ten times, and PRV infection in the sgRNA-expressing cells was evaluated in the present study. The results showed that subculturing cells has no effect on Cas9-mediated cleavage of PRV. Different passages of PX459-PRV cells can stably express sgRNA to facilitate Cas9/sgRNA cleavage on the UL30 gene of PRV, resulting in a pronounced inhibition of PRV infection. Studies to elucidate the mechanism of PRV escape are currently in progress.

Multiplex CRISPR/Cas9-mediated knockout of soybean LNK2 advances flowering time

Flowering time is an important agronomic trait for soybean yield and adaptation. However, the genetic basis of soybean adaptation to diverse latitudes is still not clear. Four NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED 2(LNK2) homeologs of Arabidopsis thaliana LNK2 were identified in soybean. Three single-guide RNAs were designed for editing the four LNK2 genes. A transgene-free homozygous quadruple mutant of the LNK2 genes was developed using the CRISPR(clustered regularly interspaced short palindromic repeats)/Cas9(CRISPR-associated protein 9). Under long-day(LD) conditions, the quadruple mutant flowered significantly earlier than the wild-type(WT). Quantitative real-time PCR(q RT-PCR)revealed that transcript levels of LNK2 were significantly lower in the quadruple mutant than in the WT under LD conditions. LNK2 promoted the expression of the legume-specific E1 gene and repressed the expression of FT2 a. Genetic markers were developed to identify LNK2 mutants for soybean breeding.These results indicate that CRISPR/Cas9-mediated targeted mutagenesis of four LNK2 genes shortens flowering time in soybean. Our findings identify novel components in flowering-time control in soybean and may be beneficial for further soybean breeding in high-latitude environments.

CRISPR/Cas9-mediated NlInR2 mutants:Analyses of residual mRNA and truncated proteins

CRISPR/Cas9 technology is a powerful genome manipulation tool in insects.However,little is known about whether mRNA and protein of a target gene are completely cleared in homozygous mutants.This study generated homozygous mutants of the insulin receptor gene 2(NlInR2)in the brown planthopper(Nilaparvata lugens)using CRISPR/Cas9 genome editing.Both frameshift mutants,E5_D17 and E6_I7,differentiated towards long wings,but there were differences in wing morphology,with E5_D17 showing wing deformities.Subsequent investigations revealed the presence of residual expression of NlInR2 mRNA in both mutants,as well as the occurrence of spliceosomes featuring exon skipping splicing in E5_D17.Additionally,the E5_D17 exhibited the detection of N-terminally truncated NlInR2 protein.RNA interference experiments indicated that the knockdown of NlInR2 expression in the E5_D17 mutant line increased the proportion of wing deformities from 11.1 to 65.6%,suggesting that the residual NlInR2 mRNA of the E5_D17 mutant might have retained some genetic functions.Our results imply that systematic characterization of residual protein expression or function in CRISPR/Cas9-generated mutant lines is necessary for phenotypic interpretation.

利用CRISPR/Cas9系统创制水稻品种GW2基因的突变体

培育具有育种价值的GW2基因编辑的水稻优异新品种在水稻育种中具有重要意义,利用CRISPR/Cas9基因编辑技术,以生产上广泛推广应用的13份水稻品种为材料,对粒质量基因(GW2)进行定向性状改良,通过农杆菌转化创制出一批无T-DNA元件的水稻非转基因GW2突变纯合株系。结果表明:13份T0代水稻转基因中,有28.0%~59.1%植株的GW2基因发生了突变,纯合突变株数量占总突变株数量的35.0%,双等位突变株数量占总突变株数量的14.2%,杂合突变株数量占总突变株数量的50.8%。此外,不同水稻品种发生的突变类型也略有不同。对13份T2代非转基因水稻GW2突变纯合株进行千粒质量性状的考种分析。与对应的野生型亲本品种相比,纯合突变水稻植株的千粒质量显著提高10.81%~58.22%。本研究结果极大地丰富了GW2的突变类型,为不同水稻品种的高产稳产创造了重要的种质资源,同时也为利用基因编辑提高水稻产量提供了有价值的育种信息。

CRISPR/Cas9技术在热带作物育种中的应用研究进展

在热带地区种植的香蕉、番木瓜、甘蔗、木薯、天然橡胶、油棕等热带作物,是我国农业的重要组成部分,不仅为我们的日常生活和工农业生产提供了重要的原材料,而且为我国热带与亚热带地区的主要农业产量和经济增长做出了贡献。然而,这些作物的现代分子育种受其生物学特性和遗传复杂性的严重阻碍,多倍化、杂合性、无性繁殖、童期长和植株高大等问题导致热带作物的传统杂交育种周期长、难度大、进展慢。基因编辑技术的发展为热带作物育种带来了新途径和新机遇。CRISPR/Cas9系统介导的基因组编辑技术以其更高的靶向效率、多功能性和易用性,已被广泛应用于植物基因组编辑育种中。近年来,该技术在香蕉、木薯、天然橡胶、甘蔗等热带作物上也实现了广泛应用。本文介绍了基于CRISPR-Cas9系统的基因组编辑、CRISPR-Cas9在热带作物改良中的应用进展以及所面临的挑战和问题,同时对热带作物基因编辑育种方面提出建议,以期为后续研究提供思路,并为进一步开发应用该技术以有效改良热带作物的植物性状提供参考。