Tomato Spotted Wilt Virus-Based CRISPR/Cas Delivery Systems For Targeted Genome Editing In Multiple Crops

CRISPR/Cas genome-editing tools provide unprecedented opportunities for basic plant biology research and crop breeding.However,the lack of robust delivery methods and the technical difficulties in plant genetic transformation have limited the widespread adoption of these revolutionary technologies in plant science.While plant delivery of CRISPR/Cas nucleases usually relies on transgenic methods,but in recent years,plant viral vectors,especially RNA viral vectors,provide a promising approach for non-transgenic delivery of nucleases.Negative-stranded RNA viruses exhibit great potential as delivery vectors due to their huge cargo capacity,great genetic stability,and no risk of viral genome integration.In this study,we describe the development of a viral delivery system based on tomato spotted wilt virus(TSWV),a negative-stranded RNA bunyavirus with an extraordinarily large host range.We have generated biocontainable vectors for confined use in the laboratory and demonstrated stable,efficient delivery of entire CRISPR/Cas cassettes in various host species and genotypes.The main results of this study are summarized as follows:1)Establishment of an efficient CRISPR/Cas nuclease delivery system based on biocontainable TSWV vectorwe generated TSWV infectious c DNA clones,and substituted the GP gene encoding the Glycoprotein precursor in the TSWV M genome and NSs gene encoding the Non-structural protein in the S genome with the red fluorescent protein(RFP)gene and the green fluorescent protein(GFP)gene,respectively;meanwhile,we synthesized the full-length L complementary DNA(c DNA)containing the RNA-dependent RNA polymerase(Rd Rp)coding sequence optimized for tobacco codon usages and removed the potential transcription cleavage sites therein to facilitate recombinant viral rescue.After agroinfiltrationmediated delivery of the p L,p M-and p S-derived constructs into Nicotiana benthamiana leaves,recombinant viruses expressing two fluorescent markers could be isolated from 100% of the infiltrated plants.On this basis,the RFP gene was replaced by Cas9 or Cas12a(Cpf1)gene,and their corresponding CRISPR guide RNAs(cr RNA or sg RNA,collectively denoted g RNA)were inserted upstream of the GFP start codon in the S genome,so as to construct a TSWV vector which can express CRISPR/Cas nuclease components and can be tracked virus infection by a visual GFP marker.The expression of GFP,as well as Cas9/Cas12 a proteins,were verified in the N.benthamiana tissues infected with the TSWV vector,and efficient gene editing were produced at the corresponding targets.When multiple tandem copies of sg RNA or cr RNA were inserted into the TSWV vectors,the virus delivery systems yielded multiplexed editing in systemically infected leaf tissues at frequencies comparable to those induced by their respective single-g RNA vectors.Further studies confirmed that the recombinant viruses with the GP and NSs genes replaced could not produce enveloped virions,and systematically infected plants in the form of naked nucleocapsids.More importantly,the recombinant virus vector lacking glycoprotein could not be transmitted by the vector insect Frankliniella occidentalis,and thus had high environmental security.After eight successive passages of recombinant virus vector through mechanical transmission in N.benthamiana,the foreign inserts were maintained stably in progeny virus genomes,and the infectivity and gene editing efficiency did not change significantly,which indicated that the vector had excellent cargo carrying capacity and genome stability.2)TSWV-based CRISPR/Cas delivery systems for genome editing in multiple crop species and genotypesUnder laboratory conditions,TSWV is readily transmitted by mechanical inoculation.We agroinoculated N.benthamiana to recover various TSWV-based vectors in leaf saps,which were then used as inoculum to mechanically inoculate various crop plants including tobacco(Nicotiana tabacum),tomato(Solanum lycopersicum),Habanero pepper(Capsicum chinense),sweet pepper(C.annuum),ground cherry(Physalis alkekengi;also known as Chinese lantern),and peanut(Arachis hypogaea)plants.The recombinant viruses systematically infected these crop hosts,and their CRISPR/Cas nuclease produced effective editing at the corresponding target,with editing efficiencies of 51%-79% depending on the specific target.Furthermore,10 different varieties each of sweet pepper and peanut were inoculated with the corresponding TSWV vector,and it was found that the editing vectors could infect each variety effectively,and the nucleases delivered by them yielded similar target gene editing frequencies,which demonstrate that developed TSWV-based vectors are broadly applicable to diverse crop hosts for somatic genome editing in a genotype-independent manner.3)The regeneration of virus-free,edited plants and analysis of the transmission pattern of viral vector-induced mutationsTSWV is excluded from the host meristem or germline cells,the somatic editing produced on infected plants can’t be directly transmitted to progeny.To recover heritable mutations,N.benthamiana,tobacco and tomato young leaves infected with recombinant TSWV vectors were used as explants for tissue culture in an induction medium without any selection.Moreover,different antiviral agents were added into the shoot induction media to test their effects in promoting virus removal.Ribavirin treatment eliminated the virus from100% of the regenerated plants and significantly increased the proportion of regenerated plants with double allelic/ homozygous mutations in N.benthamiana,tobacco and tomato.The virusremoved regenerated plants developed normally and were completely fertile,whereas the regenerated plants without virus removal exhibited severe virus symptoms and did not produce seeds or produced only a few seeds.To investigate the heritability and inheritance pattern of the mutations in regenerant plants,taking 19 regenerated plant lines(M0 generation)containing PDS gene editing as examples,the genotypes and phenotypes of their self-pollination progeny(M1 generation)were analyzed.Genotype determination of M1 offspring showed that all of the heterozygous and bi-allelic/homozygous mutations were stably transmitted to the M1 generation,nevertheless,chimeric mutations could not be inherited.Further statistical analysis of the phenotypic segregation ratios also confirmed that transmission of the heterozygous and bi-allelic mutations followed classical Mendelian laws.In summary,this study reports a CRISPR/Cas non-transgenic delivery system based on TSWV,which has the advantages of simplicity,stability,high efficiency,broad-spectrum,safety and controllability.Based on a wide range of hosts and genotype-independent editing manners,TSWV delivery system should provide a promising tool to overcome gene delivery bottlenecks for genome-editing various crop species and elite varieties with reasonable regeneration ability.