A method for the production and expedient screening of CRISPR/Cas9-mediated non-transgenic mutant plants.
ABSTRACT: Developing CRISPR/Cas9-mediated non-transgenic mutants in asexually propagated perennial crop plants is challenging but highly desirable. Here, we report a highly useful method using an Agrobacterium-mediated transient CRISPR/Cas9 gene expression system to create non-transgenic mutant plants without the need for sexual segregation. We have also developed a rapid, cost-effective, and high-throughput mutant screening protocol based on Illumina sequencing followed by high-resolution melting (HRM) analysis. Using tetraploid tobacco as a model species and the phytoene desaturase (PDS) gene as a target, we successfully created and expediently identified mutant plants, which were verified as tetra-allelic mutants. We produced pds mutant shoots at a rate of 47.5% from tobacco leaf explants, without the use of antibiotic selection. Among these pds plants, 17.2% were confirmed to be non-transgenic, for an overall non-transgenic mutation rate of 8.2%. Our method is reliable and effective in creating non-transgenic mutant plants without the need to segregate out transgenes through sexual reproduction. This method should be applicable to many economically important, heterozygous, perennial crop species that are more difficult to regenerate.
Project description:Despite recent progress, the application of CRISPR/Cas9 in perennial plants still has many obstacles to overcome. Our previous results with CRISPR/Cas9 in apple and pear indicated the frequent production of phenotypic and genotypic chimeras, after editing of the <i>phytoene desaturase</i> (<i>PDS</i>) gene conferring albino phenotype. Therefore, our first objective was to determine if adding an adventitious regeneration step from leaves of the primary transgenic plants (T0) would allow a reduction in chimerism. Among hundreds of adventitious buds regenerated from a variegated T0 line, 89% were homogeneous albino. Furthermore, the analysis of the target zone sequences of twelve of these regenerated lines (RT0 for "regenerated T0" lines) indicated that 99% of the RT0 alleles were predicted to produce a truncated target protein and that 67% of RT0 plants had less heterogeneous editing profiles than the T0. Base editors are CRISPR/Cas9-derived new genome-editing tools that allow precise nucleotide substitutions without double-stranded breaks. Hence, our second goal was to demonstrate the feasibility of CRISPR/Cas9 base editing in apple and pear using two easily scorable genes: <i>acetolactate synthase</i>-<i>ALS</i> (conferring resistance to chlorsulfuron) and <i>PDS</i>. The two guide RNAs under MdU3 and MdU6 promoters were coupled into a cytidine base editor harboring a cytidine deaminase fused to a nickase Cas9. Using this vector; we induced C-to-T DNA substitutions in the target genes; leading to discrete variation in the amino-acid sequence and generating new alleles. By co-editing <i>ALS</i> and <i>PDS</i> genes; we successfully obtained chlorsulfuron resistant and albino lines in pear. Overall; our work indicates that a regeneration step can efficiently reduce the initial chimerism and could be coupled with the application of base editing to create accurate genome edits in perennial plants.
Project description:Targeted genome engineering has emerged as an alternative to classical plant breeding and transgenic methods to improve crop plants. Among other methods (zinc finger nucleases or TAL effector nucleases) the CRISPR-Cas system proved to be the most effective, convenient and least expensive method. In this study, we optimized the conditions of application of this system on apple and explored its feasibility on pear. As a proof of concept, we chose to knock-out the Phytoene Desaturase (PDS) and Terminal Flower 1 (TFL1) genes. To improve the edition efficiency, two different single guide RNAs (gRNAs) were associated to the Cas9 nuclease for each target gene. These gRNAs were placed under the control of the U3 and U6 apple promoters. Characteristic albino phenotype was obtained for 85% of the apple transgenic lines targeted in MdPDS gene. Early flowering was observed in 93% of the apple transgenic lines targeted in MdTFL1.1 gene and 9% of the pear transgenic lines targeted in PcTFL1.1. Sequencing of the target zones in apple and pear CRISPR-PDS and CRISPR-TFL1.1 transgenic lines showed that the two gRNAs induced mutations but at variable frequencies. In most cases, Cas9 nuclease cut the DNA in the twenty targeted base pairs near the protospacer adjacent motif and insertions were more frequent than deletions or substitutions. The most frequent edition profile of PDS as well as TFL1.1 genes was chimeric biallelic. Analysis of a sample of potential off-target sequences of the CRISPR-TFL1.1 construct indicated the absence of edition in cases of three mismatches. In addition, transient transformation with the CRISPR-PDS construct produced two T-DNA free edited apple lines. Our overall results indicate that, despite the frequent occurrence of chimerism, the CRISPR-Cas 9 system is a powerful and precise method to induce targeted mutagenesis in the first generation of apple and pear transgenic lines.
Project description:Although the CRISPR/Cas9 system has been widely used for crop breeding, its application for the genetic improvement of trees has been limited, partly because of the outcrossing nature and substantial genomic heterozygosity of trees. Shanxin yang (Populus davidiana × P. bolleana), is a commercially important poplar clone that is widely grown in northern China. An established transformation protocol for this interspecific hybrid enables researchers to simultaneously investigate the efficiency and specificity of the CRISPR/Cas9-mediated manipulation of a highly heterozygous genome. Using the phytoene desaturase gene (PDS) as an example, we revealed that the CRISPR/Cas9 system could efficiently edit the Shanxin yang genome. Two sgRNAs were designed and incorporated into a single binary vector containing the Cas9 expression cassette. Among 62 independent transgenic lines, 85.5% exhibited an exclusively albino phenotype, revealing the total loss of PDS function. The Illumina sequencing results confirmed the targeted mutation of PdbPDS homologs induced by CRISPR/Cas9, and small insertions/deletions were the most common mutations. Biallelic and homozygous knockout mutations were detected at both target sites of the T0 transformants. Off-target activity was detected for sgRNA2 with a frequency of 3.2%. Additionally, the SNP interference of targeting specificity was assessed based on the sequence variation among PdbPDS homologs. A single mismatch at 19- or 10-bp from the PAM was tolerated by the CRISPR/Cas9 system. Therefore, multiple homologous genes were simultaneously edited despite the presence of a mismatch between the sgRNA and the target site. The establishment of a viable CRISPR/Cas9-based strategy for editing the Shanxin yang genome will not only accelerate the breeding process, but may also be relevant for other economically or scientifically important non-model plants species.
Project description:Background:Gene editing using CRISPR/Cas9 is a simple and powerful tool for elucidating genetic controls and for crop improvement and its use has been reported in a growing number of important food crops, including recently Fragaria. In order to inform application of the technology in Fragaria, we targeted the visible endogenous marker gene PDS (phytoene desaturase) in diploid Fragaria vesca ssp. vesca 'Hawaii 4' and octoploid F.?×?ananassa 'Calypso'. Results:Agrobacterium-mediated transformation of leaf and petiole explants was used for efficient stable integration of constructs expressing plant codon-optimised Cas9 and single guide sequences under control of the Arabidopsis U6-26 consensus promoter and terminator or Fragaria vesca U6III regulatory sequences. More than 80% ('Hawaii 4') and 50% ('Calypso') putative transgenic shoot lines (multiple shoots derived from a single callus) exhibited mutant phenotypes. Of mutant shoot lines selected for molecular analysis, approximately 75% ('Hawaii 4') and 55% ('Calypso') included albino regenerants with bi-allelic target sequence variants. Our results indicate the PDS gene is functionally diploid in 'Calypso'. Conclusion:We demonstrate that CRISPR/Cas9 may be used to generate biallelic mutants at high frequency within the genomes of diploid and octoploid strawberry. The methodology, observations and comprehensive data set presented will facilitate routine application of this technology in Fragaria to single and multiple gene copy targets where mutant phenotypes cannot be identified visually.
Project description:Genome editing in plants has been boosted tremendously by the development of CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) technology. This powerful tool allows substantial improvement in plant traits in addition to those provided by classical breeding. Here, we demonstrate the development of virus resistance in cucumber (Cucumis sativus L.) using Cas9/subgenomic RNA (sgRNA) technology to disrupt the function of the recessive eIF4E (eukaryotic translation initiation factor 4E) gene. Cas9/sgRNA constructs were targeted to the N' and C' termini of the eIF4E gene. Small deletions and single nucleotide polymorphisms (SNPs) were observed in the eIF4E gene targeted sites of transformed T1 generation cucumber plants, but not in putative off-target sites. Non-transgenic heterozygous eif4e mutant plants were selected for the production of non-transgenic homozygous T3 generation plants. Homozygous T3 progeny following Cas9/sgRNA that had been targeted to both eif4e sites exhibited immunity to Cucumber vein yellowing virus (Ipomovirus) infection and resistance to the potyviruses Zucchini yellow mosaic virus and Papaya ring spot mosaic virus-W. In contrast, heterozygous mutant and non-mutant plants were highly susceptible to these viruses. For the first time, virus resistance has been developed in cucumber, non-transgenically, not visibly affecting plant development and without long-term backcrossing, via a new technology that can be expected to be applicable to a wide range of crop plants.
Project description:BACKGROUND:Genome editing tools are important for functional genomics research and biotechnology applications. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) system for gene knockout has emerged as the most effective genome-editing tool. It has previously been reported that, in rice plants, knockdown of the Os8N3 gene resulted in enhanced resistance to Xanthomonas oryzae pv. oryzae (Xoo), while displaying abnormal pollen development. RESULTS:The CRISPR/Cas9 system was employed to knockout rice Os8N3, in order to confer enhanced resistance to Xoo. Analysis of the genotypes and edited Os8N3 in T0, T1, T2, and T3 transgenic rice plants showed that the mutations were transmitted to subsequent generations, and homozygous mutants displayed significantly enhanced resistance to Xoo. Stable transmission of CRISPR/Cas9-mediated Os8N3 gene editing without the transferred DNA (T-DNA) was confirmed by segregation in the T1 generation. With respect to many investigated agronomic traits including pollen development, there was no significant difference between homozygous mutants and non-transgenic control plants under greenhouse growth conditions. CONCLUSION:Data from this study indicate that the CRISPR/Cas9-mediated Os8N3 edition can be successfully employed for non-transgenic crop improvements.
Project description:MAIN CONCLUSION:An improved CRISPR/Cas9 system with the Arabidopsis UBQ10 promoter-driven Cas9 exhibits consistently high mutation efficiency in Arabidopsis and M. truncatula. CRISPR/Cas9 is a powerful genome editing technology that has been applied in several crop species for trait improvement due to its simplicity, versatility, and specificity. However, the mutation efficiency of CRISPR/Cas9 in Arabidopsis and M. truncatula (Mt) is still challenging and inconsistent. To analyze the functionality of the CRISPR/Cas9 system in two model dicot species, four different promoter-driven Cas9 systems to target phytoene desaturase (PDS) genes were designed. Agrobacterium-mediated transformation was used for the delivery of constructed vectors to host plants. Phenotypic and genotypic analyses revealed that the Arabidopsis UBQ10 promoter-driven Cas9 significantly improves the mutation efficiency to 95% in Arabidopsis and 70% in M. truncatula. Moreover, the UBQ10-Cas9 system yielded 11% homozygous mutants in the T1 generation in Arabidopsis. Sequencing analyses of mutation events indicated that single-nucleotide insertions are the most frequent events in Arabidopsis, whereas multi-nucleotide deletions are dominant in bi-allelic and mono-allelic homozygous mutants in M. truncatula. Taken together, the UBQ10 promoter facilitates the best improvement in the CRISPR/Cas9 efficiency in PDS gene editing, followed by the EC1.2 promoter. Consistently, the improved UBQ10-Cas9 vector highly enhanced the mutation efficiency by four-fold over the commonly used 35S promoter in both dicot species.
Project description:BACKGROUND:Soybean (Glycine max) is an economically important oil and protein crop. Plant height is a key trait that significantly impacts the yield of soybean; however, research on the molecular mechanisms associated with soybean plant height is lacking. The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR-associated system 9) system is a recently developed technology for gene editing that has been utilized to edit the genomes of crop plants. RESULTS:Here, we designed four gRNAs to mutate four LATE ELONGATED HYPOCOTYL (LHY) genes in soybean. In order to test whether the gRNAs could perform properly in transgenic soybean plants, we first tested the CRISPR construct in transgenic soybean hairy roots using Agrobacterium rhizogenes strain K599. Once confirmed, we performed stable soybean transformation and obtained 19 independent transgenic soybean plants. Subsequently, we obtained one T1 transgene-free homozygous quadruple mutant of GmLHY by self-crossing. The phenotypes of the T2-generation transgene-free quadruple mutant plants were observed, and the results showed that the quadruple mutant of GmLHY displayed reduced plant height and shortened internodes. The levels of endogenous gibberellic acid (GA3) in Gmlhy1a1b2a2b was lower than in the wild type (WT), and the shortened internode phenotype could be rescued by treatment with exogenous GA3. In addition, the relative expression levels of GA metabolic pathway genes in the quadruple mutant of GmLHY were significantly decreased in comparison to the WT. These results suggest that GmLHY encodes an MYB transcription factor that affects plant height through mediating the GA pathway in soybean. We also developed genetic markers for identifying mutants for application in breeding studies. CONCLUSIONS:Our results indicate that CRISPR/Cas9-mediated targeted mutagenesis of four GmLHY genes reduces soybean plant height and shortens internodes from 20 to 35?days after emergence (DAE). These findings provide insight into the mechanisms underlying plant height regulatory networks in soybean.
Project description:Many of the recessive virus-resistance genes in plants encode eukaryotic translation initiation factors (eIFs), including eIF4E, eIF4G, and related proteins. Notably, eIF4E and its isoform eIF(iso)4E are pivotal for viral infection and act as recessive resistance genes against various potyviruses in a wide range of plants. In this study, we used Clustered Regularly Interspaced Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated targeted mutagenesis to test whether novel sequence-specific mutations at eIF4E1 in Solanum lycopersicum (tomato) cv. Micro-Tom could confer enhanced resistance to potyviruses. This approach produced heritable homozygous mutations in the transgene-free E1 generation. Sequence analysis of eIF4E1 from E0 transgenic plants expressing Cas9 and eIF4E-sgRNA transcripts identified chimeric deletions ranging from 11 to 43 bp. Genotype analysis of the eIF4E1-edited lines in E0, E1, and E2 transgenic tomato plants showed that the mutations were transmitted to subsequent generations. When homozygous mutant lines were tested for resistance to potyviruses, they exhibited no resistance to tobacco etch virus (TEV). Notably, however, several mutant lines showed no accumulation of viral particles upon infection with pepper mottle virus (PepMoV). These results indicate that site-specific mutation of tomato eIF4E1 successfully conferred enhanced resistance to PepMoV. Thus, this study demonstrates the feasibility of the use of CRISPR/Cas9 approach to accelerate breeding for trait improvement in tomato plants.
Project description:BACKGROUND:The type II clustered, regularly interspaced, short palindromic repeat (CRISPR)/ CRISPR-associated protein 9 (Cas9) system is a novel molecular tool for site-specific genome modification. The CRISPR-Cas9 system was recently introduced into plants by transient or stable transformation. FINDINGS:Here, we report gene targeting in rice via the Agrobacterium tumefaciens-mediated CRISPR-Cas9 system. Three 20-nt CRISPR RNAs were designed to pair with diverse sites followed by the protospacer adjacent motif (PAM) of the rice herbicide resistance gene BEL. After integrating the single-guide RNA (sgRNA) and Cas9 cassette in a single binary vector, transgenic rice plants harboring sgRNA:Cas9 were generated by A. tumefaciens-mediated stable transformation. By analyzing the targeting site on the genome of corresponding transgenic plants, the mutations were determined. The mutagenesis efficiency was varied from ~2% to ~16%. Furthermore, phenotypic analysis revealed that the biallelic mutated transgenic plant was sensitive to bentazon. CONCLUSIONS:Our results indicate that the agricultural trait could be purposely modified by sgRNA:Cas9-induced gene targeting. CRISPR-Cas9 system could be exploited as a powerful tool for trait improvements in crop breeding.