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Gene editing by CRISPR/Cas9 in the obligatory outcrossing Medicago sativa


Main conclusion

The CRISPR/Cas9 technique was successfully used to edit the genome of the obligatory outcrossing plant species Medicago sativa L. (alfalfa).

RNA-guided genome engineering using Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/Cas9 technology enables a variety of applications in plants. Successful application and validation of the CRISPR technique in a multiplex genome, such as that of M. sativa (alfalfa) will ultimately lead to major advances in the improvement of this crop. We used CRISPR/Cas9 technique to mutate squamosa promoter binding protein like 9 (SPL9) gene in alfalfa. Because of the complex features of the alfalfa genome, we first used droplet digital PCR (ddPCR) for high-throughput screening of large populations of CRISPR-modified plants. Based on the results of genome editing rates obtained from the ddPCR screening, plants with relatively high rates were subjected to further analysis by restriction enzyme digestion/PCR amplification analyses. PCR products encompassing the respective small guided RNA target locus were then sub-cloned and sequenced to verify genome editing. In summary, we successfully applied the CRISPR/Cas9 technique to edit the SPL9 gene in a multiplex genome, providing some insights into opportunities to apply this technology in future alfalfa breeding. The overall efficiency in the polyploid alfalfa genome was lower compared to other less-complex plant genomes. Further refinement of the CRISPR technology system will thus be required for more efficient genome editing in this plant.


Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system is becoming the preferred approach for gene editing in many organisms (Wang et al. 2016). CRISPR are composed of repeated sequences intermixed with different non-repetitive sequences, and are found in many species of bacteria and archaea, where CRISPR/Cas system functions as an adaptive immune response to viral infection (Bolotin et al. 2005; Barrangou et al. 2007). A requirement of the Cas9 nuclease activity is to recognize a small motif known as the protospacer-associated motif (PAM), which is present in the foreign sequence, but not in the CRISPR locus (Marraffini and Sontheimer 2010; Anders et al. 2014; Sternberg et al. 2014). Currently, the Cas9 of Streptococcus pyogenes along with the PAM sequence “NGG” are the most commonly used in CRISPR-mediated genome editing (Dominguez et al. 2016).

CRISPR/Cas9 has been widely used in gene editing in plant systems, as evidenced by the near simultaneous publication of at least five reports in this area in 2013 (Feng et al. 2013; Li et al. 2013; Nekrasov et al. 2013; Shan et al. 2013; Xie and Yang 2013). Based on the general principles of homologous recombination and non-homologous end joining (NHEJ), CRISPR/Cas9-mediated genome editing machinery can target endogenous genes or transgenes to generate double-strand breaks (DSBs), and in the process introduce small deletions, targeted insertions and genome modifications (Fig. 1a). To optimize CRISPR/Cas9-based gene editing in plants, an improved construct was developed in which both the targeting vector and target locus are activated simultaneously via induction of double-strand breaking (DSB) during plant development (Fauser et al. 2012). The combination of both Cas9 and small guided RNA (sgRNA) in one vector allowed for improved plant transformation efficiency with the CRISPR/Cas9 construct.

Fig. 1

a Illustration of CRISPR/Cas9-induced genome editing in plant system, one mechanism is through homology recombination and the other one is through non-homology end joining. b Schematic representation of plant CRISPR/Cas9 vector used in this study for dicotyledon plants transformation. dp Cas9, dicot plants Cas9 protein. The used promoters are Ubi (ubiquitin), AtU6 and CaMV 35S promoter, which are used to drive the expression of Cas9, sgRNA, and Bar resistance gene, respectively

While designing CRISPR/Cas constructs and introducing them to plants have become routine, the bottleneck is in screening and genotyping large numbers of putative CRISPR-modified plants. The current available detection methods for gene editing include Surveyor assay (Guschin et al. 2010), subcloning of the putative-affected genomic locus (Perez et al. 2008), high-resolution melting curve (HRM) analysis (Thomas et al. 2014), and next generation sequencing (NGS) (Guell et al. 2014; Schmid-Burgk et al. 2014). Each of the aforementioned analytical detection methods has disadvantages. For instance, the sensitivity for Surveyor assay is limited (> 1%) and quantification is relatively imprecise. Subcloning and sequencing of PCR products of mutated sites is laborious, time-consuming and expensive. HRM analysis requires special qPCR equipment. NGS involving amplicon sequencing requires both specific equipment and specialized bioinformatics analysis. Recently, digital PCR was reported to be an effective method for detecting CRISPR-mediated genome editing (Miyaoka et al. 2014; Mock et al. 2015, 2016; Findlay et al. 2016). Even though gene-editing frequency (GEF)-digital PCR (-dPCR) requires access to dedicated dPCR devices, it provides high sensitivity (< 0.2%) with substantial accuracy, and it is relatively quick (results are available within 1 day) and inexpensive. All these characteristics make GEF-dPCR an effective method that could replace existing approaches for detecting CRISPR-induced genome editing, especially when screening multiplex plant genomes.

Medicago sativa L. (alfalfa) is the most productive perennial legume in the world. Besides being a valuable forage crop, alfalfa is also being contemplated as a potential bioenergy crop (Sanderson and Adler 2008). In alfalfa, miR156 plays fundamental and multifunctional roles in regulating different aspects of plant development by silencing at least seven SPL genes (SPL2, SPL3, SPL4, SPL6, SPL9, SPL12 and SPL13) (Aung et al. 2015; Gao et al. 2016; Arshad et al. 2017). Loss-of-function mutants are crucial for functional characterization of SPLs to associate each one to a specific alfalfa trait. Given the tetraploid nature of alfalfa, off-targets are usually generated by other gene silencing methods, such as RNAi and artificial miRNA silencing methods. However, advanced CRISPR technique would be expected to have a low frequency of off-targets in alfalfa, making the gene knock-down more specific.

In this report, we applied type II CRISPR/Cas9 system (Braatz et al. 2017) to generate and estimate editing in the SPL9 gene in alfalfa. Droplet digital PCR (ddPCR) was used for initial assessment of genome editing efficiency. Our results show that CRISPR/Cas9-mediated genome modifications were successfully applied in editing the tetraploid genome, but further improvements are still needed to increase the editing efficiency in alfalfa.

Materials and methods

Plant materials and growth conditions

The WT alfalfa (M. sativa L.) clone N4.4.2 (Badhan et al. 2014) was obtained from Dr. Daniel Brown (Agriculture and Agri-Food Canada, London, ON). All the alfalfa plants were grown under greenhouse conditions at 21–23 °C, 16 h light per day, light intensity of 380–450 W/m2 (approximately 500 W/m2 at high noon time), and a relative humidity of 70%. Due to the outcrossing nature of alfalfa, plants were propagated vegetatively by rooted stem cuttings to maintain the genotype throughout experiment.

sgRNA design, construction of sgRNA, Cas9 expression vector and plant transformation

To generate SPL9 CRISPR construct, the commercial CRISPR/Cas9 vector (pZG23C05) (ZGene Biotech Inc, Taibei, Taiwan) was used following the manufacturer’s protocol. The sgRNAs were designed using the web-based tool CRISPR-P (http://cbi.hzau.edu.cn/cgi-bin/CRISPR) (Lei et al. 2014). The required gene sequences were retrieved from a previously assembled alfalfa transcriptome database (Gao et al. 2016) based on the Medicago truncatula reference genome (http://medicago.jcvi.org/MTGD/?q=home) (Krishnakumar et al. 2015). According to the sgRNA rating scores generated from the web-based tool CRISPR-P (http://cbi.hzau.edu.cn/cgi-bin/CRISPR), two sgRNAs that possessed the highest scores (Online Resource S1) were selected for construct generation. Subsequently, genomic DNA fragments that span sgRNA sequences were PCR amplified to confirm the accuracy of genomic DNA sequences in alfalfa. For each target locus, a pair of DNA oligos (18–20 bp) was synthesized and annealed to generate dimers. These dimers were then ligated downstream of the sgRNA scaffolds in the plasmid vector pZG23C04 which expresses both Cas9 and sgRNA. The vector was then transferred to E. coli Top10 and plasmid DNA was extracted from positive clones and sequenced. These constructs were introduced into alfalfa according to Tian et al. (2002). Putative CRISPR transgenic plants were analyzed for genome editing by droplet digital PCR (ddPCR) (Mock et al. 2016) and subcloning of PCR products spanning the target locus followed by Sanger sequencing.

To verify the genomic DNA sequences in alfalfa plants, degenerate primers were designed based on the M. truncatula genomic sequences. Briefly, PCR products with the expected sizes were purified using a gel purification kit (Qiagen, Toronto, ON, Canada) and cloned into a pJET1.2/blunt cloning vector (Fermentas, Ottawa, ON, Canada). The positive PCR products were subjected to sequencing using a pJET1.2/blunt sequencing primer (Fermentas).

Droplet digital PCR (ddPCR)

The digital PCR was carried out following published protocols (Mock et al. 2016). Briefly, sequence-specific PCR primers and probes were designed (Table 1). The extracted plant genomic DNA was used as template for ddPCR. The assay consisted of the following components (final concentrations in 20 µl total reaction volume): ddPCR SuperMix for Probes (no dUTP) (1×), forward primer (900 nM), reverse primer (900 nM), reference probe (NHEJ-insensitive probe, HEX, 250 nM), NHEJ-sensitive probe (different fluorophore than reference; FAM, 250 nM), nuclease-free water, and ~ 40 ng gDNA was used as template. All primers and probes were designed using Primer3 plus (http://primer3plus.com) from Eurofins (Eurofins Genomics, Toronto, ON, Canada). All ddPCR assays were analyzed using the QX100 droplet reader and Quantasoft software version 1.7.4 (Bio-Rad, Hercules, CA, USA). Genome editing was calculated according to the ratio that is based on the concentrations of events per µl and it is used for the calculation of gene-editing frequency (GEF; Mock et al. 2016).

Table 1 Primers used in this study

Screening for mutations induced by CRISPR/Cas9 in SPL9

Genomic DNA of transgenic plants was extracted using the DNeasy Plant Mini Kit (Qiagen). Genomic DNA digested with either SgeI or HaeIII (Online Resource S1) was used as template in PCR amplification using PCR primers (Table 1) that were designed to amplify about a 600-bp amplicon containing the mutated SPL9 target sgRNA sequences. These fragments were used to verify CRISPR/Cas9-induced mutations in putative alfalfa SPL9 transgenic plants by Sanger sequencing.


Designing sgRNA for editing SPL9 in alfalfa

To design sgRNA for editing SPL9 gene in alfalfa, we first used CRISPR-P (http://cbi.hzau.edu.cn/cgi-bin/CRISPR) to analyze all the putative sgRNAs in SPL9 gene based on the reference genome of M. truncatula, a close relative of M. sativa, as the aforementioned CRISPR-P database did not include M. sativa genome sequences. The potential CRISPR sgRNAs in both the forward and reverse strands of the SPL9 are graphically illustrated in the specific genomic locus (Online Resource 1). For the SPL9 gene, two sgRNAs with the highest score were selected for further investigation in this study (Online Resource 1). The genomic DNA fragments that encompass the respective sgRNA sequences in both M. truncatula and M. sativa, as well as their alignment are shown in Online Resource 2. The selected sgRNAs with the highest scores were cloned into all-in-one CRISPR plasmid pZG23C05 vector (ZGene Biotech Inc.), which consists of dpCas9 under the Ubiquitin promoter, sgRNA under AtU6 promoter, and Basta gene (selectable marker) under 35S promoter (Fig. 1b). The construct was used to transform alfalfa, and a total of 96 plants containing potentially modified SPL9 genomic DNA locus were generated through tissue culture.

Droplet digital PCR for large-scale screening of transgenic alfalfa plants

Given the large number (a total of 96 plants) of putatively CRISPR-modified plants, we used GEF-dPCR method (Mock et al. 2016); for an initial screening of transgenic plants. In all the selected candidate CRISPR/Cas9-modified plants, the total events (Fig. 2a, purple colour bar) generated by ddPCR were all over 10,000, which confirms the validity of procedure as specified by the manufacture (Bio-Rad) (Fig. 2a). The blue colour bar represents the events detected with FAM probe (NHEJ-sensitive probe, FAM-positive event). Similarly, the green colour bar represents the events detected with HEX probe (NHEJ-insensitive probe, HEX-positive event) (Fig. 2a), suggesting certain genomic modifications occurred in this region. In ddPCR, the events detected using FAM probe should be smaller than those detected using HEX probe for a plant to be considered CRISPR/Cas9 modified (Mock et al. 2016). A more direct parameter to evaluate gene-editing frequency (GEF) is the concentration of the event per µl. Specifically, a smaller number represents a higher genome editing rate, as the rate from wild type (WT) is usually 1. As indicated in the data presented in Fig. 2b, the genotype M557 showed a relatively higher genome editing rate of 0.986 (1.4%). The two plants with the highest genome editing rate are MY547 (2.2%) and MY551 (2.2%) (Suppl. Table S1).

Fig. 2

Analysis of CRISPR/Cas9-modified alfalfa plants using gene-editing frequency digital PCR (GEF-dPCR). a Event generated by droplet PCR. The blue colour bar represents the events detected with FAM probe (NHEJ-sensitive probe, NHEF-affected sequences, FAM-positive event). Similarly, the green colour bar represents the events detected with HEX probe (NHEJ-insensitive probe, non-affected sequence, HEX-positive event). The purple colour bar represents the total events created and detected by droplet PCR. b GEF calculation of putative CRISPR-mediated genome editing based on the concentration of the event per µl. The smaller number represents the higher genome editing rates. Plants with relatively higher modification rates are indicated with red boxes

Screening of CRISPR-modified alfalfa plants by restriction enzyme digestion

Cas9 RNA-guided endonuclease (restriction enzyme digestion) was used to assess genome editing of SPL9 gene in transgenic alfalfa plants. Genomic DNA was first digested with HaeIII restriction enzyme, and the digested DNA was then used as a template to perform PCR amplification. As shown in Fig. 3, three representative transgenic plants (L1, L2 and L3), but not WT, showed amplification (Fig. 2c), suggesting that L1, L2 and L3 contain the edited genomic DNA sequences (DNA fragment that was undigested by HaeIII) of SPL9, and was thus amplified by PCR.

Fig. 3

Detection and investigation of putative CRISPR/Cas9-modified alfalfa plants using restriction enzyme digestion analysis. Using HaeIII-digested genomic DNA of WT and transgenic alfalfa (L1, L2 and L3) as a template, PCR amplification was carried out to screen alfalfa plants containing putative CRISPR/Cas9-mediated genomic modification

Validation of edited SPL9 locus by Sanger sequencing

To further validate the CRISPR/Cas9 editing of SPL9, the PCR products obtained using HaeIII-digested template genomic DNA were cloned into pJET1.2/blunt cloning vector. DNA was extracted from positive clones and sequenced. Relative to the WT sequence (Fig. 4, top panel), sequences of the representative mutated plants (∆1, ∆2, ∆3, ∆4, and ∆5) showed a combination of deletion and insertion/substitution mutations in SPL9 locus.

Fig. 4

Mutation detection of SPL9 genomic DNA in CRISPR/Cas9-mediated transgenic alfalfa plants. PCR products spanning the sgRNA targeting locus were sub-cloned and sequenced. The green bolded sequences represent HaeIII digestion site and the underlined ones show the PAM sequences. Deletions are indicated by dashed lines and mutations/insertions are indicated by red colour letters


The CRISPR/Cas9 technique has been used for genome editing in many important crops, including rice (Shan et al. 2013; Xu et al. 2014), wheat (Upadhyay et al. 2013), sorghum (Jiang et al. 2013), soybean (Cai et al. 2015) and maize (Liang et al. 2014; Xing et al. 2014), and thus we attempted to test the effectiveness of this genome editing technology in the tetraploid alfalfa genome. While a recent report showed that CRISPR-Cas9-mediated mutagenesis can lead to simultaneous modification of homologous genes in the polyploid genome of inbred Brassica napus (Braatz et al. 2017), this may not be pertinent in an outcrossing species such as alfalfa. Our results showed the feasibility of using CRISPR/Cas-mediated technique to modify alfalfa genome. The gene-editing frequency detected by ddPCR showed a highest rate of 2.2%, which is relatively low compared to other plant species. A modified CRISPR/Cas9 system was developed in the related species M. truncatula, which used the native Medicago U6 promoter to drive the expression of a specific sgRNA, and a total of 10.4% of transgenic plants showed the expected phenotype, indicating they were successfully edited by CRISPR (Meng et al. 2016). In our study, off-target analysis by PCR amplification and sequencing showed that the selected sgRNAs of SPL9 were very specific to the recognition site, with no off-target effects in the alfalfa genome.

The fact that alfalfa plants with silenced SPL9 have no distinct visible phenotype makes it difficult to assess the effects of the CRISPR/Cas9 mutagenesis in this case. Apart from the phenotypic evaluation, ddPCR-based estimation of concentration of the event per µl is a direct indicator of the genome editing rate. Compared to the results of M. truncatula (32 out of 309 showed symptoms) (Meng et al. 2016), it appears that the efficiency of CRISPR/Cas9-mediated editing of alfalfa genome is low. This may be due to alfalfa’s tetraploid genome with its highly repeated clusters. A report in the literature showed that a modified Cas9 enzyme could successfully mutate target genes in hairy root somatic cells of two legume species, soybean and M. truncatula with relatively higher efficiencies (Michno et al. 2015). This may be due to the tissue-specific expression analysis employed by the authors compared to the stable transformation procedure used in this study.

In the study on CRISPR/Cas9-mediated M. truncatula genome editing, 2 out of 16 albino (genome positively modified) T0 plants were monoallelic homozygous mutants and 14 were biallelic homozygous mutants (Meng et al. 2016), which is different from the results in Arabidopsis, where none of the T1 plants were homozygous for a gene modification event (Feng et al. 2014). Multigeneration analysis revealed that the CRISPR/Cas-induced gene modification could be specifically inherited to the next generations in Arabidopsis (Feng et al. 2014). It is plausible that CRISPR/Cas9-mediated alfalfa genome modification will become more efficient once optimizations are introduced, for example, using an alfalfa unique promoter to drive the expression of its specific sgRNA. In addition to the promoter optimization strategy, another crucial step is sgRNA selection, which influences both the on-target activity and off-target effects. The selection criteria, such as the secondary structure of the folded RNA and GC content, need to be evaluated specifically for increasing genome editing efficiency mediated by CRISPR/Cas9 (Doench et al. 2016). It is also critical to evaluate the quality of the selected sgRNAs using a range of calculation algorithms that are used in different software programs, instead of relying on only one.

Although the transformation of plants with CRISPR/Cas9 vector is routine, identification of CRISPR/Cas9-induced insertions/deletions in the transgenic plants is still challenging. So far, different techniques [surveyor assay, subcloning, high-resolution melting curve (HRM), NGS and ddPCR, see “Introduction”] have been suggested to screen for mutagenesis. In recent years, the more advanced NGS of amplicons was used to develop a scalable analysis pipeline to identify CRISPR/Cas9-induced mutations in hundreds of zebrafish germlines (Brocal et al. 2016). In the present study, the well-established ddPCR method was used for the first round screening of a large number of putative CRISPR/Cas9 transgenic plants. However, it cannot be ruled out that some genome editing events that are located outside the hot regions of PAM sequences may not be detected by this ddPCR technique.

In summary, this is the first report of the successful use of CRISPR/Cas9 to generate mutations in the genome of the outcrossing alfalfa plant. We showed that for identification of CRISPR/Cas9-induced insertions/deletions in alfalfa, ddPCR could be used for the initial screening of large numbers of transgenic plants, followed by the conventional endonuclease (restriction enzyme) digestion and Sanger sequencing for final confirmation and verification.

Author contribution statement

RG and AH conceived and designed the work. RG performed tissue culture and genotyping. BF and MC carried out genotyping. RG and AH analyzed the data. RG drafted the manuscript. AH secured funding and revised the manuscript.



Clustered Regularly Interspaced Short Palindromic Repeats


Droplet digital PCR


Non-homologous end joining


Protospacer-associated motif


Small guided RNA


Squamosa promoter binding protein like


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This project was funded by a Grant (J-000260) from Agriculture and Agri-Food Canada to AH. RG was the recipient of a NSERC Visiting Fellowship to a Canadian Government Laboratory.

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Correspondence to Abdelali Hannoufa.

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Gao, R., Feyissa, B.A., Croft, M. et al. Gene editing by CRISPR/Cas9 in the obligatory outcrossing Medicago sativa. Planta 247, 1043–1050 (2018). https://doi.org/10.1007/s00425-018-2866-1

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  • Alfalfa
  • CRISPR/Cas9
  • Droplet digital PCR (ddPCR)
  • Gene editing
  • Multiplex mutagenesis