Please cite this article in press as: Ali et al., Efficient Virus-Mediated Genome Editing in Plants Using the CRISPR/Cas9 System, Molecular Plant (2015), http://dx.doi.org/10.1016/j.molp.2015.02.011
Molecular Plant Letter to the Editor
Efficient Virus-Mediated Genome Editing in Plants Using the CRISPR/Cas9 System Dear Editor, Targeted genome editing in plants will not only facilitate functional genomics studies but also help to discover, expand, and create novel traits of agricultural importance (Pennisi, 2010). The most widely used approach for editing plant genomes involves generating targeted double-strand DNA breaks (DSBs) and harnessing the two main DSB repair pathways: imprecise non-homologous end joining and precise homology-directed repair (Voytas, 2013). Enzymes that specifically bind the userselected genomic sequences to create DSBs can be generated de novo as synthetic bimodular proteins containing a DNAbinding module, engineered to bind a user-defined sequence, along with a DNA-cleaving module, capable of making DSBs. Several classes of nucleases have been developed with DNAbinding domains engineered to confer sequence specificity, including homing endonucleases, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs). Customization of these genome editing platforms, however, requires protein engineering, a time-consuming and laborintensive process (Puchta and Fauser, 2014). Furthermore, delivery of genome engineering reagents into plant cells is a major barrier to the effective use of these technologies for creating novel traits (Baltes et al., 2014). The recently developed CRISPR/Cas9 system has been used in diverse eukaryotes for targeted genome editing (Cong et al., 2013). This system comprises the Cas9 endonuclease of Streptococcus pyogenes and a synthetic guide RNA (gRNA), which combines the functions of CRISPR RNA (cRNA) and trans-activating cRNA (tracrRNA). The gRNA directs the Cas9 endonuclease to a target sequence complementary to 20 nucleotides preceding the protospacer-associated motif (PAM) (NGG) required for Cas9 activity. The specificity of the system, and the fact that targeting is determined by the 20-nucleotide sequence of the gRNA, allows for unprecedented, facile genome engineering. Furthermore, the CRISPR/Cas9 system is amenable to making multiple, simultaneous targeted modifications (multiplexing). Several studies have reported the applicability of the CRISPR/Cas9 system to genome editing in planta in species such as rice, Nicotiana benthamiana, and Arabidopsis (Nekrasov et al., 2013; Shan et al., 2013). In these studies, to achieve plants with heritable modifications, it was necessary to generate transgenic lines that stably expressed the Cas9 and gRNA; progeny with targeted modifications were then recovered in subsequent generations. The production of transgenics is time consuming and, therefore, efficient delivery methods are needed to expedite and maximize the usefulness of this technology for trait discovery and development. In plants, Tobacco rattle virus (TRV) is an efficient vector for virus-induced gene silencing, facilitating functional genomics
in diverse plant species. TRV has a bipartite genome, consisting of two positive-sense single-stranded RNAs, designated RNA1 and RNA2. The RNA2 genome can be modified to carry exonic gene fragments for post-transcriptional gene silencing (DineshKumar et al., 2003). TRV was also shown to have promise as a vector for genome engineering: when RNA2 was replaced with an RNA for the Zif268:FokI ZFN, targeted genome modifications were recovered in somatic tobacco and petunia cells at an integrated reporter gene (Marton et al., 2010). However, for TRV to have the most utility as a vector for genome engineering, it would be desirable if the virus infected germline cells, making it possible to harvest mutant seed from infected plants. We attempted to develop a TRV-mediated gRNA delivery system that bypasses the requirement for transformation and/or regeneration of each user-defined target sequence, amenable to multiplexing, and in which editing efficiencies and applicability across plant species would be significantly improved. To construct this virus-mediated genome editing system, we generated Cas9-overexpressing (Cas9-OE) Nicotiana benthamiana transgenic lines. First, we optimized the human codonoptimized Cas9 sequence (Supplemental Sequence 1) for in planta expression by generating the pK2GW7.Cas9 clone (Supplemental Figure 1A), and then used Agrobacterium tumefaciens to transform N. benthamiana leaf discs. We validated the Cas9 clone by Sanger sequencing, and confirmed its proper localization by transient expression of a GFP-fusion variant in N. benthamiana leaves (Supplemental Figure 1A and 1B). In addition, we analyzed the transgenic N. benthamiana plants at the molecular level to determine the expression levels of the Cas9 transcript and protein (Supplemental Figure 1C and 1D). Next, we constructed and optimized a TRV RNA2 genomederived vector (Supplemental Sequence 6) for gRNA delivery. The TRV RNA2 constructs contained the gRNA under the control of the pea early browning virus (PEBV) promoter (PEBV::gRNA) to permit the expression of the gRNA from the virus RNA-dependent RNA polymerase (Figure 1A). Subsequently, we reconstituted the TRV virus in tobacco leaves by agroinfiltration of mixed Agrobacterium cultures harboring the RNA1 genome (pYL192) (Supplemental Sequence 7) in combination with different RNA2 vectors in which a gRNA with binding specificity for the phytoene desaturase (PDS) gene (Supplemental Sequences 2 and 3) was driven by the PEBV promoter (pRNA2.PEBV::PDS.gRNA) (Figure 1A and 1B). Ten days post-infiltration, we confirmed by RT–PCR the presence of
Published by the Molecular Plant Shanghai Editorial Office in association with Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and IPPE, SIBS, CAS.
Molecular Plant --, 1–4, -- 2015 ª The Author 2015.
1
Please cite this article in press as: Ali et al., Efficient Virus-Mediated Genome Editing in Plants Using the CRISPR/Cas9 System, Molecular Plant (2015), http://dx.doi.org/10.1016/j.molp.2015.02.011
Molecular Plant
Letter to the Editor
Figure 1. TRV-Mediated Genome Editing in N. benthamiana. (A) Schematic representation of TRV RNA1 and RNA2 genome organization and modification for targeted genome editing. RNA1 in the Agrobacterium binary vector system: LB (left border), 2Xp35S (2X CaMV 35S promoter), RdRNAP (134/194 kDa RNA-dependent RNA polymerase), MP (movement protein), 16k (cysteine rich protein), Rz (self-cleaving ribozyme), Tnos (nopaline synthase terminator), RB (right border). RNA2 in the Agrobacterium binary vector system: LB, p35S, CP (coat protein), Rz, Tnos, and RB. In RNA2, the gRNA was cloned under the pea early browning virus (PEBV) promoter (pPEBV::gRNA). (B) Experimental scheme of the TRV-mediated genome editing. A 20-nucleotide target sequence preceding the PAM sequence can be cloned, in the gRNA backbone under the PEBV promoter, in the RNA2 genome. Agrobacterium cultures carrying the engineered TRV RNA2 genome, conferring userselected sequence specificity, and the RNA1 genome are co-infiltrated into leaves of N. benthamiana overexpressing Cas9 (Cas9-OE) via agroinfection. Alternatively, multiple targets can be simultaneously edited by mixing RNA2 cultures that confer sequence specificities for multiple targets with RNA1 culture. After agroinfection, plants can be analyzed for the presence of the targeted modification; plant leaf discs carrying modified genomes can be either (legend continued on next page)
2
Molecular Plant --, 1–4, -- 2015 ª The Author 2015.
Please cite this article in press as: Ali et al., Efficient Virus-Mediated Genome Editing in Plants Using the CRISPR/Cas9 System, Molecular Plant (2015), http://dx.doi.org/10.1016/j.molp.2015.02.011
Molecular Plant
Letter to the Editor the TRV RNA1 and RNA2 genomes in the inoculated as well as systemic leaves (Supplemental Figure 2). We then assayed for genomic editing of our target sequence in both inoculated and systemic leaves using the Surveyor assay with T7EI nuclease. We detected significant levels of editing in inoculated and systemic leaves (Figure 1C). In both types of leaves, editing efficiencies were higher than those reported in previous studies. Moreover, the method design included selection of a PDS genomic target containing a recognition site for the NcoI endonuclease to facilitate the modification-detection analysis (Supplemental Sequence 3). To validate the T7EI results, NcoI restriction digestion analysis was performed. Genomic DNA was extracted and treated with NcoI followed by PCR amplification of the fragment flanking our target sequence (797 bp), and the PCR amplicons were digested with NcoI to determine the presence of sequence modification. Three bands were detected, of which two corresponded to the restriction pattern of the wild-type (WT) amplicon, and one band resistant to the NcoI cleavage indicated resistance to endonuclease activity due to sequence modification (Supplemental Figure 3). Furthermore, to corroborate our T7EI and restriction-protection assays, we PCR-cloned our target sequence from inoculated and systemic leaves, and then subjected 30 of the resultant clones to Sanger sequencing (Supplemental Figure 4). The sequencing analyses confirmed the results of our T7EI and restriction-protection assays, and demonstrated that modification ratios were high: indels were present in 17 clones derived from inoculated leaves (56%) and in 9 clones from systemic leaves (30%). Sanger sequencing chromatograms of representative clones show the sequence modifications at the intended target site in inoculated and systemic leaves, respectively (Supplemental Figures 5 and 6). To further validate our system, we designed and constructed a gRNA (Supplemental Sequence 4) molecule that targets the proliferating cell nuclear antigen (PCNA) gene (Supplemental Sequence 5). Using this construct, we were able to achieve targeted modifications in the PCNA gene in both inoculated and systemic leaves, as demonstrated by T7EI assays (Supplemental Figure 7).
is established that TRV is capable of infecting germline cells (Martin-Hernandez and Baulcombe, 2008), we tested whether the progeny of plants infected with pRNA2.PEBV::PDS.gRNAs would carry genomic modifications in the PDS gene target. We screened progeny plants and recovered two lines, from early-developed flowers, carrying sequence modification at the intended target site in the PDS gene (Supplemental Figure 9). The detection of germinal transmission only in progeny seed from early flowers indicates that TRV infection and persistence in meristematic cells might be optimized to improve the recovery of mutant plants from the seed progeny. Further studies of germinal transmission are required; improvements in the method may increase the frequency of transmission and recovery of mutant plants from the seed progeny. The virus-mediated genome editing platform we describe in this study is not limited to vectors derived from TRV; other RNA viruses could also be used to deliver the gRNA molecules to various plant species. Our demonstration that TRV can serve as a vehicle to deliver genome engineering reagents to all plant parts, including meristems, provides a general method for easily recovering seeds with the desired modifications, obviating the need for transformation and/or tissue culture. Furthermore, our virus-mediated genome editing system meets several important requirements for highly efficient and multiplexed editing: (1) TRV can systematically infect a large number of plant species, both naturally and under laboratory conditions; (2) the virus is easily introduced into plants via Agrobacterium and delivery into growing points of the plant; (3) the small genome size of TRV facilitates cloning, multiplexing, library constructions, and agroinfections; and (4) the virus RNA genome does not integrate into plant genomes. In conclusion, our work expands the utility of the CRISPR/Cas9 system for plant functional genomics and agricultural biotechnology applications.
SUPPLEMENTAL INFORMATION Supplemental Information is available at Molecular Plant Online.
One of the primary advantages of virus-mediated delivery of gRNA molecules is its amenability to multiplexing. To determine whether multiplexed genome editing is feasible in our editing platform, we co-delivered gRNA molecules targeting PCNA and PDS by mixing Agrobacterium tumefaciens bacterial cultures harboring RNA2 vector clones with specificities for PDS and PCNA genes. Two weeks post-infiltration, we assayed genomic modifications in systemic leaves using the T7EI assay (Figure 1D). Sanger sequencing analysis indicated the presence of modifications at both of the intended target sites (Supplemental Figure 8), although efficiencies were lower when more than one gRNA was used simultaneously. Since it
FUNDING King Abdullah University of Science and Technology (KAUST) supported this study.
ACKNOWLEDGMENTS We would like to thank Feng Zhang for providing the Cas9 clone. The authors declare competing financial interests. Received: February 13, 2015 Revised: February 13, 2015 Accepted: February 14, 2015 Published: March 6, 2015
regenerated to recover mutant plants or the seed progeny can be screened for the presence of the modification, thereby bypassing the need for tissue culture. The virus-mediated genome editing in this report was performed in a N. benthamiana transgenic line overexpressing Cas9. (C) T7EI-based mutation detection analysis of PDS gene in inoculated and systemic leaves of N. benthamiana. Targeted mutagenesis was detected in inoculated (lane 2) and systemic (lane 3) leaves co-infiltrated with RNA1 and RNA2 carrying a pPEBV::PDS.gRNA compared with vector control (lane 1). (D) Multiplex editing of PDS and PCNA genes: DNA samples were isolated from the leaves of N. benthamiana Cas9 overexpression plants co-infiltrated with RNA2.pPEBV::PDS.gRNA, RNA2.pPEBV::PCNA.gRNA, and RNA1 cultures. PCR products of the 797-bp fragment of the PDS gene and 1098-bp fragment of the PCNA gene were subjected to T7EI mutation detection analysis. The PCNA mutagenesis is shown in lanes 3 and 4, the PDS mutagenesis in lanes 7 and 8, and vector controls in lanes 1 and 5. Mutation efficiency (as a percentage) was calculated using ImageJ software (http://imagej.nih.gov/ij/). Arrowheads indicate restriction products.
Molecular Plant --, 1–4, -- 2015 ª The Author 2015.
3
Please cite this article in press as: Ali et al., Efficient Virus-Mediated Genome Editing in Plants Using the CRISPR/Cas9 System, Molecular Plant (2015), http://dx.doi.org/10.1016/j.molp.2015.02.011
Molecular Plant
Zahir Ali1, Aala Abul-faraj1, Lixin Li1, Neha Ghosh1, Marek Piatek1, Ali Mahjoub1, Mustapha Aouida1, Agnieszka Piatek1, Nicholas J. Baltes3, Daniel F. Voytas3, Savithramma Dinesh-Kumar2 and Magdy M. Mahfouz1,* 1
Division of Biological Sciences & Center for Desert Agriculture, 4700 King Abdullah University of Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia 2 Department of Plant Biology and Genome Center, University of California, Davis, CA 95616, USA 3 Department of Genetics, Cell Biology and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA *Correspondence: Magdy M. Mahfouz ([email protected]) http://dx.doi.org/10.1016/j.molp.2015.02.011
REFERENCES Baltes, N.J., Gil-Humanes, J., Cermak, T., Atkins, P.A., and Voytas, D.F. (2014). DNA replicons for plant genome engineering. Plant Cell 26:151–163. Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang, W., Marraffini, L.A., et al. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823.
4
Molecular Plant --, 1–4, -- 2015 ª The Author 2015.
Letter to the Editor Dinesh-Kumar, S.P., Anandalakshmi, R., Marathe, R., Schiff, M., and Liu, Y. (2003). Virus-induced gene silencing. Methods Mol. Biol. 236:287–294. Martin-Hernandez, A.M., and Baulcombe, D.C. (2008). Tobacco rattle virus 16-kilodalton protein encodes a suppressor of RNA silencing that allows transient viral entry in meristems. J. Virol. 82:4064–4071. Marton, I., Zuker, A., Shklarman, E., Zeevi, V., Tovkach, A., Roffe, S., Ovadis, M., Tzfira, T., and Vainstein, A. (2010). Nontransgenic genome modification in plant cells. Plant Physiol. 154:1079–1087. Nekrasov, V., Staskawicz, B., Weigel, D., Jones, J.D., and Kamoun, S. (2013). Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat. Biotechnol. 31:691–693. Pennisi, E. (2010). Sowing the seeds for the ideal crop. Science 327: 802–803. Puchta, H., and Fauser, F. (2014). Synthetic nucleases for genome engineering in plants: prospects for a bright future. Plant J. 78: 727–741. Shan, Q., Wang, Y., Li, J., Zhang, Y., Chen, K., Liang, Z., Zhang, K., Liu, J., Xi, J.J., Qiu, J.L., et al. (2013). Targeted genome modification of crop plants using a CRISPR-Cas system. Nat. Biotechnol. 31:686–688. Voytas, D.F. (2013). Plant genome engineering with sequence-specific nucleases. Annu. Rev. Plant Biol. 64:327–350.
Molecular Plant Letter to the Editor
Efficient Virus-Mediated Genome Editing in Plants Using the CRISPR/Cas9 System Dear Editor, Targeted genome editing in plants will not only facilitate functional genomics studies but also help to discover, expand, and create novel traits of agricultural importance (Pennisi, 2010). The most widely used approach for editing plant genomes involves generating targeted double-strand DNA breaks (DSBs) and harnessing the two main DSB repair pathways: imprecise non-homologous end joining and precise homology-directed repair (Voytas, 2013). Enzymes that specifically bind the userselected genomic sequences to create DSBs can be generated de novo as synthetic bimodular proteins containing a DNAbinding module, engineered to bind a user-defined sequence, along with a DNA-cleaving module, capable of making DSBs. Several classes of nucleases have been developed with DNAbinding domains engineered to confer sequence specificity, including homing endonucleases, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs). Customization of these genome editing platforms, however, requires protein engineering, a time-consuming and laborintensive process (Puchta and Fauser, 2014). Furthermore, delivery of genome engineering reagents into plant cells is a major barrier to the effective use of these technologies for creating novel traits (Baltes et al., 2014). The recently developed CRISPR/Cas9 system has been used in diverse eukaryotes for targeted genome editing (Cong et al., 2013). This system comprises the Cas9 endonuclease of Streptococcus pyogenes and a synthetic guide RNA (gRNA), which combines the functions of CRISPR RNA (cRNA) and trans-activating cRNA (tracrRNA). The gRNA directs the Cas9 endonuclease to a target sequence complementary to 20 nucleotides preceding the protospacer-associated motif (PAM) (NGG) required for Cas9 activity. The specificity of the system, and the fact that targeting is determined by the 20-nucleotide sequence of the gRNA, allows for unprecedented, facile genome engineering. Furthermore, the CRISPR/Cas9 system is amenable to making multiple, simultaneous targeted modifications (multiplexing). Several studies have reported the applicability of the CRISPR/Cas9 system to genome editing in planta in species such as rice, Nicotiana benthamiana, and Arabidopsis (Nekrasov et al., 2013; Shan et al., 2013). In these studies, to achieve plants with heritable modifications, it was necessary to generate transgenic lines that stably expressed the Cas9 and gRNA; progeny with targeted modifications were then recovered in subsequent generations. The production of transgenics is time consuming and, therefore, efficient delivery methods are needed to expedite and maximize the usefulness of this technology for trait discovery and development. In plants, Tobacco rattle virus (TRV) is an efficient vector for virus-induced gene silencing, facilitating functional genomics
in diverse plant species. TRV has a bipartite genome, consisting of two positive-sense single-stranded RNAs, designated RNA1 and RNA2. The RNA2 genome can be modified to carry exonic gene fragments for post-transcriptional gene silencing (DineshKumar et al., 2003). TRV was also shown to have promise as a vector for genome engineering: when RNA2 was replaced with an RNA for the Zif268:FokI ZFN, targeted genome modifications were recovered in somatic tobacco and petunia cells at an integrated reporter gene (Marton et al., 2010). However, for TRV to have the most utility as a vector for genome engineering, it would be desirable if the virus infected germline cells, making it possible to harvest mutant seed from infected plants. We attempted to develop a TRV-mediated gRNA delivery system that bypasses the requirement for transformation and/or regeneration of each user-defined target sequence, amenable to multiplexing, and in which editing efficiencies and applicability across plant species would be significantly improved. To construct this virus-mediated genome editing system, we generated Cas9-overexpressing (Cas9-OE) Nicotiana benthamiana transgenic lines. First, we optimized the human codonoptimized Cas9 sequence (Supplemental Sequence 1) for in planta expression by generating the pK2GW7.Cas9 clone (Supplemental Figure 1A), and then used Agrobacterium tumefaciens to transform N. benthamiana leaf discs. We validated the Cas9 clone by Sanger sequencing, and confirmed its proper localization by transient expression of a GFP-fusion variant in N. benthamiana leaves (Supplemental Figure 1A and 1B). In addition, we analyzed the transgenic N. benthamiana plants at the molecular level to determine the expression levels of the Cas9 transcript and protein (Supplemental Figure 1C and 1D). Next, we constructed and optimized a TRV RNA2 genomederived vector (Supplemental Sequence 6) for gRNA delivery. The TRV RNA2 constructs contained the gRNA under the control of the pea early browning virus (PEBV) promoter (PEBV::gRNA) to permit the expression of the gRNA from the virus RNA-dependent RNA polymerase (Figure 1A). Subsequently, we reconstituted the TRV virus in tobacco leaves by agroinfiltration of mixed Agrobacterium cultures harboring the RNA1 genome (pYL192) (Supplemental Sequence 7) in combination with different RNA2 vectors in which a gRNA with binding specificity for the phytoene desaturase (PDS) gene (Supplemental Sequences 2 and 3) was driven by the PEBV promoter (pRNA2.PEBV::PDS.gRNA) (Figure 1A and 1B). Ten days post-infiltration, we confirmed by RT–PCR the presence of
Published by the Molecular Plant Shanghai Editorial Office in association with Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and IPPE, SIBS, CAS.
Molecular Plant --, 1–4, -- 2015 ª The Author 2015.
1
Please cite this article in press as: Ali et al., Efficient Virus-Mediated Genome Editing in Plants Using the CRISPR/Cas9 System, Molecular Plant (2015), http://dx.doi.org/10.1016/j.molp.2015.02.011
Molecular Plant
Letter to the Editor
Figure 1. TRV-Mediated Genome Editing in N. benthamiana. (A) Schematic representation of TRV RNA1 and RNA2 genome organization and modification for targeted genome editing. RNA1 in the Agrobacterium binary vector system: LB (left border), 2Xp35S (2X CaMV 35S promoter), RdRNAP (134/194 kDa RNA-dependent RNA polymerase), MP (movement protein), 16k (cysteine rich protein), Rz (self-cleaving ribozyme), Tnos (nopaline synthase terminator), RB (right border). RNA2 in the Agrobacterium binary vector system: LB, p35S, CP (coat protein), Rz, Tnos, and RB. In RNA2, the gRNA was cloned under the pea early browning virus (PEBV) promoter (pPEBV::gRNA). (B) Experimental scheme of the TRV-mediated genome editing. A 20-nucleotide target sequence preceding the PAM sequence can be cloned, in the gRNA backbone under the PEBV promoter, in the RNA2 genome. Agrobacterium cultures carrying the engineered TRV RNA2 genome, conferring userselected sequence specificity, and the RNA1 genome are co-infiltrated into leaves of N. benthamiana overexpressing Cas9 (Cas9-OE) via agroinfection. Alternatively, multiple targets can be simultaneously edited by mixing RNA2 cultures that confer sequence specificities for multiple targets with RNA1 culture. After agroinfection, plants can be analyzed for the presence of the targeted modification; plant leaf discs carrying modified genomes can be either (legend continued on next page)
2
Molecular Plant --, 1–4, -- 2015 ª The Author 2015.
Please cite this article in press as: Ali et al., Efficient Virus-Mediated Genome Editing in Plants Using the CRISPR/Cas9 System, Molecular Plant (2015), http://dx.doi.org/10.1016/j.molp.2015.02.011
Molecular Plant
Letter to the Editor the TRV RNA1 and RNA2 genomes in the inoculated as well as systemic leaves (Supplemental Figure 2). We then assayed for genomic editing of our target sequence in both inoculated and systemic leaves using the Surveyor assay with T7EI nuclease. We detected significant levels of editing in inoculated and systemic leaves (Figure 1C). In both types of leaves, editing efficiencies were higher than those reported in previous studies. Moreover, the method design included selection of a PDS genomic target containing a recognition site for the NcoI endonuclease to facilitate the modification-detection analysis (Supplemental Sequence 3). To validate the T7EI results, NcoI restriction digestion analysis was performed. Genomic DNA was extracted and treated with NcoI followed by PCR amplification of the fragment flanking our target sequence (797 bp), and the PCR amplicons were digested with NcoI to determine the presence of sequence modification. Three bands were detected, of which two corresponded to the restriction pattern of the wild-type (WT) amplicon, and one band resistant to the NcoI cleavage indicated resistance to endonuclease activity due to sequence modification (Supplemental Figure 3). Furthermore, to corroborate our T7EI and restriction-protection assays, we PCR-cloned our target sequence from inoculated and systemic leaves, and then subjected 30 of the resultant clones to Sanger sequencing (Supplemental Figure 4). The sequencing analyses confirmed the results of our T7EI and restriction-protection assays, and demonstrated that modification ratios were high: indels were present in 17 clones derived from inoculated leaves (56%) and in 9 clones from systemic leaves (30%). Sanger sequencing chromatograms of representative clones show the sequence modifications at the intended target site in inoculated and systemic leaves, respectively (Supplemental Figures 5 and 6). To further validate our system, we designed and constructed a gRNA (Supplemental Sequence 4) molecule that targets the proliferating cell nuclear antigen (PCNA) gene (Supplemental Sequence 5). Using this construct, we were able to achieve targeted modifications in the PCNA gene in both inoculated and systemic leaves, as demonstrated by T7EI assays (Supplemental Figure 7).
is established that TRV is capable of infecting germline cells (Martin-Hernandez and Baulcombe, 2008), we tested whether the progeny of plants infected with pRNA2.PEBV::PDS.gRNAs would carry genomic modifications in the PDS gene target. We screened progeny plants and recovered two lines, from early-developed flowers, carrying sequence modification at the intended target site in the PDS gene (Supplemental Figure 9). The detection of germinal transmission only in progeny seed from early flowers indicates that TRV infection and persistence in meristematic cells might be optimized to improve the recovery of mutant plants from the seed progeny. Further studies of germinal transmission are required; improvements in the method may increase the frequency of transmission and recovery of mutant plants from the seed progeny. The virus-mediated genome editing platform we describe in this study is not limited to vectors derived from TRV; other RNA viruses could also be used to deliver the gRNA molecules to various plant species. Our demonstration that TRV can serve as a vehicle to deliver genome engineering reagents to all plant parts, including meristems, provides a general method for easily recovering seeds with the desired modifications, obviating the need for transformation and/or tissue culture. Furthermore, our virus-mediated genome editing system meets several important requirements for highly efficient and multiplexed editing: (1) TRV can systematically infect a large number of plant species, both naturally and under laboratory conditions; (2) the virus is easily introduced into plants via Agrobacterium and delivery into growing points of the plant; (3) the small genome size of TRV facilitates cloning, multiplexing, library constructions, and agroinfections; and (4) the virus RNA genome does not integrate into plant genomes. In conclusion, our work expands the utility of the CRISPR/Cas9 system for plant functional genomics and agricultural biotechnology applications.
SUPPLEMENTAL INFORMATION Supplemental Information is available at Molecular Plant Online.
One of the primary advantages of virus-mediated delivery of gRNA molecules is its amenability to multiplexing. To determine whether multiplexed genome editing is feasible in our editing platform, we co-delivered gRNA molecules targeting PCNA and PDS by mixing Agrobacterium tumefaciens bacterial cultures harboring RNA2 vector clones with specificities for PDS and PCNA genes. Two weeks post-infiltration, we assayed genomic modifications in systemic leaves using the T7EI assay (Figure 1D). Sanger sequencing analysis indicated the presence of modifications at both of the intended target sites (Supplemental Figure 8), although efficiencies were lower when more than one gRNA was used simultaneously. Since it
FUNDING King Abdullah University of Science and Technology (KAUST) supported this study.
ACKNOWLEDGMENTS We would like to thank Feng Zhang for providing the Cas9 clone. The authors declare competing financial interests. Received: February 13, 2015 Revised: February 13, 2015 Accepted: February 14, 2015 Published: March 6, 2015
regenerated to recover mutant plants or the seed progeny can be screened for the presence of the modification, thereby bypassing the need for tissue culture. The virus-mediated genome editing in this report was performed in a N. benthamiana transgenic line overexpressing Cas9. (C) T7EI-based mutation detection analysis of PDS gene in inoculated and systemic leaves of N. benthamiana. Targeted mutagenesis was detected in inoculated (lane 2) and systemic (lane 3) leaves co-infiltrated with RNA1 and RNA2 carrying a pPEBV::PDS.gRNA compared with vector control (lane 1). (D) Multiplex editing of PDS and PCNA genes: DNA samples were isolated from the leaves of N. benthamiana Cas9 overexpression plants co-infiltrated with RNA2.pPEBV::PDS.gRNA, RNA2.pPEBV::PCNA.gRNA, and RNA1 cultures. PCR products of the 797-bp fragment of the PDS gene and 1098-bp fragment of the PCNA gene were subjected to T7EI mutation detection analysis. The PCNA mutagenesis is shown in lanes 3 and 4, the PDS mutagenesis in lanes 7 and 8, and vector controls in lanes 1 and 5. Mutation efficiency (as a percentage) was calculated using ImageJ software (http://imagej.nih.gov/ij/). Arrowheads indicate restriction products.
Molecular Plant --, 1–4, -- 2015 ª The Author 2015.
3
Please cite this article in press as: Ali et al., Efficient Virus-Mediated Genome Editing in Plants Using the CRISPR/Cas9 System, Molecular Plant (2015), http://dx.doi.org/10.1016/j.molp.2015.02.011
Molecular Plant
Zahir Ali1, Aala Abul-faraj1, Lixin Li1, Neha Ghosh1, Marek Piatek1, Ali Mahjoub1, Mustapha Aouida1, Agnieszka Piatek1, Nicholas J. Baltes3, Daniel F. Voytas3, Savithramma Dinesh-Kumar2 and Magdy M. Mahfouz1,* 1
Division of Biological Sciences & Center for Desert Agriculture, 4700 King Abdullah University of Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia 2 Department of Plant Biology and Genome Center, University of California, Davis, CA 95616, USA 3 Department of Genetics, Cell Biology and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA *Correspondence: Magdy M. Mahfouz ([email protected]) http://dx.doi.org/10.1016/j.molp.2015.02.011
REFERENCES Baltes, N.J., Gil-Humanes, J., Cermak, T., Atkins, P.A., and Voytas, D.F. (2014). DNA replicons for plant genome engineering. Plant Cell 26:151–163. Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang, W., Marraffini, L.A., et al. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823.
4
Molecular Plant --, 1–4, -- 2015 ª The Author 2015.
Letter to the Editor Dinesh-Kumar, S.P., Anandalakshmi, R., Marathe, R., Schiff, M., and Liu, Y. (2003). Virus-induced gene silencing. Methods Mol. Biol. 236:287–294. Martin-Hernandez, A.M., and Baulcombe, D.C. (2008). Tobacco rattle virus 16-kilodalton protein encodes a suppressor of RNA silencing that allows transient viral entry in meristems. J. Virol. 82:4064–4071. Marton, I., Zuker, A., Shklarman, E., Zeevi, V., Tovkach, A., Roffe, S., Ovadis, M., Tzfira, T., and Vainstein, A. (2010). Nontransgenic genome modification in plant cells. Plant Physiol. 154:1079–1087. Nekrasov, V., Staskawicz, B., Weigel, D., Jones, J.D., and Kamoun, S. (2013). Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat. Biotechnol. 31:691–693. Pennisi, E. (2010). Sowing the seeds for the ideal crop. Science 327: 802–803. Puchta, H., and Fauser, F. (2014). Synthetic nucleases for genome engineering in plants: prospects for a bright future. Plant J. 78: 727–741. Shan, Q., Wang, Y., Li, J., Zhang, Y., Chen, K., Liang, Z., Zhang, K., Liu, J., Xi, J.J., Qiu, J.L., et al. (2013). Targeted genome modification of crop plants using a CRISPR-Cas system. Nat. Biotechnol. 31:686–688. Voytas, D.F. (2013). Plant genome engineering with sequence-specific nucleases. Annu. Rev. Plant Biol. 64:327–350.
