This article was downloaded by: [New York University] On: 05 June 2015, At: 01:40 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Plant Signaling & Behavior Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kpsb20
Activity and Specificity of TRV-Mediated Gene Editing in Plants a
a
a
Zahir Ali , Aala Abul-faraj , Marek Piatek & Magdy M. Mahfouz
a
a
Laboratory for Genome Engineering, Division of Biological Sciences & Center for Desert Agriculture, 4700 King Abdullah University of Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia Accepted author version posted online: 03 Jun 2015.
Click for updates To cite this article: Zahir Ali, Aala Abul-faraj, Marek Piatek & Magdy M. Mahfouz (2015): Activity and Specificity of TRVMediated Gene Editing in Plants, Plant Signaling & Behavior, DOI: 10.1080/15592324.2015.1044191 To link to this article: http://dx.doi.org/10.1080/15592324.2015.1044191
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Short Communication
Activity and Specificity of TRV-Mediated Gene Editing in Plants Zahir Ali1, Aala Abul-faraj1, Marek Piatek1, and Magdy M. Mahfouz1, * 1
Laboratory for Genome Engineering, Division of Biological Sciences & Center for Desert Agriculture, 4700
King Abdullah University of Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia Key words Plant genome engineering, CRISPR/Cas9 system, synthetic site-specific nucleases (SSNs), viral-mediated genome editing, TRV *
Downloaded by [New York University] at 01:40 05 June 2015
Correspondence to: Magdy M. Mahfouz, Email: [email protected]
Abstract Plant trait engineering requires efficient targeted genome-editing technologies. Clustered regularly interspaced palindromic repeats (CRISPRs)/ CRISPR associated (Cas) type II system is used for targeted genome-editing applications across eukaryotic species including plants. Delivery of genome engineering reagents and recovery of mutants remain challenging tasks for in planta applications. Recently, we reported the development of Tobacco rattle virus (TRV)-mediated genome editing in Nicotiana benthamiana. TRV infects the growing points and possesses small genome size; which facilitate cloning, multiplexing, and agroinfections. Here, we report on the persistent activity and specificity of the TRV-mediated CRISPR/Cas9 system for targeted modification of the Nicotiana benthamiana genome. Our data reveal the persistence of the TRV- mediated Cas9 activity for up to 30 days post-agroinefection. Further, our data indicate that TRV-mediated genome editing exhibited no offtarget activities at potential off-targets indicating the precision of the system for plant genome engineering. Taken together, our data establish the feasibility and exciting possibilities of using virus-mediated CRISPR/Cas9 for targeted engineering of plant genomes.
1
TEXT Efficient technologies for targeted engineering of plant genomes are highly needed to discover and develop novel traits in key plant species important for food security. Site-Specific nucleases (SSNs) have been used to generate targeted double strand breaks (DSBs) and harnessing the DNA repair machinery of the imprecise nonhomologous end-joining (NHEJ) and precise homology-directed repair (HDR).1-4 Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have been used for targeted editing of plant genomes.1,5-7 Customization of ZFNs and TALENs require protein engineering for each user-selected targeted, a resource intensive and time-consuming process.8 Different repeat assembly protocols have been developed for TALENs engineering but the requirement of using two TALENs monomer to bind to the sense and antisense
Downloaded by [New York University] at 01:40 05 June 2015
strands simultaneously complicates its application in plants.3 Recently, bacterial and archaeal natural immunity system that targets and destroys invading nucleic acids has been adapted for genome engineering across eukaryotic species. The clustered regularly interspaced palindromic repeat (CRISPR)/ CRISPR associated (Cas) 9 system has been used in diverse plant species such as rice, Nicotiana benthamiana, and Arabidopsis for targeted genome editing.9-11 The CRISPR/Cas9 system is comprised of the Cas9 endonuclease of Streptococcus pyogenes and a synthetic guide RNA (gRNA), which combines functions of CRISPR RNA (cRNA) and transactivating cRNA (tracrRNA) to direct the Cas9 protein to the DNA target sequence preceding the protospacerassociated motif (PAM) (NGG).12,13 Because the specificity of the system is determined by the 20-nucleotide sequence of the gRNA, it allows for unprecedented and facile genome engineering. Further, the CRISPR/Cas9 system is used to simultaneously edit multiple genomic targets.12 Delivery of genome engineering reagents into plant cells is a major barrier for efficient and effective use of these technologies for targeted improvement of crop traits. Tobacco rattle virus (TRV) is used as an efficient vector for virus-induced gene silencing (VIGS) for functional genomics studies in diverse plant species.14,15 TRV has a bipartite RNA1 and RNA2 genomes, and the RNA2 genome can be modified to carry exonic gene fragments for post-transcriptional VIGS.16 The cargo capacity of TRV is limited to 2-3 kb, and cannot be used to deliver Cas9 endonuclease into plants but it can be used to deliver one or more gRNAs. Recently, we developed TRV as vector to systemically deliver gRNA and demonstrated high efficiency of targeted modification in Nicotiana benthamiana.17 Here, we report on the activity and specificity of the TRV-mediated CRISPR/Cas9 system for targeted genome editing. To determine whether the TRV was capable of producing and delivering the gRNA molecules systemically and the persistence of genomic modification in growing 2
tissues, the TRV virus was delivered by agroinfiltration and reconstituted in the leaves of N.benthamiana expressing Cas9. This was performed using mixed Agrobacterium cultures harboring the RNA1 genome (pYL192) in combination with an RNA2 vector, in which a gRNA with binding specificity for the PDS gene was driven by the PEBV promoter (pRNA2.PEBV::PDS.gRNA) (Fig 1A). A gRNA empty vector clone was used as the negative control. Samples were collected at 7, 15 and 30 days post-infiltration (dpi), and targeted editing of the PDS target sequence was assessed in three independent plants by PCR amplifying a 797 bp fragment flanking the target site. Then, PCR products were directly subjected to the T7EI assay. Our results showed high levels of genome editing at 7, 15 and 30 days (Fig. 1B). The modification efficiency was analyzed using ImageJ software as described previously (http://imageJ.nih.gov/ij/).17 The high efficiency of TRV-mediated CRISPR/Cas9
Downloaded by [New York University] at 01:40 05 June 2015
editing indicated a persistent activity of the system, which is important for the modification of germline cells and recovery of mutant seed progeny. Since TRV is capable of infecting whole plant parts including meristematic tissues, we tested whether the seed progeny of plants infected with pRNA2.PEBV::PDS.gRNAs would carry genomic modifications in the PDS gene target.18 Three early-matured seed capsules next to the infiltrated leaves were collected as one pool . Small leaf discs from 50 progeny plants of each pool were collected in one tube as one pool for a total of four pools including a WT control. The corresponding 404 bp DNA PCR fragments were subjected to NcoI restriction digestion to assess for targeted sequence modification in these pools. An NcoI enzyme-resistant band (404 bp) appeared only in pools derived from plants infected with the pRNA2:pEBV::gRNA.PDS construct compared to the WT plants, indicating the presence of genomic modification in seed progeny (Fig. 1C). To confirm these data, the PCR product was cloned into a topo cloning vector and the resultant clones were subjected to Sanger sequencing, which confirmed the presence of modification at the intended target site (Fig. 1D). These data indicate the feasibility of recovering plants carrying the targeted modification, albeit at very low efficiency, and bypassing the need for transformation and tissue culture (Fig.1D). The detection of germinal transmission only in early flowers indicates that TRV infection and persistence in meristematic cells need to be optimized to improve the recovery of mutated plants from the seed progeny. We did not recover plants carrying the targeted mutagenesis from capsules produced later in development. Further improvements might increase the frequency of germinal transmission and recovery of mutant plants from the seed progeny.17,18 A primary concern in applications of CRISPR/Cas9 genome editing is off-target activities.19,20 Although this issue is less important in plant than in human applications, we attempted to determine whether our system 3
exhibited off-target activities. To identify candidate unintended targets of genome editing, the draft genome of N. benthamiana was screened for imperfect matches (i.e., allowing several mismatches) to the 20-nucleotide gRNA sequence (Figure 2A), and 13 candidates were then subjected to T7EI and restriction-protection assays. Consistent with previous reports, no genomic modifications were detected at any of the predicted unintended targets (Fig. 2B).4,10,21 Therefore, we concluded that either our system exhibits no off-target activities, or that any such activities occurred at levels too low to be detected by the modification-detection assays used. In conclusion, our work demonstrates the persistence of TRV-mediated CRISPR/Cas9 editing and the possibility of optimizing this method to recover progeny plants carrying the targeted modifications thereby bypassing the need for tissue culture or repeated transformation. This method will expand the utility of the
Downloaded by [New York University] at 01:40 05 June 2015
CRISPR/Cas9 system for plant functional genomics and targeted improvements of crop traits. Further, the use of heterozygous Cas9 overexpressing plants with this facile and versatile genome-editing platform allows the engineering and production of plants free of foreign DNA. This might overcome the regulatory hurdles that impede the commercialization of engineered plants. COMPETING FINANCIAL INTERESTS The authors declare competing financial interests. ACKNOWLEDGMENTS We would like to thank members of the laboratory for genome engineering at KAUST for helpful discussions and comments. This study is supported by King Abdullah University of Science and Technology (KAUST).
4
Downloaded by [New York University] at 01:40 05 June 2015
References
1.
Voytas, D.F. Plant genome engineering with sequence-specific nucleases. Annual review of plant biology 64, 327-350 (2013).
2.
Puchta, H. & Fauser, F. Synthetic nucleases for genome engineering in plants: prospects for a bright future. The Plant journal : for cell and molecular biology 78, 727-741 (2014).
3.
Mahfouz, M.M., Piatek, A. & Stewart, C.N., Jr. Genome engineering via TALENs and CRISPR/Cas9 systems: challenges and perspectives. Plant biotechnology journal 12, 1006-1014 (2014).
4.
Zhang, H., et al. The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation. Plant biotechnology journal 12, 797807 (2014).
5.
Shukla, V.K., et al. Precise genome modification in the crop species Zea mays using zinc-finger nucleases. Nature 459, 437-441 (2009).
6.
Townsend, J.A., et al. High-frequency modification of plant genes using engineered zinc-finger nucleases. Nature 459, 442-445 (2009).
7.
Li, L., et al. Characterization and DNA-binding specificities of Ralstonia TAL-like effectors. Molecular plant 6, 1318-1330 (2013).
8.
Aouida, M., Piatek, M.J., Bangarusamy, D.K. & Mahfouz, M.M. Activities and specificities of homodimeric TALENs in Saccharomyces cerevisiae. Current genetics 60, 61-74 (2014).
9.
Fauser, F., Schiml, S. & Puchta, H. Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana. The Plant journal : for cell and molecular biology 79, 348-359 (2014).
10.
Nekrasov, V., Staskawicz, B., Weigel, D., Jones, J.D. & Kamoun, S. Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nature biotechnology 31, 691-693 (2013).
11.
Shan, Q., et al. Targeted genome modification of crop plants using a CRISPR-Cas system. Nature biotechnology 31, 686-688 (2013).
12.
Cong, L., et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819-823 (2013).
13.
Piatek, A., et al. RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors. Plant biotechnology journal (2014).
14.
Liu, Y., Schiff, M. & Dinesh-Kumar, S.P. Virus-induced gene silencing in tomato. The Plant journal : for cell and molecular biology 31, 777-786 (2002).
15.
Macfarlane, S.A. Tobraviruses--plant pathogens and tools for biotechnology. Molecular plant pathology 11, 577-583 (2010).
16.
Senthil-Kumar, M. & Mysore, K.S. Tobacco rattle virus-based virus-induced gene silencing in Nicotiana benthamiana. Nature protocols 9, 1549-1562 (2014). 5
Ali, Z., et al. Efficient Virus-Mediated Genome Editing in Plants using the CRISPR/Cas9 System. Molecular plant (2015).
18.
Martin-Hernandez, A.M. & Baulcombe, D.C. Tobacco rattle virus 16-kilodalton protein encodes a suppressor of RNA silencing that allows transient viral entry in meristems. Journal of virology 82, 4064-4071 (2008).
19.
Kuscu, C., Arslan, S., Singh, R., Thorpe, J. & Adli, M. Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. Nature biotechnology 32, 677-683 (2014).
20.
Wu, X., et al. Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nature biotechnology 32, 670-676 (2014).
21.
Feng, Z., et al. Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America 111, 4632-4637 (2014).
Downloaded by [New York University] at 01:40 05 June 2015
17.
6
Downloaded by [New York University] at 01:40 05 June 2015
Figure 1 Persistence of TRV-mediated CRISPR/Cas9 targeted mutagenesis of the PDS3 gene. (A) Establishment of TRV infection in B14 plants, Nicotiana benthamiana Cas9 overexpression line. Agrobacterium cultures containing RNA1 and engineered RNA2 harboring gRNA for targeting PDS gene were mixed 1: 1 (OD600 0.1 each) and co-infiltrated to two fully expanded true leaves. Systemic leaves were collected 7, 15 and 30 days post-infiltration (dpi). (B) T7EI assay for indels detection. Genomic DNA was extracted from the systemic leaves and purified PCR product (200ng) of PDS fragment flanking the targeted locus was subjected to T7EI analysis. All three leaves collected at different time points showed high efficiency of targeted mutagenesis (43– 61 %) compared to vector control systemic leaves collected at 30 dpi. (C) Analysis of progeny plants for the presence of targeted modification using NcoI recognition site loss assay. DNA was extracted from progeny plants (50 seedlings pooled in one tube) and PCR was performed with a primer set to amplify 404 bp fragment encompassing the target site. Purified PCR product (300 ng) was treated with NcoI and separated on 2% agarose gel. Progeny pool 1 and 2 clearly showed a resistant DNA fragment of 404 bp indicating the targeted mutagenesis. (D) Alignment of Sanger sequencing reads showing the presence of indels at the PDS
7
target sequence. Numbers to the right of sequence alignment indicates the number of nucleotides deleted by
Downloaded by [New York University] at 01:40 05 June 2015
targeting the PDS genomic target.
8
Downloaded by [New York University] at 01:40 05 June 2015
Figure 2 TRV-based CRISPR/Cas9 system exhibited no apparent off-target effects in N. benthamiana genome. (A) Table showing combinatorics approach to identify potential off-target binding of PDS3 gRNA in N. benthamiana genome. The putative off-target binding sites were subjected to further annotation, where sequences were split into two groups of conserved and not conserved NcoI restriction site directly preceding the PAM sequence and sites containing mutation in seed and non-seed sequence. Based on dissimilarities of 1 to 7 nucleotides with PDS3 gRNA, a total of 4265 potential off targets were detected, out of which 375 have NcoI site proximal to PAM sequence. (B) T7EI assay for the presence of indels at potential off target sites. DNA fragment flanking potential 13 targets were amplified by PCR with their respective primers. TRV-mediated
9
CRISPR/Cas9 system exhibited no detectable activities at all tested targets using the T7EI assays. The respective
Downloaded by [New York University] at 01:40 05 June 2015
contig number of each of 13 potential off target site are represented on the top of gel
10
Plant Signaling & Behavior Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kpsb20
Activity and Specificity of TRV-Mediated Gene Editing in Plants a
a
a
Zahir Ali , Aala Abul-faraj , Marek Piatek & Magdy M. Mahfouz
a
a
Laboratory for Genome Engineering, Division of Biological Sciences & Center for Desert Agriculture, 4700 King Abdullah University of Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia Accepted author version posted online: 03 Jun 2015.
Click for updates To cite this article: Zahir Ali, Aala Abul-faraj, Marek Piatek & Magdy M. Mahfouz (2015): Activity and Specificity of TRVMediated Gene Editing in Plants, Plant Signaling & Behavior, DOI: 10.1080/15592324.2015.1044191 To link to this article: http://dx.doi.org/10.1080/15592324.2015.1044191
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Short Communication
Activity and Specificity of TRV-Mediated Gene Editing in Plants Zahir Ali1, Aala Abul-faraj1, Marek Piatek1, and Magdy M. Mahfouz1, * 1
Laboratory for Genome Engineering, Division of Biological Sciences & Center for Desert Agriculture, 4700
King Abdullah University of Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia Key words Plant genome engineering, CRISPR/Cas9 system, synthetic site-specific nucleases (SSNs), viral-mediated genome editing, TRV *
Downloaded by [New York University] at 01:40 05 June 2015
Correspondence to: Magdy M. Mahfouz, Email: [email protected]
Abstract Plant trait engineering requires efficient targeted genome-editing technologies. Clustered regularly interspaced palindromic repeats (CRISPRs)/ CRISPR associated (Cas) type II system is used for targeted genome-editing applications across eukaryotic species including plants. Delivery of genome engineering reagents and recovery of mutants remain challenging tasks for in planta applications. Recently, we reported the development of Tobacco rattle virus (TRV)-mediated genome editing in Nicotiana benthamiana. TRV infects the growing points and possesses small genome size; which facilitate cloning, multiplexing, and agroinfections. Here, we report on the persistent activity and specificity of the TRV-mediated CRISPR/Cas9 system for targeted modification of the Nicotiana benthamiana genome. Our data reveal the persistence of the TRV- mediated Cas9 activity for up to 30 days post-agroinefection. Further, our data indicate that TRV-mediated genome editing exhibited no offtarget activities at potential off-targets indicating the precision of the system for plant genome engineering. Taken together, our data establish the feasibility and exciting possibilities of using virus-mediated CRISPR/Cas9 for targeted engineering of plant genomes.
1
TEXT Efficient technologies for targeted engineering of plant genomes are highly needed to discover and develop novel traits in key plant species important for food security. Site-Specific nucleases (SSNs) have been used to generate targeted double strand breaks (DSBs) and harnessing the DNA repair machinery of the imprecise nonhomologous end-joining (NHEJ) and precise homology-directed repair (HDR).1-4 Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have been used for targeted editing of plant genomes.1,5-7 Customization of ZFNs and TALENs require protein engineering for each user-selected targeted, a resource intensive and time-consuming process.8 Different repeat assembly protocols have been developed for TALENs engineering but the requirement of using two TALENs monomer to bind to the sense and antisense
Downloaded by [New York University] at 01:40 05 June 2015
strands simultaneously complicates its application in plants.3 Recently, bacterial and archaeal natural immunity system that targets and destroys invading nucleic acids has been adapted for genome engineering across eukaryotic species. The clustered regularly interspaced palindromic repeat (CRISPR)/ CRISPR associated (Cas) 9 system has been used in diverse plant species such as rice, Nicotiana benthamiana, and Arabidopsis for targeted genome editing.9-11 The CRISPR/Cas9 system is comprised of the Cas9 endonuclease of Streptococcus pyogenes and a synthetic guide RNA (gRNA), which combines functions of CRISPR RNA (cRNA) and transactivating cRNA (tracrRNA) to direct the Cas9 protein to the DNA target sequence preceding the protospacerassociated motif (PAM) (NGG).12,13 Because the specificity of the system is determined by the 20-nucleotide sequence of the gRNA, it allows for unprecedented and facile genome engineering. Further, the CRISPR/Cas9 system is used to simultaneously edit multiple genomic targets.12 Delivery of genome engineering reagents into plant cells is a major barrier for efficient and effective use of these technologies for targeted improvement of crop traits. Tobacco rattle virus (TRV) is used as an efficient vector for virus-induced gene silencing (VIGS) for functional genomics studies in diverse plant species.14,15 TRV has a bipartite RNA1 and RNA2 genomes, and the RNA2 genome can be modified to carry exonic gene fragments for post-transcriptional VIGS.16 The cargo capacity of TRV is limited to 2-3 kb, and cannot be used to deliver Cas9 endonuclease into plants but it can be used to deliver one or more gRNAs. Recently, we developed TRV as vector to systemically deliver gRNA and demonstrated high efficiency of targeted modification in Nicotiana benthamiana.17 Here, we report on the activity and specificity of the TRV-mediated CRISPR/Cas9 system for targeted genome editing. To determine whether the TRV was capable of producing and delivering the gRNA molecules systemically and the persistence of genomic modification in growing 2
tissues, the TRV virus was delivered by agroinfiltration and reconstituted in the leaves of N.benthamiana expressing Cas9. This was performed using mixed Agrobacterium cultures harboring the RNA1 genome (pYL192) in combination with an RNA2 vector, in which a gRNA with binding specificity for the PDS gene was driven by the PEBV promoter (pRNA2.PEBV::PDS.gRNA) (Fig 1A). A gRNA empty vector clone was used as the negative control. Samples were collected at 7, 15 and 30 days post-infiltration (dpi), and targeted editing of the PDS target sequence was assessed in three independent plants by PCR amplifying a 797 bp fragment flanking the target site. Then, PCR products were directly subjected to the T7EI assay. Our results showed high levels of genome editing at 7, 15 and 30 days (Fig. 1B). The modification efficiency was analyzed using ImageJ software as described previously (http://imageJ.nih.gov/ij/).17 The high efficiency of TRV-mediated CRISPR/Cas9
Downloaded by [New York University] at 01:40 05 June 2015
editing indicated a persistent activity of the system, which is important for the modification of germline cells and recovery of mutant seed progeny. Since TRV is capable of infecting whole plant parts including meristematic tissues, we tested whether the seed progeny of plants infected with pRNA2.PEBV::PDS.gRNAs would carry genomic modifications in the PDS gene target.18 Three early-matured seed capsules next to the infiltrated leaves were collected as one pool . Small leaf discs from 50 progeny plants of each pool were collected in one tube as one pool for a total of four pools including a WT control. The corresponding 404 bp DNA PCR fragments were subjected to NcoI restriction digestion to assess for targeted sequence modification in these pools. An NcoI enzyme-resistant band (404 bp) appeared only in pools derived from plants infected with the pRNA2:pEBV::gRNA.PDS construct compared to the WT plants, indicating the presence of genomic modification in seed progeny (Fig. 1C). To confirm these data, the PCR product was cloned into a topo cloning vector and the resultant clones were subjected to Sanger sequencing, which confirmed the presence of modification at the intended target site (Fig. 1D). These data indicate the feasibility of recovering plants carrying the targeted modification, albeit at very low efficiency, and bypassing the need for transformation and tissue culture (Fig.1D). The detection of germinal transmission only in early flowers indicates that TRV infection and persistence in meristematic cells need to be optimized to improve the recovery of mutated plants from the seed progeny. We did not recover plants carrying the targeted mutagenesis from capsules produced later in development. Further improvements might increase the frequency of germinal transmission and recovery of mutant plants from the seed progeny.17,18 A primary concern in applications of CRISPR/Cas9 genome editing is off-target activities.19,20 Although this issue is less important in plant than in human applications, we attempted to determine whether our system 3
exhibited off-target activities. To identify candidate unintended targets of genome editing, the draft genome of N. benthamiana was screened for imperfect matches (i.e., allowing several mismatches) to the 20-nucleotide gRNA sequence (Figure 2A), and 13 candidates were then subjected to T7EI and restriction-protection assays. Consistent with previous reports, no genomic modifications were detected at any of the predicted unintended targets (Fig. 2B).4,10,21 Therefore, we concluded that either our system exhibits no off-target activities, or that any such activities occurred at levels too low to be detected by the modification-detection assays used. In conclusion, our work demonstrates the persistence of TRV-mediated CRISPR/Cas9 editing and the possibility of optimizing this method to recover progeny plants carrying the targeted modifications thereby bypassing the need for tissue culture or repeated transformation. This method will expand the utility of the
Downloaded by [New York University] at 01:40 05 June 2015
CRISPR/Cas9 system for plant functional genomics and targeted improvements of crop traits. Further, the use of heterozygous Cas9 overexpressing plants with this facile and versatile genome-editing platform allows the engineering and production of plants free of foreign DNA. This might overcome the regulatory hurdles that impede the commercialization of engineered plants. COMPETING FINANCIAL INTERESTS The authors declare competing financial interests. ACKNOWLEDGMENTS We would like to thank members of the laboratory for genome engineering at KAUST for helpful discussions and comments. This study is supported by King Abdullah University of Science and Technology (KAUST).
4
Downloaded by [New York University] at 01:40 05 June 2015
References
1.
Voytas, D.F. Plant genome engineering with sequence-specific nucleases. Annual review of plant biology 64, 327-350 (2013).
2.
Puchta, H. & Fauser, F. Synthetic nucleases for genome engineering in plants: prospects for a bright future. The Plant journal : for cell and molecular biology 78, 727-741 (2014).
3.
Mahfouz, M.M., Piatek, A. & Stewart, C.N., Jr. Genome engineering via TALENs and CRISPR/Cas9 systems: challenges and perspectives. Plant biotechnology journal 12, 1006-1014 (2014).
4.
Zhang, H., et al. The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation. Plant biotechnology journal 12, 797807 (2014).
5.
Shukla, V.K., et al. Precise genome modification in the crop species Zea mays using zinc-finger nucleases. Nature 459, 437-441 (2009).
6.
Townsend, J.A., et al. High-frequency modification of plant genes using engineered zinc-finger nucleases. Nature 459, 442-445 (2009).
7.
Li, L., et al. Characterization and DNA-binding specificities of Ralstonia TAL-like effectors. Molecular plant 6, 1318-1330 (2013).
8.
Aouida, M., Piatek, M.J., Bangarusamy, D.K. & Mahfouz, M.M. Activities and specificities of homodimeric TALENs in Saccharomyces cerevisiae. Current genetics 60, 61-74 (2014).
9.
Fauser, F., Schiml, S. & Puchta, H. Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana. The Plant journal : for cell and molecular biology 79, 348-359 (2014).
10.
Nekrasov, V., Staskawicz, B., Weigel, D., Jones, J.D. & Kamoun, S. Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nature biotechnology 31, 691-693 (2013).
11.
Shan, Q., et al. Targeted genome modification of crop plants using a CRISPR-Cas system. Nature biotechnology 31, 686-688 (2013).
12.
Cong, L., et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819-823 (2013).
13.
Piatek, A., et al. RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors. Plant biotechnology journal (2014).
14.
Liu, Y., Schiff, M. & Dinesh-Kumar, S.P. Virus-induced gene silencing in tomato. The Plant journal : for cell and molecular biology 31, 777-786 (2002).
15.
Macfarlane, S.A. Tobraviruses--plant pathogens and tools for biotechnology. Molecular plant pathology 11, 577-583 (2010).
16.
Senthil-Kumar, M. & Mysore, K.S. Tobacco rattle virus-based virus-induced gene silencing in Nicotiana benthamiana. Nature protocols 9, 1549-1562 (2014). 5
Ali, Z., et al. Efficient Virus-Mediated Genome Editing in Plants using the CRISPR/Cas9 System. Molecular plant (2015).
18.
Martin-Hernandez, A.M. & Baulcombe, D.C. Tobacco rattle virus 16-kilodalton protein encodes a suppressor of RNA silencing that allows transient viral entry in meristems. Journal of virology 82, 4064-4071 (2008).
19.
Kuscu, C., Arslan, S., Singh, R., Thorpe, J. & Adli, M. Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. Nature biotechnology 32, 677-683 (2014).
20.
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Figure 1 Persistence of TRV-mediated CRISPR/Cas9 targeted mutagenesis of the PDS3 gene. (A) Establishment of TRV infection in B14 plants, Nicotiana benthamiana Cas9 overexpression line. Agrobacterium cultures containing RNA1 and engineered RNA2 harboring gRNA for targeting PDS gene were mixed 1: 1 (OD600 0.1 each) and co-infiltrated to two fully expanded true leaves. Systemic leaves were collected 7, 15 and 30 days post-infiltration (dpi). (B) T7EI assay for indels detection. Genomic DNA was extracted from the systemic leaves and purified PCR product (200ng) of PDS fragment flanking the targeted locus was subjected to T7EI analysis. All three leaves collected at different time points showed high efficiency of targeted mutagenesis (43– 61 %) compared to vector control systemic leaves collected at 30 dpi. (C) Analysis of progeny plants for the presence of targeted modification using NcoI recognition site loss assay. DNA was extracted from progeny plants (50 seedlings pooled in one tube) and PCR was performed with a primer set to amplify 404 bp fragment encompassing the target site. Purified PCR product (300 ng) was treated with NcoI and separated on 2% agarose gel. Progeny pool 1 and 2 clearly showed a resistant DNA fragment of 404 bp indicating the targeted mutagenesis. (D) Alignment of Sanger sequencing reads showing the presence of indels at the PDS
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target sequence. Numbers to the right of sequence alignment indicates the number of nucleotides deleted by
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targeting the PDS genomic target.
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Figure 2 TRV-based CRISPR/Cas9 system exhibited no apparent off-target effects in N. benthamiana genome. (A) Table showing combinatorics approach to identify potential off-target binding of PDS3 gRNA in N. benthamiana genome. The putative off-target binding sites were subjected to further annotation, where sequences were split into two groups of conserved and not conserved NcoI restriction site directly preceding the PAM sequence and sites containing mutation in seed and non-seed sequence. Based on dissimilarities of 1 to 7 nucleotides with PDS3 gRNA, a total of 4265 potential off targets were detected, out of which 375 have NcoI site proximal to PAM sequence. (B) T7EI assay for the presence of indels at potential off target sites. DNA fragment flanking potential 13 targets were amplified by PCR with their respective primers. TRV-mediated
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CRISPR/Cas9 system exhibited no detectable activities at all tested targets using the T7EI assays. The respective
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contig number of each of 13 potential off target site are represented on the top of gel
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