- Email: [email protected]
Non-transgenic Plant Genome Editing Using Purified Sequence-Specific Nucleases
Accepted Manuscript Non-transgenic plant genome editing using purified sequence-specific nucleases Song Luo, Jin Li, Thomas J. Stoddard, Nicholas J. Baltes, Zachary L. Demorest, Benjamin M. Clasen, Andrew Coffman, Adam Retterath, Luc Mathis, Daniel F. Voytas, Feng Zhang PII:
S1674-2052(15)00265-8
DOI:
10.1016/j.molp.2015.05.012
Reference:
MOLP 145
To appear in:
MOLECULAR PLANT
Received Date: 9 February 2015 Revised Date:
13 May 2015
Accepted Date: 27 May 2015
Please cite this article as: Luo S., Li J., Stoddard T.J., Baltes N.J., Demorest Z.L., Clasen B.M., Coffman A., Retterath A., Mathis L., Voytas D.F., and Zhang F. (2015). Non-transgenic plant genome editing using purified sequence-specific nucleases. Mol. Plant. doi: 10.1016/j.molp.2015.05.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Title: Non-transgenic plant genome editing using purified sequence-specific nucleases
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Song Luo1*, Jin Li1, Thomas J. Stoddard1, Nicholas J.
Authors/ Affiliations:
Baltes1, Zachary L. Demorest1, Benjamin M. Clasen1, Andrew Coffman1, Adam Retterath1, Luc Mathis1, Daniel F. Voytas1, Feng Zhang1
[email protected]
Non-transgenic genome editing in plants.
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EP
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Running Title:
M AN U
Contact:
SC
1. Cellectis Plant Sciences. 600 County Road D West, Suite 8. New Brighton, MN 55112. USA.
1
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Non-transgenic plant genome editing using purified sequence-specific nucleases
Song Luo, Jin Li, Thomas J. Stoddard, Nicholas J. Baltes, Zachary L. Demorest, Benjamin M. Clasen,
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Andrew Coffman, Adam Retterath, Luc Mathis, Daniel F. Voytas, Feng Zhang Dear Editor,
Sequence-specific nucleases, including zinc-finger nucleases, meganucleases, TAL effector nucleases (TALENs) and CRISPR/Cas systems, have been used to introduce targeted mutations in a wide range of plant species (Voytas, 2013; Baltes et al., 2015). However, delivery of these nucleases using traditional
SC
transformation methods (e.g., particle bombardment, Agrobacterium or protoplast transformation) may result in undesired genetic alterations due to random insertion of nuclease-encoding DNA into the host
M AN U
genome. Random integration of DNA is a particular concern for trait improvement and gene function studies, because it may lead to unintended gene inactivation or it may alter expression of host genes. Furthermore, for crop varieties created using biotechnology, the presence of foreign DNA is a trigger for regulation by many governmental agencies (Voytas et al. 2014). Therefore, methods to modify plant genomes that do not require DNA delivery would have value in both commercial and academic settings. Here we demonstrate non-transgenic plant genome engineering by introducing sequence-specific nucleases as purified protein. This approach enabled targeted mutagenesis of endogenous sequences
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within plant cells, while avoiding integration of foreign DNA into the genome. The advent of genome engineering as a means of creating genetic variation in plants has raised questions regarding how the resulting, modified plants will be regulated by governmental authorities
EP
(Jones, 2015). Whereas some plants carrying NHEJ-induced mutations will not be regulated in the US (Waltz et al., 2012), the delivery of nucleic acid to a cell is sufficient to trigger regulation in some jurisdictions. The ‘nucleic acid’ trigger limits the use of nuclease platforms such a CRISPR/Cas for
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creating genetic variation relevant to agriculture, because a guide RNA is required by CRISPR/Cas for DNA targeting. To reduce concerns regarding the use of nucleic acids to modify plant cells, we sought to deliver active, sequence-specific nucleases as purified protein. We consider this method as a nontransgenic approach for targeted genome engineering. To determine if protein can be delivered to plant cells, we transformed Nicotiana tabacum protoplasts with purified EGFP protein using polyethylene glycol (PEG). Shortly following transformation, cells were washed and treated with trypsin to inactivate extracellular EGFP. Cells were then analyzed for EGFP expression using flow cytometry. We observed a transformation frequency of
2
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27.7% (Supplemental Figure 1), confirming that protein can be delivered to protoplasts (Wu et al, 2003), albeit at lower frequencies than plasmid DNA (Supplemental Figure 2). Next, we sought to optimize delivery of sequence-specific nucleases to plant cells using the meganuclease I-SceI. We reasoned that because meganucleases are the smallest of the nucleases used for
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genome engineering, they should be most amenable to direct protein delivery. Activity of I-SceI in plant cells was assessed using a yellow fluorescent protein (YFP) reporter plasmid. Cleavage of the reporter by I-SceI was expected to stimulate single-strand annealing (SSA) between two direct repeats in the coding sequence, thereby restoring YFP gene function. The YFP reporter plasmid, along with I-SceI protein,
SC
were delivered to Nicotiana tabacum protoplasts by PEG-mediated transformation (Supplemental Methods). Two days after transformation, protoplasts were assessed for YFP expression by flow
cytometry. Among the protoplasts to which I-SceI protein and the YFP reporter plasmid were delivered,
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4.4% had detectable levels of YFP expression (Supplemental Figure 2). To control for basal recombination, protoplasts were transformed with the YFP reporter plasmid alone; no protoplasts with YFP expression were detected (0.0%). These results suggest that meganuclease protein can be delivered to plant cells; however, they do not rule out the possibility that I-SceI cleaved the YFP reporter extracellularly prior to DNA uptake. To address this concern, we sequentially transformed protoplasts with YFP reporter plasmid and then I-SceI protein. Protoplasts that were transformed with YFP reporter
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plasmid were washed several times before being transformed with I-SceI protein. When the reporter plasmid and I-SceI protein were delivered sequentially, YFP expression was observed in 2.7% of the cells, indicating that I-SceI protein was successfully delivered to plant cell nuclei (Supplemental Figure 2).
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To determine if I-SceI is active against an endogenous target (instead of an episomal target), we transformed meganuclease protein into protoplasts that were isolated from a transgenic line of N. tabacum plants containing a single genomic I-SceI recognition site (Pacher et al, 2007). To increase the likelihood
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of capturing NHEJ-induced mutations, protoplasts were co-transformed with a plasmid encoding 3’ repair exonuclease 2 (Trex2) – a protein previously shown to increase the mutagenic efficiency of meganucleases (Certo et al, 2012). As a positive control, we transformed protoplasts with plasmid DNA encoding I-SceI. Cell viability was assessed two days post transformation – no differences were observed between cells transformed with DNA and cells transformed with protein. To determine if I-SceI was active at its endogenous target site, genomic DNA was isolated, and a 301 bp fragment encompassing the I-SceI recognition site was amplified by PCR. The PCR product was then subjected to 454 pyrosequencing. I-SceI protein activity was determined by analyzing sequencing reads for mutations at the
3
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predicted cleavage site. Mutagenesis frequency was calculated as the proportion of sequencing reads with NHEJ-induced mutations out of the total sequencing reads. The mutagenesis frequency of I-SceI and control treatments is summarized in Supplemental Table 1. Protoplasts delivered I-SceI as DNA yielded a 15% mutagenesis frequency, and when combined
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with DNA encoding Trex2, the mutagenesis frequency increased to 59.2%. When I-SceI was delivered as protein, the mutagenesis activity was below the limit of detection. However, when I-SceI protein was codelivered with Trex2 DNA, a 7.7% mutagenesis frequency was observed, suggesting that I-SceI is indeed cleaving its target site. To verify that translocation of I-SceI protein from extracellular to intracellular is
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not facilitated by the co-transformation of DNA, we transformed cells with a mixture of I-SceI protein and DNA encoding YFP - no evidence of NHEJ repair was found in 12,502 reads. Examples of I-SceI protein-induced mutations are shown in Supplemental Figure 3. Collectively, these data demonstrate that
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active meganuclease protein can be delivered to plant cell nuclei to generate targeted, chromosomal double-strand breaks. Furthermore, these data suggest that delivery of protein results in lower rates of mutagenesis compared to plasmid DNA. This observation is most likely due to differences in transformation efficiencies between DNA and protein (87.6% compared to 27.7%, respectively [Supplemental Figures 2 and 3]), and the extended length of time that protein is present when expressed from plasmid DNA.
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TALENs are a relatively recent addition to the arsenal of sequence-specific nucleases, and they offer clear advantages over meganucleases for genome engineering: they are simple to construct and have high activity and target site specificity. One disadvantage of TALENs, however, is their size: TALEN monomers are approximately six times larger than meganucleases (~950 vs ~165 amino acids). We next
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sought to determine if TALEN protein can be delivered to plant cells. Two recombinant TALEN monomers (ALS2T1L and ALS2T1R) were constructed that target a sequence ~306 bp downstream of the acetolactate synthase 2 gene (NbALS2) of N. benthamiana (Figure 1A). The TALEN monomers were
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cloned into a bacterial-expression plasmid (pQE-80L-Kan), expressed in BL21 cells, and purified using an N-terminal His tag (Figure 1B). To ensure that the purified TALEN protein was active against its corresponding target sequence, equal amounts of ALS2T1L and ALS2T1R protein were mixed with a PCR fragment containing the TALEN recognition site (NbALS2). As a negative control, TALEN proteins were mixed with a PCR product that did not contain the recognition site (NbALS1). Cleaved PCR product was only observed in the sample containing the TALEN proteins and the PCR product with the TALEN recognition site (Figure 1C), indicating that the purified TALENs were functional against their target sequence in vitro.
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To determine if TALEN protein can be delivered to plant cell nuclei, N. benthamiana protoplasts were transformed with purified ALS2T1L and ALS2T1R protein. To avoid delivery of nucleic acids, Trex2 plasmid DNA was not codelivered. Protoplasts were harvested 48 hours after transformation, and genomic DNA was isolated. A 253 bp fragment encompassing the TALEN recognition site was amplified
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by PCR and subjected to 454 pyro-sequencing. The mutagenesis frequency of ALS2T1L and ALS2T1R and control treatments is summarized in Figure 1D. When plasmid DNA encoding ALS2T1L and
ALS2TR was delivered to protoplasts, 18.6% of the sequencing reads carried mutations; when TALEN protein was delivered, we observed a 1.4% mutagenesis frequency. Whereas the delivery of protein
large TALEN proteins to plant cell nuclei is achievable.
SC
resulted in lower frequencies of mutagenesis compared to plasmid DNA, it was evident that delivery of
Our results demonstrate that sequence-specific nucleases (both meganuclease and TALENs) can
M AN U
be delivered to plant cells as purified protein. The nuclease proteins were effectively translocated to the nucleus where they cleaved host DNA at a predetermined, endogenous locus. Subsequent repair by NHEJ of the nuclease-induced DSB resulted in mutations detectable by DNA sequencing. However, compared to transformation of DNA, delivery of purified nucleases results in lower mutagenesis frequencies. Future efforts to increase mutagenesis frequencies can be directed at increasing protein transformation efficiency, and increasing the mutagenic activity of the sequence-specific nuclease of interest. Using the
without use of nucleic acids.
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References:
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approach described in this study, a plant containing targeted genome modifications can be attained
Baltes, N. and Voytas, D (2015) Enabling plant synthetic biology through genome engineering.
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Trends Biotechnol. 33, 120-131.
Certo, M., Gwiazda, K., Kuhar, R., Sather, B., Curinga, G., Mandt, T., Brault, M., Lambert, A., Baxter, S., Jacoby, K., et al. (2012). Coupling endonucleases with DNA end–processing enzymes to drive gene disruption. Nature Methods. 9, 973-975. Gaj, T., Guo, J., Kato, Y., Sirk, S. and Barbas, C. (2012). Targeted gene knockout by direct delivery of zinc-finger nuclease proteins. Nature Methods. 9, 805-807.
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Hospital, F. (2009). Challenges for effective marker-assisted selection in plants. Genetica. 136, 303-310. Jones, H. (2015) Regulatory uncertainty over genome editing. Nature Plants. 1, 1-3.
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Liu, J., Gaj, T., Patterson, J., Sirk, S. and Barbas, C. (2014). Cell-Penetrating Peptide-Mediated Delivery of TALEN Proteins via Bioconjugation for Genome Engineering. PLoS ONE. 9, e85755.
Matzke, A. and Matzke, M. (1998). Position effects and epigenetic silencing of plant transgenes.
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Curr Opin Plant Biol. 1, 142-148.
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Voytas, D. (2013). Plant Genome Engineering with Sequence-Specific Nucleases. Annu. Rev. Plant Biol. 64, 327-350.
Wu, Y., Wood, M., Tao, Y. and Katagiri, F. (2003). Direct delivery of bacterial avirulence proteins into resistant Arabidopsis protoplasts leads to hypersensitive cell death. Plant J. 33, 131137.
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Figure legend
Figure 1. Plant genome editing by direct delivery of TALEN protein. (A) Illustration of the ALS2T1L and ALS2T1R target site within the Nicotiana benthamiana
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acetolactate synthase 2 gene (NbALS2).
(B) Diagram of the TALEN bacterial expression plasmid used for TALEN expression and
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purification, and an illustration of a TALE protein not bound to DNA (left). SDS-PAGE gel of purified ALS2T1L and ALS2T1R (right). Black arrows indicate the full-size TALEN protein.
(C) In vitro TALEN specificity and activity assay. Black arrows indicate the cleavage product. TALENs refer to purified ALS2T1L and ALS2T1R protein. (D) Mutation frequency at the TALEN target site measured by 454-pyrosequencing (top). Examples of mutations from protoplasts delivered TALEN protein (bottom).
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Supplemental Online Methods Sequence-specific nucleases: cloning, expression and purification The meganuclease, I-SceI, was purchased from New England Biolabs (Cat# R0694). Plasmids
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for producing the recombinant TALENs were provided by Cellectis Bioresearch (8, rue de la Croix Jarry, 75013 PARIS). TALEN coding sequences were cloned into the protein expression vector pQE-80L-Kan (Qiagen, Cat# 32943). A SV40 nuclear localization signal was added to the N-terminus of the TALEN protein. The TALEN pair (ALS2T1L and ALS2T1R) is specific for a
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site downstream of N. benthamiana ALS2 gene. E. coli strain BL21 (NEB Cat# C2530H) was used for protein expression. His-tag protein expression and purification was carried out as
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described in the Qiagen Ni-NTA Spin kit manual.
Commercial and homemade sequence-specific nucleases were dialyzed before transformation to remove detergent and salt. Briefly, 20 µl of nuclease was placed on a Millipore 0.025µm VSWP filter (CAT# VSWP02500). The filter was floated on MMG buffer (0.4 M mannitol, 4 mM MES, 15 mM MgCl2, pH 5.8) at 4°C for 1 hour. After the enzyme solution was
further use.
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completely equilibrated by MMG buffer, it was transferred to a new tube and kept on ice until
Protoplast isolation and transformation
Tobacco seeds were sterilized by washing them successively in 100% ethanol, 50% bleach and
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then sterile distilled water. The sterilized seeds were planted on MS agarose medium supplemented with iron. Protoplasts were isolated from young expanded leaves using the
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protocol described by Zhang et al. (Plant Physiology 161: 20-27, 2013). Protein was delivered to plant cells by PEG-mediated protoplast transformation as
described by Yoo et al. (Nature Protocols 2:1565-1572, 2007) with modifications. Briefly, 10 µg protein or 15 µg DNA was mixed with 200,000 protoplasts at room temperature in 200 µl of 0.4 M mannitol, 4 mM MES, 15 mM MgCl2, pH 5.8. Then, an equal volume of 40% PEG-4000, 0.2 M mannitol, 100 mM CaCl2, pH 5.8 was added to protoplasts and immediately mixed. The mixture was incubated in the dark for 30 minutes before washing once with 0.45 M mannitol, 10 mM CaCl2. For the EGFP protein transformation, an additional 5 minute wash with 0.025% 7
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Trypsin in 0.45 M mannitol and, 10 mM CaCl2 was applied. The protoplasts were then washed with K3G1 medium twice before moving the cells to 1 ml of K3G1 in a petri dish for long-term
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culture.
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Supplemental Figure legend
Supplemental Figure 1. Quantification of the transformation efficiency of purified EGFP
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protein in protoplasts. Commercially available EGFP protein (BioVision, Cat# 4999-100) was transformed into Nicotiana tabacum protoplasts at a 1 µM concentration using polyethylene glycol (PEG). To remove extracellular EGFP protein, cells were washed in a solution containing trypsin at a concentration of 0.025%. Cells were analyzed by flow cytometry.
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Supplemental Figure 2. Single-strand annealing (SSA) assay for I-SceI delivered as protein. Percentage of YFP-positive cells as determined by flow cytometry. Nicotiana tabacum protoplasts were transformed with 35S:YFP plasmid DNA as a transformation control. As a negative control, protoplasts were transformed with the SSA construct YF(I-SceI)FP. Protoplasts
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were also transformed with I-SceI protein plus the reporter construct (lower right), I-SceI plasmid DNA plus the reporter construct (lower left), and sequentially transformed with the
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reporter construct and then I-SceI protein (upper right).
Supplemental Figure 3. Representative sequence analysis of the integrated I-SceI targeting site. Multiple deletions induced by I-SceI/Trex2 and NHEJ repair are aligned to the cleavage site (top, WT).
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Supplemental Table 1. I-SceI-induced mutagenesis. # seq reads total # seq
NHEJ mutagenesis
mutations
reads analyzed
frequency (%)
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Treatment
with
0
11,130
0.0
Trex2 (DNA)
1*
9,264
0.0
Trex2 (DNA)
5458
9,222
59.2
I-SceI (DNA)
2881
19,261
1142
14,921
I-SceI (DNA) +
Trex2 (DNA) I-SceI (protein)
1*
I-SceI (protein) + YFP (DNA)
0
15.0
7.7
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I-SceI (protein) +
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H2O
14,362
0.0
12,502
0.0
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∗ Sequence contained a single nucleotide substitution near the predicted site of cleavage.
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S1674-2052(15)00265-8
DOI:
10.1016/j.molp.2015.05.012
Reference:
MOLP 145
To appear in:
MOLECULAR PLANT
Received Date: 9 February 2015 Revised Date:
13 May 2015
Accepted Date: 27 May 2015
Please cite this article as: Luo S., Li J., Stoddard T.J., Baltes N.J., Demorest Z.L., Clasen B.M., Coffman A., Retterath A., Mathis L., Voytas D.F., and Zhang F. (2015). Non-transgenic plant genome editing using purified sequence-specific nucleases. Mol. Plant. doi: 10.1016/j.molp.2015.05.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Title: Non-transgenic plant genome editing using purified sequence-specific nucleases
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Song Luo1*, Jin Li1, Thomas J. Stoddard1, Nicholas J.
Authors/ Affiliations:
Baltes1, Zachary L. Demorest1, Benjamin M. Clasen1, Andrew Coffman1, Adam Retterath1, Luc Mathis1, Daniel F. Voytas1, Feng Zhang1
[email protected]
Non-transgenic genome editing in plants.
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Running Title:
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Contact:
SC
1. Cellectis Plant Sciences. 600 County Road D West, Suite 8. New Brighton, MN 55112. USA.
1
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Non-transgenic plant genome editing using purified sequence-specific nucleases
Song Luo, Jin Li, Thomas J. Stoddard, Nicholas J. Baltes, Zachary L. Demorest, Benjamin M. Clasen,
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Andrew Coffman, Adam Retterath, Luc Mathis, Daniel F. Voytas, Feng Zhang Dear Editor,
Sequence-specific nucleases, including zinc-finger nucleases, meganucleases, TAL effector nucleases (TALENs) and CRISPR/Cas systems, have been used to introduce targeted mutations in a wide range of plant species (Voytas, 2013; Baltes et al., 2015). However, delivery of these nucleases using traditional
SC
transformation methods (e.g., particle bombardment, Agrobacterium or protoplast transformation) may result in undesired genetic alterations due to random insertion of nuclease-encoding DNA into the host
M AN U
genome. Random integration of DNA is a particular concern for trait improvement and gene function studies, because it may lead to unintended gene inactivation or it may alter expression of host genes. Furthermore, for crop varieties created using biotechnology, the presence of foreign DNA is a trigger for regulation by many governmental agencies (Voytas et al. 2014). Therefore, methods to modify plant genomes that do not require DNA delivery would have value in both commercial and academic settings. Here we demonstrate non-transgenic plant genome engineering by introducing sequence-specific nucleases as purified protein. This approach enabled targeted mutagenesis of endogenous sequences
TE D
within plant cells, while avoiding integration of foreign DNA into the genome. The advent of genome engineering as a means of creating genetic variation in plants has raised questions regarding how the resulting, modified plants will be regulated by governmental authorities
EP
(Jones, 2015). Whereas some plants carrying NHEJ-induced mutations will not be regulated in the US (Waltz et al., 2012), the delivery of nucleic acid to a cell is sufficient to trigger regulation in some jurisdictions. The ‘nucleic acid’ trigger limits the use of nuclease platforms such a CRISPR/Cas for
AC C
creating genetic variation relevant to agriculture, because a guide RNA is required by CRISPR/Cas for DNA targeting. To reduce concerns regarding the use of nucleic acids to modify plant cells, we sought to deliver active, sequence-specific nucleases as purified protein. We consider this method as a nontransgenic approach for targeted genome engineering. To determine if protein can be delivered to plant cells, we transformed Nicotiana tabacum protoplasts with purified EGFP protein using polyethylene glycol (PEG). Shortly following transformation, cells were washed and treated with trypsin to inactivate extracellular EGFP. Cells were then analyzed for EGFP expression using flow cytometry. We observed a transformation frequency of
2
ACCEPTED MANUSCRIPT
27.7% (Supplemental Figure 1), confirming that protein can be delivered to protoplasts (Wu et al, 2003), albeit at lower frequencies than plasmid DNA (Supplemental Figure 2). Next, we sought to optimize delivery of sequence-specific nucleases to plant cells using the meganuclease I-SceI. We reasoned that because meganucleases are the smallest of the nucleases used for
RI PT
genome engineering, they should be most amenable to direct protein delivery. Activity of I-SceI in plant cells was assessed using a yellow fluorescent protein (YFP) reporter plasmid. Cleavage of the reporter by I-SceI was expected to stimulate single-strand annealing (SSA) between two direct repeats in the coding sequence, thereby restoring YFP gene function. The YFP reporter plasmid, along with I-SceI protein,
SC
were delivered to Nicotiana tabacum protoplasts by PEG-mediated transformation (Supplemental Methods). Two days after transformation, protoplasts were assessed for YFP expression by flow
cytometry. Among the protoplasts to which I-SceI protein and the YFP reporter plasmid were delivered,
M AN U
4.4% had detectable levels of YFP expression (Supplemental Figure 2). To control for basal recombination, protoplasts were transformed with the YFP reporter plasmid alone; no protoplasts with YFP expression were detected (0.0%). These results suggest that meganuclease protein can be delivered to plant cells; however, they do not rule out the possibility that I-SceI cleaved the YFP reporter extracellularly prior to DNA uptake. To address this concern, we sequentially transformed protoplasts with YFP reporter plasmid and then I-SceI protein. Protoplasts that were transformed with YFP reporter
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plasmid were washed several times before being transformed with I-SceI protein. When the reporter plasmid and I-SceI protein were delivered sequentially, YFP expression was observed in 2.7% of the cells, indicating that I-SceI protein was successfully delivered to plant cell nuclei (Supplemental Figure 2).
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To determine if I-SceI is active against an endogenous target (instead of an episomal target), we transformed meganuclease protein into protoplasts that were isolated from a transgenic line of N. tabacum plants containing a single genomic I-SceI recognition site (Pacher et al, 2007). To increase the likelihood
AC C
of capturing NHEJ-induced mutations, protoplasts were co-transformed with a plasmid encoding 3’ repair exonuclease 2 (Trex2) – a protein previously shown to increase the mutagenic efficiency of meganucleases (Certo et al, 2012). As a positive control, we transformed protoplasts with plasmid DNA encoding I-SceI. Cell viability was assessed two days post transformation – no differences were observed between cells transformed with DNA and cells transformed with protein. To determine if I-SceI was active at its endogenous target site, genomic DNA was isolated, and a 301 bp fragment encompassing the I-SceI recognition site was amplified by PCR. The PCR product was then subjected to 454 pyrosequencing. I-SceI protein activity was determined by analyzing sequencing reads for mutations at the
3
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predicted cleavage site. Mutagenesis frequency was calculated as the proportion of sequencing reads with NHEJ-induced mutations out of the total sequencing reads. The mutagenesis frequency of I-SceI and control treatments is summarized in Supplemental Table 1. Protoplasts delivered I-SceI as DNA yielded a 15% mutagenesis frequency, and when combined
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with DNA encoding Trex2, the mutagenesis frequency increased to 59.2%. When I-SceI was delivered as protein, the mutagenesis activity was below the limit of detection. However, when I-SceI protein was codelivered with Trex2 DNA, a 7.7% mutagenesis frequency was observed, suggesting that I-SceI is indeed cleaving its target site. To verify that translocation of I-SceI protein from extracellular to intracellular is
SC
not facilitated by the co-transformation of DNA, we transformed cells with a mixture of I-SceI protein and DNA encoding YFP - no evidence of NHEJ repair was found in 12,502 reads. Examples of I-SceI protein-induced mutations are shown in Supplemental Figure 3. Collectively, these data demonstrate that
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active meganuclease protein can be delivered to plant cell nuclei to generate targeted, chromosomal double-strand breaks. Furthermore, these data suggest that delivery of protein results in lower rates of mutagenesis compared to plasmid DNA. This observation is most likely due to differences in transformation efficiencies between DNA and protein (87.6% compared to 27.7%, respectively [Supplemental Figures 2 and 3]), and the extended length of time that protein is present when expressed from plasmid DNA.
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TALENs are a relatively recent addition to the arsenal of sequence-specific nucleases, and they offer clear advantages over meganucleases for genome engineering: they are simple to construct and have high activity and target site specificity. One disadvantage of TALENs, however, is their size: TALEN monomers are approximately six times larger than meganucleases (~950 vs ~165 amino acids). We next
EP
sought to determine if TALEN protein can be delivered to plant cells. Two recombinant TALEN monomers (ALS2T1L and ALS2T1R) were constructed that target a sequence ~306 bp downstream of the acetolactate synthase 2 gene (NbALS2) of N. benthamiana (Figure 1A). The TALEN monomers were
AC C
cloned into a bacterial-expression plasmid (pQE-80L-Kan), expressed in BL21 cells, and purified using an N-terminal His tag (Figure 1B). To ensure that the purified TALEN protein was active against its corresponding target sequence, equal amounts of ALS2T1L and ALS2T1R protein were mixed with a PCR fragment containing the TALEN recognition site (NbALS2). As a negative control, TALEN proteins were mixed with a PCR product that did not contain the recognition site (NbALS1). Cleaved PCR product was only observed in the sample containing the TALEN proteins and the PCR product with the TALEN recognition site (Figure 1C), indicating that the purified TALENs were functional against their target sequence in vitro.
4
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To determine if TALEN protein can be delivered to plant cell nuclei, N. benthamiana protoplasts were transformed with purified ALS2T1L and ALS2T1R protein. To avoid delivery of nucleic acids, Trex2 plasmid DNA was not codelivered. Protoplasts were harvested 48 hours after transformation, and genomic DNA was isolated. A 253 bp fragment encompassing the TALEN recognition site was amplified
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by PCR and subjected to 454 pyro-sequencing. The mutagenesis frequency of ALS2T1L and ALS2T1R and control treatments is summarized in Figure 1D. When plasmid DNA encoding ALS2T1L and
ALS2TR was delivered to protoplasts, 18.6% of the sequencing reads carried mutations; when TALEN protein was delivered, we observed a 1.4% mutagenesis frequency. Whereas the delivery of protein
large TALEN proteins to plant cell nuclei is achievable.
SC
resulted in lower frequencies of mutagenesis compared to plasmid DNA, it was evident that delivery of
Our results demonstrate that sequence-specific nucleases (both meganuclease and TALENs) can
M AN U
be delivered to plant cells as purified protein. The nuclease proteins were effectively translocated to the nucleus where they cleaved host DNA at a predetermined, endogenous locus. Subsequent repair by NHEJ of the nuclease-induced DSB resulted in mutations detectable by DNA sequencing. However, compared to transformation of DNA, delivery of purified nucleases results in lower mutagenesis frequencies. Future efforts to increase mutagenesis frequencies can be directed at increasing protein transformation efficiency, and increasing the mutagenic activity of the sequence-specific nuclease of interest. Using the
without use of nucleic acids.
EP
References:
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approach described in this study, a plant containing targeted genome modifications can be attained
Baltes, N. and Voytas, D (2015) Enabling plant synthetic biology through genome engineering.
AC C
Trends Biotechnol. 33, 120-131.
Certo, M., Gwiazda, K., Kuhar, R., Sather, B., Curinga, G., Mandt, T., Brault, M., Lambert, A., Baxter, S., Jacoby, K., et al. (2012). Coupling endonucleases with DNA end–processing enzymes to drive gene disruption. Nature Methods. 9, 973-975. Gaj, T., Guo, J., Kato, Y., Sirk, S. and Barbas, C. (2012). Targeted gene knockout by direct delivery of zinc-finger nuclease proteins. Nature Methods. 9, 805-807.
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Hospital, F. (2009). Challenges for effective marker-assisted selection in plants. Genetica. 136, 303-310. Jones, H. (2015) Regulatory uncertainty over genome editing. Nature Plants. 1, 1-3.
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Liu, J., Gaj, T., Patterson, J., Sirk, S. and Barbas, C. (2014). Cell-Penetrating Peptide-Mediated Delivery of TALEN Proteins via Bioconjugation for Genome Engineering. PLoS ONE. 9, e85755.
Matzke, A. and Matzke, M. (1998). Position effects and epigenetic silencing of plant transgenes.
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Curr Opin Plant Biol. 1, 142-148.
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Voytas, D. (2013). Plant Genome Engineering with Sequence-Specific Nucleases. Annu. Rev. Plant Biol. 64, 327-350.
Wu, Y., Wood, M., Tao, Y. and Katagiri, F. (2003). Direct delivery of bacterial avirulence proteins into resistant Arabidopsis protoplasts leads to hypersensitive cell death. Plant J. 33, 131137.
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Figure legend
Figure 1. Plant genome editing by direct delivery of TALEN protein. (A) Illustration of the ALS2T1L and ALS2T1R target site within the Nicotiana benthamiana
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acetolactate synthase 2 gene (NbALS2).
(B) Diagram of the TALEN bacterial expression plasmid used for TALEN expression and
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purification, and an illustration of a TALE protein not bound to DNA (left). SDS-PAGE gel of purified ALS2T1L and ALS2T1R (right). Black arrows indicate the full-size TALEN protein.
(C) In vitro TALEN specificity and activity assay. Black arrows indicate the cleavage product. TALENs refer to purified ALS2T1L and ALS2T1R protein. (D) Mutation frequency at the TALEN target site measured by 454-pyrosequencing (top). Examples of mutations from protoplasts delivered TALEN protein (bottom).
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Supplemental Online Methods Sequence-specific nucleases: cloning, expression and purification The meganuclease, I-SceI, was purchased from New England Biolabs (Cat# R0694). Plasmids
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for producing the recombinant TALENs were provided by Cellectis Bioresearch (8, rue de la Croix Jarry, 75013 PARIS). TALEN coding sequences were cloned into the protein expression vector pQE-80L-Kan (Qiagen, Cat# 32943). A SV40 nuclear localization signal was added to the N-terminus of the TALEN protein. The TALEN pair (ALS2T1L and ALS2T1R) is specific for a
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site downstream of N. benthamiana ALS2 gene. E. coli strain BL21 (NEB Cat# C2530H) was used for protein expression. His-tag protein expression and purification was carried out as
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described in the Qiagen Ni-NTA Spin kit manual.
Commercial and homemade sequence-specific nucleases were dialyzed before transformation to remove detergent and salt. Briefly, 20 µl of nuclease was placed on a Millipore 0.025µm VSWP filter (CAT# VSWP02500). The filter was floated on MMG buffer (0.4 M mannitol, 4 mM MES, 15 mM MgCl2, pH 5.8) at 4°C for 1 hour. After the enzyme solution was
further use.
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completely equilibrated by MMG buffer, it was transferred to a new tube and kept on ice until
Protoplast isolation and transformation
Tobacco seeds were sterilized by washing them successively in 100% ethanol, 50% bleach and
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then sterile distilled water. The sterilized seeds were planted on MS agarose medium supplemented with iron. Protoplasts were isolated from young expanded leaves using the
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protocol described by Zhang et al. (Plant Physiology 161: 20-27, 2013). Protein was delivered to plant cells by PEG-mediated protoplast transformation as
described by Yoo et al. (Nature Protocols 2:1565-1572, 2007) with modifications. Briefly, 10 µg protein or 15 µg DNA was mixed with 200,000 protoplasts at room temperature in 200 µl of 0.4 M mannitol, 4 mM MES, 15 mM MgCl2, pH 5.8. Then, an equal volume of 40% PEG-4000, 0.2 M mannitol, 100 mM CaCl2, pH 5.8 was added to protoplasts and immediately mixed. The mixture was incubated in the dark for 30 minutes before washing once with 0.45 M mannitol, 10 mM CaCl2. For the EGFP protein transformation, an additional 5 minute wash with 0.025% 7
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Trypsin in 0.45 M mannitol and, 10 mM CaCl2 was applied. The protoplasts were then washed with K3G1 medium twice before moving the cells to 1 ml of K3G1 in a petri dish for long-term
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culture.
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Supplemental Figure legend
Supplemental Figure 1. Quantification of the transformation efficiency of purified EGFP
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protein in protoplasts. Commercially available EGFP protein (BioVision, Cat# 4999-100) was transformed into Nicotiana tabacum protoplasts at a 1 µM concentration using polyethylene glycol (PEG). To remove extracellular EGFP protein, cells were washed in a solution containing trypsin at a concentration of 0.025%. Cells were analyzed by flow cytometry.
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Supplemental Figure 2. Single-strand annealing (SSA) assay for I-SceI delivered as protein. Percentage of YFP-positive cells as determined by flow cytometry. Nicotiana tabacum protoplasts were transformed with 35S:YFP plasmid DNA as a transformation control. As a negative control, protoplasts were transformed with the SSA construct YF(I-SceI)FP. Protoplasts
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were also transformed with I-SceI protein plus the reporter construct (lower right), I-SceI plasmid DNA plus the reporter construct (lower left), and sequentially transformed with the
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reporter construct and then I-SceI protein (upper right).
Supplemental Figure 3. Representative sequence analysis of the integrated I-SceI targeting site. Multiple deletions induced by I-SceI/Trex2 and NHEJ repair are aligned to the cleavage site (top, WT).
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Supplemental Table 1. I-SceI-induced mutagenesis. # seq reads total # seq
NHEJ mutagenesis
mutations
reads analyzed
frequency (%)
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Treatment
with
0
11,130
0.0
Trex2 (DNA)
1*
9,264
0.0
Trex2 (DNA)
5458
9,222
59.2
I-SceI (DNA)
2881
19,261
1142
14,921
I-SceI (DNA) +
Trex2 (DNA) I-SceI (protein)
1*
I-SceI (protein) + YFP (DNA)
0
15.0
7.7
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I-SceI (protein) +
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14,362
0.0
12,502
0.0
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∗ Sequence contained a single nucleotide substitution near the predicted site of cleavage.
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