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ScienceDirect Plant genome editing by novel tools: TALEN and other sequence specific nucleases Thorben Sprink1, Janina Metje2 and Frank Hartung1 Genome editing technologies using sequence specific nucleases (SSNs) became a tremendously powerful and precise tool for reverse genetic approaches and applied biology. Transcription activator-like effector nucleases (TALENs) in particular, consisting of a free designable DNA binding domain and a nuclease, have been exploited today by a huge number of approaches in many different organisms. The convenience of designing the DNA binding domain and straightforward protocols for their assembly, as well as the broad number of applications in different scientific fields made it Natures method of the year 2011. TALENs act as molecular scissors by introducing double strand breaks (DSBs) to the DNA at a given location. The DSBs are subsequently repaired by the cell itself using different repair pathways such as nonhomologous end joining (NHEJ) or homologous recombination (HR). These mechanisms can lead to deletions, insertions, replacements or larger chromosomal rearrangements. By offering a template DNA it is possible to channel the repair in direction of HR. In this article we review the recent findings in the field of SSN approaches with emphasis on plants. Addresses 1 Julius Ku¨hn Institut, Institute for Biosafety in Plant Biotechnology, Erwin Baur-Str. 27, 06484 Quedlinburg, Germany 2 Max Plank Institute for Biophysical Chemistry, Research Group Autophagy, Am Fassberg 11, 37077 Go¨ttingen, Germany Corresponding author: Hartung, Frank ([email protected])
Current Opinion in Biotechnology 2015, 32:47–53 This review comes from a themed issue on Plant biotechnology Edited by Inge Broer and George N Skaracis
http://dx.doi.org/10.1016/j.copbio.2014.11.010 0958-1669/# 2014 Elsevier Ltd. All right reserved.
given location (for a glossary see Box 1). In the past, targeted genome modifications in plants were far away from any reasonable efficiency [1], but since it became possible to introduce double strand breaks (DSBs) into the DNA at a defined specific site using SSNs such as zinc finger nucleases (ZFNs) [2] or meganucleases [3], the efficiency has been raised tremendously (for recent review see [4]). Unfortunately, both techniques possess an unfortunate drawback, as the precise design of the DNA binding site is somewhat limited due to the architecture of these SSNs. These things changed dramatically since Boch et al. and others unveiled the code for transcription activator-like effectors (TALE) in 2009 [5,6]. In 2010 the first transcription activator-like effector nucleases (TALENs) combining the DNA binding part of a TALE and an unspecific nuclease were published by the group of Dan Voytas [7,8]. Like ZFNs, TALENs are based on a fusion of a designed DNA binding domain with the nuclease FokI allowing the introduction of a DSB at a given location. Such a DSB activates two main repair pathways in the cell, non homologous end joining (NHEJ) and homologous recombination (HR) (Figure 1). NHEJ is mostly imprecise, leading to insertions or deletions at the repair site and in most cases, a disrupted gene sequence leading to a loss of function mutation. Real gene targeting can be achieved by HR where the DSB is repaired by a homologous template such as the sister chromatid, the homologous chromosome or an additionally presented homologous template DNA [9]. The newest achievement in the field of SSNs is the use of clustered regularly interspaced short palindromic repeats (CRISPRs) fused with a Cas9 nuclease [10], reviewed by Kamoun et al. in this issue. In this review we focus on the recent developments in the field of SSNs, focusing on TALENs, ZFNs and meganucleases during the last two years.
Plant genome editing by SSNs
Introduction Over the past decades, agriculture has had to change to meet the challenge of a growing number of people in the world in a shrinking crop area. New technologies in breeding like the green revolution and hybrid plants helped to increase agricultural productivity. Recent developments in genome engineering using sequence specific nucleases (SSNs) to regulate the site of genomic modification could help to speed up breeding processes by altering specific genes at a chosen site or by introduction of new traits at a www.sciencedirect.com
So far several groups have successfully modified plant genes in vivo by using TALENs to induce DSBs and repair by NHEJ. One of the broadest approaches to edit endogenous plant genes has been done by Shan et al. In total, they successfully targeted seven genes in Brachypodium and four in rice by specific TALEN pairs. The resulting mutations were predominantly short deletions of 1–20 nt, indicating DNA repair by the NHEJ pathway [9,11]. In addition to this Shan et al. also deleted larger regions of rice DNA through application of two TALEN pairs with specific cutting sites more than thousand nucleotides apart. This approach shows the potential usefulness of SSNs for selective deletion or Current Opinion in Biotechnology 2015, 32:47–53
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Box 1 SSNs: Sequence specific nucleases, are a combination of a DNA recognition part and a nuclease part. Typical SSNs are CRISPR/Cas9, TALENs, ZFNs and meganucleases. CRISPR/Cas9 (CRISPR associated (Cas) system): Clustered regularly interspaced short palindromic repeats allow bacteria an adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNA) and trans activating crRNA (tracrRNA) to guide the silencing of invading nucleic acids. Three types of CRISPR/Cas system exist. Type II in which Cas9 is guided by a crRNA–tracrRNA target identification to cut DNA at the identified region, is used in an altered form for genome engineering in a couple of organism. TALENs: Transcription activator like effector nucleases are a combination of the catalytic domain of an endonuclease most frequently FokI fused with the DNA binding domain derived from transcription activator like effectors from Xanthomonas spec. TALENs consist of multiple repeats of a 33–35 amino acids long sequence containing a so-called repeat variable diresidue (RVD) which are the amino acids nos. 12 and 13. Each repeat binds to a certain single base pair. TALENs are commonly used as a pair to introduce double strand breaks (DSBs) to the DNA, as FokI only introduces DSBs as a dimer. RVD: Repeat variable diresidue, the 12th and 13th position in a TALE domain, defining which base pair will preferential be bound (HD=C, NG=T, NI=A, NS=A, NN=G/A NK=G). ZFN: Zinc finger nucleases are a combination of a zinc finger DNA binding domain with an endonuclease most frequently FokI. In the zinc finger DNA binding domain the a-helices define which three base pairs will be recognized. In nature there are >1000 zinc finger domains known. Typical ZFNs consists of minimal 3 zinc finger domains each recognizing 3 bp. Nickase: An enzyme that introduces single strand breaks (nicks) to the DNA. A FokI dimer with one inactivated FokI is often used as a nickase in SSN approaches. Meganuclease: Naturally occurring endonucleases from different organisms such as bacteria, fungi and algae. They have a long DNA recognition site with variable size differing between 12 and 30 bp. Naturally occurring meganucleases can be modified by introducing changes to the amino acids found in the recognition site of the protein to adapt for restriction of a specifically chosen sequence. DSB: A double strand break is a form of DNA damage, in which both DNA strands are broken. DSBs are the result of for example, endonuclease restriction or natural influences such as irradiation or chemicals. During meiosis, DSBs are specially introduced to the DNA by SPO11 to initiate recombination processes. cNHEJ: Canonical or classical non-homologous end joining is the first major DSB repair mechanism in somatic cells. It is called non homologous because the break ends are ligated without the use of a homologous sequence such as the sister chromatid or the homologous chromosome. The breaks are either directly ligated or short microhomologies on the single stranded overhangs are used for the ligation. NHEJ is often imprecise, leading to small insertions or deletions at the repaired site. aNHEJ: Alternative non-homologous end joining, is a pathway which uses microhomologies present on both sites of the DSB, aNHEJ often leads to larger deletions at the site of DSB repair. HR: Homologous recombination is the second major DSB repair mechanism using various pathways leading in some cases to the formation of double holliday junctions, for example crossover between homologous chromosomes in meiosis. In contrast to NHEJ, HR uses a homologous template to repair the DSB, this is often the sister chromatid or the homologous chromosome. In an SSN approach homologous sequences can be additionally introduced to act as a repair template to insert single or multiple transgenes as well as to induce single nucleotide changes to a specific gene.
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rearrangement of genes or even gene clusters. Such deletion or rearrangement of gene clusters such as R-genes has been achieved in Arabidopsis by Qi et al. [12]. They used ZFNs for this approach and successfully introduced large deletions (from 4.5 kb up to 9 Mb) in seven independent endogenous loci in Arabidopsis in a range of 0.1–3% of the cases, therefore proving the applicability of large gene deletions by SSNs in plants. Qi et al. also found inversions or duplications of such large genomic regions but in a frequency below 0.1%. Recent studies in the field focused on gene editing in crop plants, such as Glycine max [13]. Haun et al. disrupted two fatty desaturase genes (FAD2-1A and FAD2-1B) using TALENs, to achieve a soybean with an improved number of monosaturated oils with longer shelf life. They managed to create a homozygous knockout line for both genes, but lacking the TALEN construct by crossing the transgenic plants with their wild type parents. Wang et al. were able to present a TALEN approach addressing three homoeoalleles in hexaploid bread wheat by a single TALEN pair addressing a conserved domain in the three alleles, to improve heritable resistance to powdery mildew [14]. They managed to create TALEN-free resistant plants in three generations by selfing one plant carrying a heterozygous mutation for each homoeoallele, showing the large opportunities for breeding programs offered by SSNs. Two other groups using TALEN approaches focused on the genome modification of barley, another important crop. Wendt et al. used a one vector system and immature embryos focusing on an endogenous barley gene promoter. They reached a transformation rate of 20%, mostly resulting in NHEJ events leading to small deletions [15]. Gurushidze et al. focused on the knockout of GFP in transgenic haploid barley cells using a slightly different approach in which both TALEN arms are brought separately into the cells. They also reached up to 20% frequency of mutation induction in their transgenic lines. Interestingly, they found deletions in the GFP sequence even in plants where only one of the two TALEN arms was stably integrated, showing a transient activity of the respective other TALEN in the plants [16]. In a proof of concept experiment using TALENs, Lor et al. created knockout lines of the PROCERA gene in tomato, showing that it is possible to create tomato plants which contain only the desired mutations, and not the TALEN construct. For this approach they used an estrogen inducible promoter to control the risk of TALEN cytotoxicity. Spraying of plants (or parts of them) with estradiol resulted in a very low amount of cells carrying the mutation. Lor et al. subsequently immersed the cotyledons of T1 plants, three times in an estradiol containing solution at weekly intervals. Using this modified protocol they managed to produce plants containing www.sciencedirect.com
Plant genome editing via sequence specific nucleases Sprink, Metje and Hartung 49
Figure 1
Double strand break (DSB) initiation DSB (a)
Homologous recombination (HR)
(b)
Non-homologous end joining (NHEJ)
Strand invasion
End capture
Strand annealing > 100 bp
Various HR pathway resolutions
Single strand annealing (SSA)
Gene addition/ replacement/ adaption
some bp
Classical nonhomologous end joining (cNHEJ) Alternative non-homologous end joining(aNHEJ)
Gene disruption by deletion or insertions of a few bps Current Opinion in Biotechnology
The two main cellular DSB repair-pathways. (a) Homologous recombination (HR) which is divided into the resolution of double holliday junctions (dHJ), created after strand invasion of the broken strand, by various dHJ resolution pathways and in the relatively simple single strand annealing (SSA) pathway where a tandem homology on both ends of the break is used for repair. This can be a repeated sequence as well as an external DNA fragment transferred into the cell. The common outcomes of the pathways are gene addition, replacement or gene adaptation. (b) Non homologous end joining (NHEJ). NHEJ can be divided into two main branches, the classical (also called canonical) NHEJ, which ligates broken DNA ends together without any homology and the alternative non homologous end joining, which ligates broken DNA ends using microhomologies of two nucleotides on the single stranded break ends. The common outcome in both cases is a disruption of the gene by deletions or insertions of a few nucleotides.
the desired mutations and could show that these mutations were stably inherited and the TALEN construct was lost by segregation [17].
In planta gene targeting by SSNs In contrast to repair by NHEJ which mostly leads to loss of function mutations, the application of HR for gene targeting is more challenging. Fauser et al. used a meganuclease based approach for guiding DSB repair to HR by adapting a system formerly used in Drosophila for in vivo gene targeting [18]. They created individual lines transformed either with a target sequence (a truncated GUS gene flanked by two ISceI sites) or a donor sequence (the rest of the GUS gene including homology to the target construct also flanked by two ISceI sites). After creating homozygous lines for both targets they crossed the lines and the resulting progeny www.sciencedirect.com
was then crossed further to a line expressing ISceI. The final outcome was a successful gene targeting via HR with approximately 1% efficiency. Remarkably, additional integration of the target elsewhere in the genome by NHEJ was not found in any of the analyzed plants. Zhang and colleagues had a closer look at the events of a TALEN pair targeting the duplicated acetolactate synthase genes (SurA and SurB) in N. tabacum, compared to a ZFN targeting the same gene pair [19]. They pointed out that there are more possible TALEN target sites (223) than ZFN target sites (3) in the gene pair, making TALEN the more flexible approach. Coeval showing that TALENs are more specific compared to ZFNs since a single nucleotide mismatch between SurA and SurB in one TALEN pair was leading to a fourfold decrease in activity for cutting SurB. A ZFN containing two single nucleotide mismatches showed the same Current Opinion in Biotechnology 2015, 32:47–53
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efficiency for both genes. By offering a target with long homology arms to SurB, they were able to trigger HR into SurA and SurB, showing that in some cases the outcome is a combination of HR and NHEJ, probably due to repeated binding of the TALEN pair. Recently, Dan Voytas group published a new method for genome engineering in plant cells using SSNs (ZFNs, TALENs and CRISPR/Cas9) which guides the DNA repair to the HR pathway [20]. They used geminivirus based replicons (GVRs) for the delivery of SSNs as well as a DNA repair template into cells. They developed both two and one vector systems, and showed that GVR replication is more powerful when a transacting replication protein (REP) is present. It was possible to tremendously increase the number of transcripts using the GVR system, thereby enhancing the number of gene targeting events by two orders of magnitude compared to conventional Agrobacterium driven T-DNA delivery. Interestingly, they found a lower amount of NHEJ events in the cells. This effect was caused by a combination of targeted DSBs, high replication of repair template and a pleiotropic effect of REP. With this technique it is possible to regenerate plants with a desired change in the DNA sequence in less than 6 weeks.
TALENs in metazoa In metazoans, like in plants, SSNs are providing big opportunities for specific and easy genome editing. TALENs were recently used in a broad range of metazoans from simple organisms like Drosophila [21], Xenopus [22], Branchiostoma [23], and Zebrafish [24] to more complex vertebrates like mice [25], rats [26], pigs [27] and non-human primates [28]. In most of these organisms TALENs have been used to disrupt genes by NHEJ to study gene knockouts or establish the system in the organisms such as Branchiostoma [23], but human disease models can also be studied using TALENs. TALENs in metazoa can be used in cell lines as well as in whole organisms without access to embryonic stem cells. To introduce TALEN into organisms, fertilized one-cell embryos are microinjected with TALEN mRNA into the male pronucleus and/or into the cytoplasm [29,30]. Cell lines are transfected by either electroporation or commercial available transfection reagents (for a review see [31]). Using TALEN-targeted fibroblast cell lines as nuclear donors for somatic cell nuclear transfer is also possible [32]. In contrast to plants, animal systems have been used to introduce larger constructs into genome by HR. Moghaddassi et al. managed to introduce the 11.5 kb long gene for human serum albumin (HSA) into the gene sequence of bovine serum albumin (BSA) with a high efficiency (11%). They disrupted the gene for BSA and the cattle produced HSA instead [32]. Since methods developed first in animal systems are often adapted to
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plants [18] such adaptation of genes could be performed in plants in the near future too.
Modifications of the SSN-system Based on former experiences with ZFN, several groups modified the standard system consisting of two TALEbinding modules each fused to an unspecific FokI nuclease acting as a dimer [7,33,34]. One of the obvious modifications to reduce the size of TALENs, was the construction of a single chain TALEN in which the two FokI subunits are fused together by a 95 amino acid polypeptide linker [35]. Such a modification makes it much easier to design new TALENs as one has to choose only one binding site instead of two. A second very interesting modification is the inactivation of one of the two present FokI subunits, resulting in a so called nickase as only one FokI protein introduces a single strand DNA break whereas the other is still present in the dimer but remains inactive on the DNA. These kind of nickases have been developed successfully for ZFN and recently also for TALENs and CRISPR/Cas9, and used in animal systems or plants [36–38]. In a slightly different approach Beurdeley et al. fused the homing endonuclease I-TevI C-terminal, to a TALE binding scaffold which eventually resulted in a combination of a single chain TALEN and a nickase function of this enzyme called compact TALEN (cTALEN) [39]. Besides the smaller size and easier design of this cTALEN it has been shown in general that the introduction of a nick instead of a DSB alters the ratio between NHEJ and HR reasonably in the direction of HR [37,38,40]. This usually leads to much less cytotoxicity of nickases in comparison to DSB inducing nucleases [40]. Coeval with these changes, the backbone architecture of the TALENs is still undergoing some adaptations. Sakuma et al. alternated the non RVD region of the DNA binding domain of the TALENs by changing two amino acids flanking the RVD [41]. These changes improved the activity of the TALEN pair in most cases using endogenous genes as well as eGFP in transgenic zebrafish. Zheng et al. dispensed the last half RVD of the TALENs without lowering the efficiency of two TALEN pairs used for GUS genes in N. benthamiana by showing that it does not matter if the last half of RVD is enhanced to a full RVD or just left away; this should simplify the assembly of TALENs [42]. Simultaneously with the number of structural changes, the requirements for the TALEN design are changing to keep up to date with a number of bioinformatic-tools to help the operator create effective TALENs and avoid pitfalls in the design. Most assembly methods have individual requirements in the design of TALEN pairs. Booher and Bogdanove recently published a short overview of the commonly used bioinformatic tools [43]. Most www.sciencedirect.com
Plant genome editing via sequence specific nucleases Sprink, Metje and Hartung 51
of them are open access and free to use. They listed tools for many of the commonly used assembly protocols such as golden gate by Cermak et al., Mussolino et al. and other free available assembly sets [33,44].
First commercial applications One commercial application of targeted genome engineering using SSNs is the stacking of already used traits in crop plants, which is currently a tricky task due to segregation of traits in the progeny. Two companies have already published their first results for trait stacking using SSNs. D’ Halluin et al. (Bayer crop science) designed a meganuclease for adding two herbicide resistance genes into a cotton line containing a Cry2Ae and a bar gene. Most identified events were single events in which the traits were correctly stacked without additional insertion of the resistance gene in the genome or insertion of the meganuclease gene at all. In the progeny the stacked genes showed inheritance in a normal Mendelian manner as a single locus [45]. Another trait stacking approach was done in maize by Ainley et al. (Dow AgroSciences, Sangamo BioSciences) using ZFNs [46]. They designed so called trait landing pads (TLPs) adjacent to a transgene, by adding a novel combination of known ZFN recognition sites from other species to a transgene, thereby enabling the stacking of multiple genes to a given locus only limited by the number of TLPs. A transgenic maize line containing a pat gene with adjacent TLP was bombarded with a ZFNpair cutting inside the TLP and a donor DNA with a region homologous to the TLP region containing a new TLP site. They could show that it is possible to use ZFN for trait stacking, but mentioned that a limitation of the technology today is the use of selectable markers [46].
Conclusions A lot of successful genome editing work in plants and other organisms has been done using TALENs and other SSNs like CRISPR/Cas9. As both techniques are in principle leading to the same result, a targeted DSB in the genome of the organism, practical considerations as well as individual requirements of the commonly used systems will decide which of them can be used for a specific purpose. The existing system of SSNs and any newly developed one will definitely have an enormous impact on plant breeding in research and development. These SSN systems provide researchers and plant breeders with a fast growing toolbox which can be rapidly adapted to address specific questions. The ability to introduce a specific alteration in the genome will open up the door to precisely modify or introduce a desired trait in an elite plant used for breeding or production. In addition to this, the possibility of stacking such events can be used for multiresistant trait development and production of different pharmaceutically relevant proteins in a much more controlled manner. Nevertheless, there is www.sciencedirect.com
still some work to do, as to date nobody can predict how efficiently an SSN will cut at the specifically chosen position. The mechanisms influencing the effectiveness of the respective SSN system should be addressed in future approaches. Several works have been performed with selected crop plants, though most of it is still proof of concept experiments. Further analyses should be done, addressing multiple species and cultivars to show if the underlying mechanisms are common for all plants or not. In addition to this, a comprehensive screening for off target activity in crop plants with large genomes would be helpful to address issues of effectiveness, which might be connected to such unintended side effects. Since a few years there is a worldwide discussion about the application of TALENs and other SSNs in terms of whether such techniques are under the scope of GMO regulation or not [47]. A clear decision has not been made but several institutions like the European Food Safety Authority (EFSA) or the European Academies Science Advisory Council (EASAC) have made suggestions how to handle this technique [48,49]. The US department of Agriculture decides in a case-by-case manner and stated on requests that small mutations induced by meganucleases and ZFNs fall outside their scope of regulation (for details see the USDA website topic: letters of inquiry) [50]. A case statement concerning plants produced by TALEN or CRISPR/Cas9 technique is still missing but is expected in the near future. In summary a common view of the application of SSNs in light of GMO regulation is: A DSB induction by an SSN followed by cellular repair without the integration of foreign recombinant DNA (e.g. mismatch repair and most kinds of NHEJ involving only point mutations and small insertion/deletions) is categorized as ZFN-1. ZFN-2 is the same as ZFN-1 but accompanied by a non integrated template DNA which drives repair into a certain direction involving intended nucleotide changes. These two techniques should not be regulated as GMO-technique as the resulting organism does not contain recombinant DNA and is indistinguishable from organisms obtained by conventional mutagenesis using chemicals or irradiation [48,51,52]. The third category of SSN-technique, ZFN-3, aims for a directed and specific gene insertion or exchange using homologous recombination (see Figure 1a) and therefore the majority of experts agree that this will result in an organism containing recombinant DNA and must be regulated as a GMO [48,51,52].
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest We apologize to all colleagues for whose we were not being able to cite, all relevant and earlier papers because of space limitations and Current Opinion in Biotechnology 2015, 32:47–53
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the focus of the article on recent research. We would like to thank Allan West for thorough reading of the manuscript. This work was partly funded by the Deutsche Forschungsgemeinschaft (DFG) SPP1384.
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33. Cermak T, Doyle EL, Christian M, Wang L, Zhang Y, Schmidt C, Baller JA, Somia NV, Bogdanove AJ, Voytas DF: Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res 2011, 39:e82. 34. Li T, Huang S, Jiang WZ, Wright D, Spalding MH, Weeks DP, Yang B: TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain. Nucleic Acids Res 2011, 39:359-372. 35. Sun N, Zhao HM: A single-chain TALEN architecture for genome engineering. Mol Biosyst 2014, 10:446-453. 36. Kim E, Kim S, Kim DH, Choi B-S, Choi I-Y, Kim J-S: Precision genome engineering with programmable DNA-nicking enzymes. Genome Res 2012, 22:1327-1333. 37. Fauser F, Schiml S, Puchta H: Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana. Plant J 2014, 79:348-359.
43. Booher NJ, Bogdanove AJ: Tools for TAL effector design and target prediction. Methods 2014, 69:121-127. 44. Mussolino C, Morbitzer R, Lu¨tge F, Dannemann N, Lahaye T, Cathomen T: A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity. Nucleic Acids Res 2011, 39:9283-9293. 45. D’Halluin K, Vanderstraeten C, Van Hulle J, Rosolowska J, Van Den Brande I, Pennewaert A, D’Hont K, Bossut M, Jantz D, Ruiter R et al.: Targeted molecular trait stacking in cotton through targeted double-strand break induction. Plant Biotechnol J 2013, 11:933-941. 46. Ainley WM, Sastry-Dent L, Welter ME, Murray MG, Zeitler B, Amora R, Corbin DR, Miles RR, Arnold NL, Strange TL et al.: Trait stacking via targeted genome editing. Plant Biotechnol J 2013, 11:1126-1134. A study presenting recent findings on trait stacking in crop plants using ZFNs or meganucleases. The authors present a way of combining traits with each other by using introducing known binding sites of SSNs adjacent to the trait.
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41. Sakuma T, Ochiai H, Kaneko T, Mashimo T, Tokumasu D, Sakane Y, Suzuki K-i, Miyamoto T, Sakamoto N, Matsuura S et al.: Repeating pattern of non-RVD variations in DNA-binding modules enhances TALEN activity. Sci Rep 2013, 3:3379. 42. Zheng CK, Wang CL, Zhang XP, Wang FJ, Qin TF, Zhao KJ: The last half-repeat of transcription activator-like effector (TALE) is dispensable and thereby TALE-based technology can be simplified. Mol Plant Pathol 2014, 15:690-697.
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Current Opinion in Biotechnology 2015, 32:47–53
ScienceDirect Plant genome editing by novel tools: TALEN and other sequence specific nucleases Thorben Sprink1, Janina Metje2 and Frank Hartung1 Genome editing technologies using sequence specific nucleases (SSNs) became a tremendously powerful and precise tool for reverse genetic approaches and applied biology. Transcription activator-like effector nucleases (TALENs) in particular, consisting of a free designable DNA binding domain and a nuclease, have been exploited today by a huge number of approaches in many different organisms. The convenience of designing the DNA binding domain and straightforward protocols for their assembly, as well as the broad number of applications in different scientific fields made it Natures method of the year 2011. TALENs act as molecular scissors by introducing double strand breaks (DSBs) to the DNA at a given location. The DSBs are subsequently repaired by the cell itself using different repair pathways such as nonhomologous end joining (NHEJ) or homologous recombination (HR). These mechanisms can lead to deletions, insertions, replacements or larger chromosomal rearrangements. By offering a template DNA it is possible to channel the repair in direction of HR. In this article we review the recent findings in the field of SSN approaches with emphasis on plants. Addresses 1 Julius Ku¨hn Institut, Institute for Biosafety in Plant Biotechnology, Erwin Baur-Str. 27, 06484 Quedlinburg, Germany 2 Max Plank Institute for Biophysical Chemistry, Research Group Autophagy, Am Fassberg 11, 37077 Go¨ttingen, Germany Corresponding author: Hartung, Frank ([email protected])
Current Opinion in Biotechnology 2015, 32:47–53 This review comes from a themed issue on Plant biotechnology Edited by Inge Broer and George N Skaracis
http://dx.doi.org/10.1016/j.copbio.2014.11.010 0958-1669/# 2014 Elsevier Ltd. All right reserved.
given location (for a glossary see Box 1). In the past, targeted genome modifications in plants were far away from any reasonable efficiency [1], but since it became possible to introduce double strand breaks (DSBs) into the DNA at a defined specific site using SSNs such as zinc finger nucleases (ZFNs) [2] or meganucleases [3], the efficiency has been raised tremendously (for recent review see [4]). Unfortunately, both techniques possess an unfortunate drawback, as the precise design of the DNA binding site is somewhat limited due to the architecture of these SSNs. These things changed dramatically since Boch et al. and others unveiled the code for transcription activator-like effectors (TALE) in 2009 [5,6]. In 2010 the first transcription activator-like effector nucleases (TALENs) combining the DNA binding part of a TALE and an unspecific nuclease were published by the group of Dan Voytas [7,8]. Like ZFNs, TALENs are based on a fusion of a designed DNA binding domain with the nuclease FokI allowing the introduction of a DSB at a given location. Such a DSB activates two main repair pathways in the cell, non homologous end joining (NHEJ) and homologous recombination (HR) (Figure 1). NHEJ is mostly imprecise, leading to insertions or deletions at the repair site and in most cases, a disrupted gene sequence leading to a loss of function mutation. Real gene targeting can be achieved by HR where the DSB is repaired by a homologous template such as the sister chromatid, the homologous chromosome or an additionally presented homologous template DNA [9]. The newest achievement in the field of SSNs is the use of clustered regularly interspaced short palindromic repeats (CRISPRs) fused with a Cas9 nuclease [10], reviewed by Kamoun et al. in this issue. In this review we focus on the recent developments in the field of SSNs, focusing on TALENs, ZFNs and meganucleases during the last two years.
Plant genome editing by SSNs
Introduction Over the past decades, agriculture has had to change to meet the challenge of a growing number of people in the world in a shrinking crop area. New technologies in breeding like the green revolution and hybrid plants helped to increase agricultural productivity. Recent developments in genome engineering using sequence specific nucleases (SSNs) to regulate the site of genomic modification could help to speed up breeding processes by altering specific genes at a chosen site or by introduction of new traits at a www.sciencedirect.com
So far several groups have successfully modified plant genes in vivo by using TALENs to induce DSBs and repair by NHEJ. One of the broadest approaches to edit endogenous plant genes has been done by Shan et al. In total, they successfully targeted seven genes in Brachypodium and four in rice by specific TALEN pairs. The resulting mutations were predominantly short deletions of 1–20 nt, indicating DNA repair by the NHEJ pathway [9,11]. In addition to this Shan et al. also deleted larger regions of rice DNA through application of two TALEN pairs with specific cutting sites more than thousand nucleotides apart. This approach shows the potential usefulness of SSNs for selective deletion or Current Opinion in Biotechnology 2015, 32:47–53
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Box 1 SSNs: Sequence specific nucleases, are a combination of a DNA recognition part and a nuclease part. Typical SSNs are CRISPR/Cas9, TALENs, ZFNs and meganucleases. CRISPR/Cas9 (CRISPR associated (Cas) system): Clustered regularly interspaced short palindromic repeats allow bacteria an adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNA) and trans activating crRNA (tracrRNA) to guide the silencing of invading nucleic acids. Three types of CRISPR/Cas system exist. Type II in which Cas9 is guided by a crRNA–tracrRNA target identification to cut DNA at the identified region, is used in an altered form for genome engineering in a couple of organism. TALENs: Transcription activator like effector nucleases are a combination of the catalytic domain of an endonuclease most frequently FokI fused with the DNA binding domain derived from transcription activator like effectors from Xanthomonas spec. TALENs consist of multiple repeats of a 33–35 amino acids long sequence containing a so-called repeat variable diresidue (RVD) which are the amino acids nos. 12 and 13. Each repeat binds to a certain single base pair. TALENs are commonly used as a pair to introduce double strand breaks (DSBs) to the DNA, as FokI only introduces DSBs as a dimer. RVD: Repeat variable diresidue, the 12th and 13th position in a TALE domain, defining which base pair will preferential be bound (HD=C, NG=T, NI=A, NS=A, NN=G/A NK=G). ZFN: Zinc finger nucleases are a combination of a zinc finger DNA binding domain with an endonuclease most frequently FokI. In the zinc finger DNA binding domain the a-helices define which three base pairs will be recognized. In nature there are >1000 zinc finger domains known. Typical ZFNs consists of minimal 3 zinc finger domains each recognizing 3 bp. Nickase: An enzyme that introduces single strand breaks (nicks) to the DNA. A FokI dimer with one inactivated FokI is often used as a nickase in SSN approaches. Meganuclease: Naturally occurring endonucleases from different organisms such as bacteria, fungi and algae. They have a long DNA recognition site with variable size differing between 12 and 30 bp. Naturally occurring meganucleases can be modified by introducing changes to the amino acids found in the recognition site of the protein to adapt for restriction of a specifically chosen sequence. DSB: A double strand break is a form of DNA damage, in which both DNA strands are broken. DSBs are the result of for example, endonuclease restriction or natural influences such as irradiation or chemicals. During meiosis, DSBs are specially introduced to the DNA by SPO11 to initiate recombination processes. cNHEJ: Canonical or classical non-homologous end joining is the first major DSB repair mechanism in somatic cells. It is called non homologous because the break ends are ligated without the use of a homologous sequence such as the sister chromatid or the homologous chromosome. The breaks are either directly ligated or short microhomologies on the single stranded overhangs are used for the ligation. NHEJ is often imprecise, leading to small insertions or deletions at the repaired site. aNHEJ: Alternative non-homologous end joining, is a pathway which uses microhomologies present on both sites of the DSB, aNHEJ often leads to larger deletions at the site of DSB repair. HR: Homologous recombination is the second major DSB repair mechanism using various pathways leading in some cases to the formation of double holliday junctions, for example crossover between homologous chromosomes in meiosis. In contrast to NHEJ, HR uses a homologous template to repair the DSB, this is often the sister chromatid or the homologous chromosome. In an SSN approach homologous sequences can be additionally introduced to act as a repair template to insert single or multiple transgenes as well as to induce single nucleotide changes to a specific gene.
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rearrangement of genes or even gene clusters. Such deletion or rearrangement of gene clusters such as R-genes has been achieved in Arabidopsis by Qi et al. [12]. They used ZFNs for this approach and successfully introduced large deletions (from 4.5 kb up to 9 Mb) in seven independent endogenous loci in Arabidopsis in a range of 0.1–3% of the cases, therefore proving the applicability of large gene deletions by SSNs in plants. Qi et al. also found inversions or duplications of such large genomic regions but in a frequency below 0.1%. Recent studies in the field focused on gene editing in crop plants, such as Glycine max [13]. Haun et al. disrupted two fatty desaturase genes (FAD2-1A and FAD2-1B) using TALENs, to achieve a soybean with an improved number of monosaturated oils with longer shelf life. They managed to create a homozygous knockout line for both genes, but lacking the TALEN construct by crossing the transgenic plants with their wild type parents. Wang et al. were able to present a TALEN approach addressing three homoeoalleles in hexaploid bread wheat by a single TALEN pair addressing a conserved domain in the three alleles, to improve heritable resistance to powdery mildew [14]. They managed to create TALEN-free resistant plants in three generations by selfing one plant carrying a heterozygous mutation for each homoeoallele, showing the large opportunities for breeding programs offered by SSNs. Two other groups using TALEN approaches focused on the genome modification of barley, another important crop. Wendt et al. used a one vector system and immature embryos focusing on an endogenous barley gene promoter. They reached a transformation rate of 20%, mostly resulting in NHEJ events leading to small deletions [15]. Gurushidze et al. focused on the knockout of GFP in transgenic haploid barley cells using a slightly different approach in which both TALEN arms are brought separately into the cells. They also reached up to 20% frequency of mutation induction in their transgenic lines. Interestingly, they found deletions in the GFP sequence even in plants where only one of the two TALEN arms was stably integrated, showing a transient activity of the respective other TALEN in the plants [16]. In a proof of concept experiment using TALENs, Lor et al. created knockout lines of the PROCERA gene in tomato, showing that it is possible to create tomato plants which contain only the desired mutations, and not the TALEN construct. For this approach they used an estrogen inducible promoter to control the risk of TALEN cytotoxicity. Spraying of plants (or parts of them) with estradiol resulted in a very low amount of cells carrying the mutation. Lor et al. subsequently immersed the cotyledons of T1 plants, three times in an estradiol containing solution at weekly intervals. Using this modified protocol they managed to produce plants containing www.sciencedirect.com
Plant genome editing via sequence specific nucleases Sprink, Metje and Hartung 49
Figure 1
Double strand break (DSB) initiation DSB (a)
Homologous recombination (HR)
(b)
Non-homologous end joining (NHEJ)
Strand invasion
End capture
Strand annealing > 100 bp
Various HR pathway resolutions
Single strand annealing (SSA)
Gene addition/ replacement/ adaption
some bp
Classical nonhomologous end joining (cNHEJ) Alternative non-homologous end joining(aNHEJ)
Gene disruption by deletion or insertions of a few bps Current Opinion in Biotechnology
The two main cellular DSB repair-pathways. (a) Homologous recombination (HR) which is divided into the resolution of double holliday junctions (dHJ), created after strand invasion of the broken strand, by various dHJ resolution pathways and in the relatively simple single strand annealing (SSA) pathway where a tandem homology on both ends of the break is used for repair. This can be a repeated sequence as well as an external DNA fragment transferred into the cell. The common outcomes of the pathways are gene addition, replacement or gene adaptation. (b) Non homologous end joining (NHEJ). NHEJ can be divided into two main branches, the classical (also called canonical) NHEJ, which ligates broken DNA ends together without any homology and the alternative non homologous end joining, which ligates broken DNA ends using microhomologies of two nucleotides on the single stranded break ends. The common outcome in both cases is a disruption of the gene by deletions or insertions of a few nucleotides.
the desired mutations and could show that these mutations were stably inherited and the TALEN construct was lost by segregation [17].
In planta gene targeting by SSNs In contrast to repair by NHEJ which mostly leads to loss of function mutations, the application of HR for gene targeting is more challenging. Fauser et al. used a meganuclease based approach for guiding DSB repair to HR by adapting a system formerly used in Drosophila for in vivo gene targeting [18]. They created individual lines transformed either with a target sequence (a truncated GUS gene flanked by two ISceI sites) or a donor sequence (the rest of the GUS gene including homology to the target construct also flanked by two ISceI sites). After creating homozygous lines for both targets they crossed the lines and the resulting progeny www.sciencedirect.com
was then crossed further to a line expressing ISceI. The final outcome was a successful gene targeting via HR with approximately 1% efficiency. Remarkably, additional integration of the target elsewhere in the genome by NHEJ was not found in any of the analyzed plants. Zhang and colleagues had a closer look at the events of a TALEN pair targeting the duplicated acetolactate synthase genes (SurA and SurB) in N. tabacum, compared to a ZFN targeting the same gene pair [19]. They pointed out that there are more possible TALEN target sites (223) than ZFN target sites (3) in the gene pair, making TALEN the more flexible approach. Coeval showing that TALENs are more specific compared to ZFNs since a single nucleotide mismatch between SurA and SurB in one TALEN pair was leading to a fourfold decrease in activity for cutting SurB. A ZFN containing two single nucleotide mismatches showed the same Current Opinion in Biotechnology 2015, 32:47–53
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efficiency for both genes. By offering a target with long homology arms to SurB, they were able to trigger HR into SurA and SurB, showing that in some cases the outcome is a combination of HR and NHEJ, probably due to repeated binding of the TALEN pair. Recently, Dan Voytas group published a new method for genome engineering in plant cells using SSNs (ZFNs, TALENs and CRISPR/Cas9) which guides the DNA repair to the HR pathway [20]. They used geminivirus based replicons (GVRs) for the delivery of SSNs as well as a DNA repair template into cells. They developed both two and one vector systems, and showed that GVR replication is more powerful when a transacting replication protein (REP) is present. It was possible to tremendously increase the number of transcripts using the GVR system, thereby enhancing the number of gene targeting events by two orders of magnitude compared to conventional Agrobacterium driven T-DNA delivery. Interestingly, they found a lower amount of NHEJ events in the cells. This effect was caused by a combination of targeted DSBs, high replication of repair template and a pleiotropic effect of REP. With this technique it is possible to regenerate plants with a desired change in the DNA sequence in less than 6 weeks.
TALENs in metazoa In metazoans, like in plants, SSNs are providing big opportunities for specific and easy genome editing. TALENs were recently used in a broad range of metazoans from simple organisms like Drosophila [21], Xenopus [22], Branchiostoma [23], and Zebrafish [24] to more complex vertebrates like mice [25], rats [26], pigs [27] and non-human primates [28]. In most of these organisms TALENs have been used to disrupt genes by NHEJ to study gene knockouts or establish the system in the organisms such as Branchiostoma [23], but human disease models can also be studied using TALENs. TALENs in metazoa can be used in cell lines as well as in whole organisms without access to embryonic stem cells. To introduce TALEN into organisms, fertilized one-cell embryos are microinjected with TALEN mRNA into the male pronucleus and/or into the cytoplasm [29,30]. Cell lines are transfected by either electroporation or commercial available transfection reagents (for a review see [31]). Using TALEN-targeted fibroblast cell lines as nuclear donors for somatic cell nuclear transfer is also possible [32]. In contrast to plants, animal systems have been used to introduce larger constructs into genome by HR. Moghaddassi et al. managed to introduce the 11.5 kb long gene for human serum albumin (HSA) into the gene sequence of bovine serum albumin (BSA) with a high efficiency (11%). They disrupted the gene for BSA and the cattle produced HSA instead [32]. Since methods developed first in animal systems are often adapted to
Current Opinion in Biotechnology 2015, 32:47–53
plants [18] such adaptation of genes could be performed in plants in the near future too.
Modifications of the SSN-system Based on former experiences with ZFN, several groups modified the standard system consisting of two TALEbinding modules each fused to an unspecific FokI nuclease acting as a dimer [7,33,34]. One of the obvious modifications to reduce the size of TALENs, was the construction of a single chain TALEN in which the two FokI subunits are fused together by a 95 amino acid polypeptide linker [35]. Such a modification makes it much easier to design new TALENs as one has to choose only one binding site instead of two. A second very interesting modification is the inactivation of one of the two present FokI subunits, resulting in a so called nickase as only one FokI protein introduces a single strand DNA break whereas the other is still present in the dimer but remains inactive on the DNA. These kind of nickases have been developed successfully for ZFN and recently also for TALENs and CRISPR/Cas9, and used in animal systems or plants [36–38]. In a slightly different approach Beurdeley et al. fused the homing endonuclease I-TevI C-terminal, to a TALE binding scaffold which eventually resulted in a combination of a single chain TALEN and a nickase function of this enzyme called compact TALEN (cTALEN) [39]. Besides the smaller size and easier design of this cTALEN it has been shown in general that the introduction of a nick instead of a DSB alters the ratio between NHEJ and HR reasonably in the direction of HR [37,38,40]. This usually leads to much less cytotoxicity of nickases in comparison to DSB inducing nucleases [40]. Coeval with these changes, the backbone architecture of the TALENs is still undergoing some adaptations. Sakuma et al. alternated the non RVD region of the DNA binding domain of the TALENs by changing two amino acids flanking the RVD [41]. These changes improved the activity of the TALEN pair in most cases using endogenous genes as well as eGFP in transgenic zebrafish. Zheng et al. dispensed the last half RVD of the TALENs without lowering the efficiency of two TALEN pairs used for GUS genes in N. benthamiana by showing that it does not matter if the last half of RVD is enhanced to a full RVD or just left away; this should simplify the assembly of TALENs [42]. Simultaneously with the number of structural changes, the requirements for the TALEN design are changing to keep up to date with a number of bioinformatic-tools to help the operator create effective TALENs and avoid pitfalls in the design. Most assembly methods have individual requirements in the design of TALEN pairs. Booher and Bogdanove recently published a short overview of the commonly used bioinformatic tools [43]. Most www.sciencedirect.com
Plant genome editing via sequence specific nucleases Sprink, Metje and Hartung 51
of them are open access and free to use. They listed tools for many of the commonly used assembly protocols such as golden gate by Cermak et al., Mussolino et al. and other free available assembly sets [33,44].
First commercial applications One commercial application of targeted genome engineering using SSNs is the stacking of already used traits in crop plants, which is currently a tricky task due to segregation of traits in the progeny. Two companies have already published their first results for trait stacking using SSNs. D’ Halluin et al. (Bayer crop science) designed a meganuclease for adding two herbicide resistance genes into a cotton line containing a Cry2Ae and a bar gene. Most identified events were single events in which the traits were correctly stacked without additional insertion of the resistance gene in the genome or insertion of the meganuclease gene at all. In the progeny the stacked genes showed inheritance in a normal Mendelian manner as a single locus [45]. Another trait stacking approach was done in maize by Ainley et al. (Dow AgroSciences, Sangamo BioSciences) using ZFNs [46]. They designed so called trait landing pads (TLPs) adjacent to a transgene, by adding a novel combination of known ZFN recognition sites from other species to a transgene, thereby enabling the stacking of multiple genes to a given locus only limited by the number of TLPs. A transgenic maize line containing a pat gene with adjacent TLP was bombarded with a ZFNpair cutting inside the TLP and a donor DNA with a region homologous to the TLP region containing a new TLP site. They could show that it is possible to use ZFN for trait stacking, but mentioned that a limitation of the technology today is the use of selectable markers [46].
Conclusions A lot of successful genome editing work in plants and other organisms has been done using TALENs and other SSNs like CRISPR/Cas9. As both techniques are in principle leading to the same result, a targeted DSB in the genome of the organism, practical considerations as well as individual requirements of the commonly used systems will decide which of them can be used for a specific purpose. The existing system of SSNs and any newly developed one will definitely have an enormous impact on plant breeding in research and development. These SSN systems provide researchers and plant breeders with a fast growing toolbox which can be rapidly adapted to address specific questions. The ability to introduce a specific alteration in the genome will open up the door to precisely modify or introduce a desired trait in an elite plant used for breeding or production. In addition to this, the possibility of stacking such events can be used for multiresistant trait development and production of different pharmaceutically relevant proteins in a much more controlled manner. Nevertheless, there is www.sciencedirect.com
still some work to do, as to date nobody can predict how efficiently an SSN will cut at the specifically chosen position. The mechanisms influencing the effectiveness of the respective SSN system should be addressed in future approaches. Several works have been performed with selected crop plants, though most of it is still proof of concept experiments. Further analyses should be done, addressing multiple species and cultivars to show if the underlying mechanisms are common for all plants or not. In addition to this, a comprehensive screening for off target activity in crop plants with large genomes would be helpful to address issues of effectiveness, which might be connected to such unintended side effects. Since a few years there is a worldwide discussion about the application of TALENs and other SSNs in terms of whether such techniques are under the scope of GMO regulation or not [47]. A clear decision has not been made but several institutions like the European Food Safety Authority (EFSA) or the European Academies Science Advisory Council (EASAC) have made suggestions how to handle this technique [48,49]. The US department of Agriculture decides in a case-by-case manner and stated on requests that small mutations induced by meganucleases and ZFNs fall outside their scope of regulation (for details see the USDA website topic: letters of inquiry) [50]. A case statement concerning plants produced by TALEN or CRISPR/Cas9 technique is still missing but is expected in the near future. In summary a common view of the application of SSNs in light of GMO regulation is: A DSB induction by an SSN followed by cellular repair without the integration of foreign recombinant DNA (e.g. mismatch repair and most kinds of NHEJ involving only point mutations and small insertion/deletions) is categorized as ZFN-1. ZFN-2 is the same as ZFN-1 but accompanied by a non integrated template DNA which drives repair into a certain direction involving intended nucleotide changes. These two techniques should not be regulated as GMO-technique as the resulting organism does not contain recombinant DNA and is indistinguishable from organisms obtained by conventional mutagenesis using chemicals or irradiation [48,51,52]. The third category of SSN-technique, ZFN-3, aims for a directed and specific gene insertion or exchange using homologous recombination (see Figure 1a) and therefore the majority of experts agree that this will result in an organism containing recombinant DNA and must be regulated as a GMO [48,51,52].
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest We apologize to all colleagues for whose we were not being able to cite, all relevant and earlier papers because of space limitations and Current Opinion in Biotechnology 2015, 32:47–53
52 Plant biotechnology
the focus of the article on recent research. We would like to thank Allan West for thorough reading of the manuscript. This work was partly funded by the Deutsche Forschungsgemeinschaft (DFG) SPP1384.
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