University of Groningen Development of genetic manipulation tools in Macrostomum lignano for dissection of molecular mechanisms of regeneration Wudarski, Jakub

University of Groningen

Development of genetic manipulation tools in Macrostomum lignano for dissection of

molecular mechanisms of regeneration

Wudarski, Jakub

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CHAPTER 4

Proof of principle for

transposon-mediated transgenesis in

_Macrostomum

lignano

Jakub Wudarski1*_{, Kirill Ustyantsev}2*_{, Eugene Berezikov}1,2

1_{European Research Institute for the Biology of Ageing, University of Groningen, University Medical Center Groningen,}

Groningen, The Netherlands.

2 Institute of Cytology and Genetics, Prospekt Lavrentyeva 10, 630090 Novosibirsk, Russia. * Authors contributed equally

ABSTRACT

With the recent progress in the field of flatworm transgenics Macrostomum lignano has become

a great model organism to address questions that were previously impossible to answer. Random integration is currently the only available method used for genetic manipulation in the flatworm. Despite its efficiency there is room and need for improvement. Transposon mediated transgenesis allows for a more controlled approach, enabling easy mapping of the integration site, large cargo insertions and possibility of mutagenesis studies. Here we present for the first time the use of piggyBac transposon to create a stable transgenic line in M. lignano. We also discuss implications and future perspectives for further development of

the transgenic techniques in this animal.

INTRODUCTION

Macrostomum lignano is a free-living flatworm that is gaining increased attention as a powerful

model organism. Thanks to its high regeneration capabilities and the availability of a robust transgenesis method it can be used as a testbed in many research areas, including stem cell biology, regeneration and ageing [1].

Although we have entered the era of CRISPR/Cas9 based genome editing, other methods are still valid. Viruses, TALENs, integrases, transposons and other approaches are widely used in many laboratories and will remain useful as long as they offer something unique as to what the growing family of CRISPR-related techniques has to offer [2, 3].

Transposons or transposable elements (TEs), are a good example of a versatile tool for genetic research. Currently, several transposon systems are commonly used, each having its

applications niche, with piggyBac (PB), Tol2, Mos1, minos and P-element being amongst the

most popular [3–6]. TEs are classified as mobile genetic elements, meaning that they can change their location and/or copy number independently from other genes. They are often viewed as genomic parasites, can comprise large percentages of a species' genome and have been identified in all known sequences of prokaryotic and eukaryotic organisms. The main classification for TEs is based on transposition mechanism that they use [7].

The class I transposable elements, also known as retrotransposons, use an RNA intermediate and most often move via ‘copy-and-paste’ mechanism, also referred to as replicative. We can distinguish two groups in this class: retrotransposons that have their retroelement main fragment flanked with Long Terminal Repeats (LTR) and non-LTR retrotransposons that lack the flanking sequences [8].

The class II TEs are often referred to as DNA transposons. The majority of class II TEs move by using a ‘cut-and-paste’ mechanism, so there is no replication of the sequence that is being moved. This process is enabled by the activity of a specific enzyme called transposase. Transposase recognizes specific binding sites present in inverted terminal repeats (ITRs) – DNA sequences flanking the mobile element – and removes the fragment to insert it into another location. This new target site is specific for the transposon and is usually only a few

nucleotide long, making the process semi-random [9]. Normally, the source sequence of the transposase is located between the ITRs. However, it can be exchanged for another sequence, which unable TE to change its location unless the appropriate transposase enzyme is supplied externally in trans [3].

The above characteristics of the class II DNA transposons make them a useful tool in genetic studies. Absence of specific sequence requirements for the insert, ease of use, high activity and high cargo capacity (i.e. can be used to insert large DNA fragments into the hosts' genome) are the main advantages of using transposons as vectors for genetic manipulation. However, this comes with the price of choosing the location for the cargo. Transposons are inserted in a semi-random manner, where the requirements for the target site are low [3]. Nevertheless, various studies, such as random wide range mutagenesis [10–12] or experiments requiring large cargo insertions [13], are easily done using transposons, and this approach is preferred to other available methods [3].

PiggyBac transposon has been first discovered and isolated from the genome of cabbage

looper moth (Trichoplusia ni) in the late 1980s [14]. It is a class II TE and was unique until

2000, when a second PB element was discovered [15]. The later advancements in genome sequencing revealed that the PB-like elements are actually widespread among animals, including moths, frogs and mammals. This lead to creating a separate PB transposon superfamily encompassing all of the similar mobile elements [16]. The main advantages of using PB over other TEs are its wide range of target organisms in which it works, ‘footprint-free’ excision, high biological security (they are unable to mobilize, unless provided with the PB transposase), high transposition efficiency and large cargo size. The main drawback is that the insertion site is semi-random and requires an TTAA site, so precise genome engineering is impossible to achieve [17].

Even though the possibility of creating transgenic lines in flatworms described in Chapter 2 of this thesis is on its own a major technological step in understanding the biology of these fascinating creatures, there is still a lot of room for improvement, and advances in this field would prove very useful. Although at the early stages in the development of

a transgenesis method in M. lignano the possibility to use Minos transposon system was

explored, it appeared that random integration of DNA construct occurred with relatively

high frequencies and no Minos-mediated integration was observed [18], and further attempts

for the development of transposon-based transgenesis were postponed. Thus, up to date random integration is the only published method to introduce foreign genetic material into

M. lignano genome [18]. The main downsides of using this method are lack of control over

the insertion location, use of irradiation during the process, and probability of multiple integrations with the possibility of inserts forming tandem repeats. These tandem repeats hinder the commonly used methods for mapping the genomic location of the inserts; they can also affect the stability of the inserts due to recombination events. Another potential drawback of the current approach is irradiation. Mild exposure to gamma rays causes double

stranded brakes in the DNA, stimulating the repair mechanism of the cell and is used to increase the efficiency of integration of transgenes in the genome [18]. However, the damage inflicted to the DNA can introduce alterations in the genome that are difficult to detect and correct for. The use of a transposon system can eliminate some of these problems. Single copy insertions into a semi-random location (e.g. TTAA) would significantly improve the control over the generation of M. lignano transgenic animals and enhance the insertional mutagenesis

toolkit that is currently available [17, 19].

Here we present a proof of principle for piggyBac-mediated transgenesis in M. lignano.

We introduced mNeonGreen [20] fluorescent protein under the control of the ubiquitous elongation factor alpha promoter that was flanked by PB ITRs and mobilized using PB transposase, demonstrating that transposon-based transgenesis can be performed in M. lignano.

RESULTS

Evaluating

piggyBac activity in vivo

The main characteristic of all transposon-based transgenesis approaches is the fact that it is a two-component system (Fig 1). Therefore, choosing the correct form in which both components would be delivered to the host cell was the first decision that needed to be made. The first component of the system, which is the gene of interest flanked by the ITRs, is almost always provided in the form of plasmid DNA. This approach has the advantage of being easy to prepare and work with. It also lowers the probability of random integration in comparison to linear DNA, a phenomenon that was previously described [18, 21]. Transposase is the second part of the mix. It can be usually supplied in three different forms: DNA, mRNA or protein, each having its pros and cons. However, in the case of PB there is no readily available protein, and we were unable to find any reliable research indicating successful transposition

using a recombinant piggyBac transposase. We were left with the choice between mRNA and

DNA. We have decided on using the transposase in the form of mRNA. This decision was made because of two reasons. The first one is the short half-life of mRNA, which prevents the transposon from being re-excised after being integrated into the genome target. The second reason was to prevent the transposase gene itself from being integrated into the genome, a risk that would have been present if the transposase in the form of DNA would have been used.

To deliver the transposon and transposase mix we have used the previously established microinjection approach [18]. There is no established guideline for the transposon to transposase ratio in the literature that we could use for our model organism, therefore we have decided to test different mixes of the transposon and transposase starting with the previously published values [6, 22]. We assumed that lowering the concentration of the plasmid will result in lower rate of random integration events, so we decided to use a 10 times lower amount of plasmid compared to the one used in our standard random integration protocol (Table 1). We have injected around 100 eggs for each transposase:transposon ratio

and counted all the eggs that were expressing the fluorescent protein as positive. We have also injected the plasmid without the transposase to estimate the transgenesis rates that were not the result of transposition. First, we tested whether PB transposase provided in the form of mRNA can excise its target from the plasmid in vivo in M. lignano. For this, we selected worms

with positive transgene expression, and used PCR and sequencing to investigate the structural changes in the injected plasmid (Fig. 2 B and C). The PCR test performed using plasmid-specific primers showed a band of around 947 bp, indicating the transposon excision from the plasmid backbone. Following this, a nested PCR was performed and the product of this PCR was sequenced. The excision site revealed that the transposase actively cuts the gene of interest and ITRs from the plasmid, leaving the canonical TTAA footprint (Fig. 2 C).

Transposase 5’ UTR 3’ UTR ITR ITR 13 bp TIR 19 bp sub-TIR 5’UTR - min 311bp 3’UTR - min 235 bp Transposase - 1785 bp GOI ITR ITR Transposase GOI ITR ITR Transposon GOI

ITR ITR Genomic DNA

Genomic DNA

CUT

PASTE

A

B

Table 1 The transposase to transposon ratios used in trial injections.

Transposase mRNA (ng/ul, helper)

Transposon KU#17 plasmid (ng/ul, donor)

Molar ratio Transposase :

Transposon eggs injected positive eggs

200 15 40 : 1 100 4 100 15 20 : 1 103 8 50 15 10 : 1 95 18 25 15 5 : 1 84 14 10 15 2 : 1 71 9 5.2 15 1 : 1 80 7 0 15 0 76 0

Figure 1.The PiggyBac

transpo-son system A. PiggyBac transposon

schematic overview. B. Transpos-able elements like piggyBac can

be ‘cut’ from their donor with the use of the enzyme transposase and ‘pasted’ into the target insertion site (TTAA).

Distinguishing random integration from transposons insertion

For further experiments we have chosen two mixes (‘blue’ and ‘pink’) because they had the transposase:transposon rate that gave the highest positive result (Fig 2. A). However, we needed to address another issue regarding the use of plasmid DNA during microinjections.

Previous research showed that M. lignano has a high rate of random integration events when

using both plasmid and linear DNA [18]. The underlying cause is yet to be established, however it might be connected to the nature of microinjections and the mechanical damage that is being imposed on the target genetic material during the process. The abovementioned complications may result in random integration that would be difficult to discriminate from transposon insertion. To be able to efficiently screen for the positive integration of the gene of interest via the transposon system and to distinguish it from random integration, we have constructed a plasmid with two different fluorescent proteins under control of two different promoters. One is placed inside ITRs and the other one outside (Fig 2 D). We have chosen mScarlet [23] and mNeonGreen [20] instead of commonly used GFP and RUBY because we were expecting lower number of copies (only one preferably) that would be integrated into the genome, which in turn might have lowered the brightness of the signal. We decided to put mScarlet outside of the PB ITRs and mNeonGreen inside the ITRs. This arrangement was dictated by the fact that the green fluorescent protein is brighter and easier to detect than the red one. When transgene integration occurs with transposition mechanism, only the sequence between ITRs is cut out from the plasmid and pasted into genome, and the rest of the plasmid DNA is lost. Thus, mScarlet expression after microinjection would indicate that the entire plasmid was either integrated or used as an extrachromosomal DNA. Therefore only mNeonGreen positive and mScarlet negative worms were selected for further analysis as potential transposon-mediated integration events.

B C 250 500 750 1000 E A D 947bp Sample 1 2 3 4 5 6

Figure 2. Confiramtion of piggyBac activity. A. Gel showing the PCR product of a plasmid from which the transposable element was cut out. B. Sequencing results showing the excision site, the canonical TTAA piggyBac

footprint is highlighted. C & D. Single and double promoter plasmids used for the injections. E. Positive worm expressing mNeonGreen under the EFa promoter inserted using the PB transposon system. Channels from left to right: brightfield, merged, FITC, dsRED.

Out of the initial 76 injections 12 eggs were expressing both of the fluorescent proteins. From the hatched worms we could select one that was mNeonGreen positive and mScarlet negative. This worm was singled out and crossed with a wild type worm to test whether the inserted gene is transmitted to the offspring. Progeny that were positive for the transgene confirmed the mNeonGreen gene transmission to the next generations and were used to establish a stable transgenic line (Fig 2 E). Transgenic worms were then used for total DNA extraction. The first approach to characterize the line was to use standard inverse PCR method. Unfortunately, we were unable to get any positive results with this approach, therefore we switched to the Genome Walker protocol [24]. This method uses specific adapters that are ligated to the ends of digested DNA and used in a subsequent PCR amplification together with insert specific primers to provide an easy-to-sequence product [18]. Using this method, we found that the integration occurred at the position 202,611 in the scaf1687 (Mlig_3_7 assembly). Interestingly, the insertion site was not the standard TTAA but TATA, indicating a non-canonical insertion (Fig. 3). Previous research has shown that this occurrence is very rare and that the mismatch in the sequence is repaired by the repair mechanism of the host cell [25]. Up to date we still miss other established and characterized lines where the transgene was introduced using the PB transposon system. Further research on the topic is necessary to determine whether this is a rare event in the case of PB activity in M. lignano.

TTAA

scaf1687:202,549-202,611 5' ITR piggyBac

ATAT

3' ITR piggyBac scaf1687:202,612-202,683

transgene pEFa:mNeonGreen

ATAT

TATA

piggyBac transposon with a transgene inserted into the genome Adaptor

GenomeWalker AdaptorGenomeWalker

3' ITR 5' ITR pEFa RV primer FWprimer А. B. C. D.

DISCUSSION

Advancements of genetic manipulation tools play an important role in our progress towards understanding any model organism. In this proof of principle study we demonstrated that

piggyBac transposon can be successfully mobilized to integrate into the genome of M. lignano

when microinjected into single-cell stage embryos together with the transposase in the form of mRNA. To establish the most optimal transposase mRNA to donor plasmid ratio we have tested several possibilities and chosen the ones that seemed the most efficient in our hands. Additionally, by using a two-promoter donor plasmid we have developed an easy selection system to discriminate unwanted random integration events. We have also shown that the insertion site can be precisely identified with the aid of the Genome Walker approach.

Having a working transposon-based transgenesis system opens new perspectives for future research. One of such possibilities are mutagenetic screens, where one line carrying a detectable cargo can be crossed with a second line carrying the transposase. The progeny of such a cross, where transposon would be mobilized and inserted in different new locations, can be screened

TTAA

scaf1687:202,549-202,611 5' ITR piggyBac

ATAT

3' ITR piggyBac scaf1687:202,612-202,683

transgene pEFa:mNeonGreen

ATAT

TATA

piggyBac transposon with a transgene inserted into the genome Adaptor

GenomeWalker AdaptorGenomeWalker

3' ITR 5' ITR pEFa RV primer FWprimer А. B. C. D.

Figure 3. Identification of the insertion site A-C. Schematic rep-resentation of the insertion that oc-curred during the transposition from the donor plasmid into the genome of

M. lignano. D. Schematic

representa-tion of the inserrepresenta-tion mechanism into the non-canonical insertion site.

for interesting phenotypes and the affected genes can be identified by mapping the insertion. The main caveat of the transposon approach presented here seems to be the low frequency of the positive genomic integration events that are transmitted to the progeny and could be fully characterized. Also the non-canonical insertion site in the established line raises the question about the efficiency of the method and calls for further optimization.

One of the modifications that can be explored is to use transposase in the form of protein. This method should increase the transmission rate. It eliminates the time gap caused by the translation of mRNA and enables the transposition to occur in the earlier developmental stage. In consequence the protein approach ensures a higher percentage of the cells carrying the transgene. It also eliminates the mRNA stability and handling problem, a common risk connected to any work concerning mRNA. The proof of principle for using transposase protein was already demonstrated for Tol2 TE in zebrafish [26]. It would therefore be of interest to

evaluate whether Tol2 transposon system can be used in M. lignano.

The development of the genetic manipulation tools in M. lignano was meant as a technological

platform for harvesting the power of this fascinating animal. It was therefore implied that that refinements and expansions of the available toolkit are bound to happen. Confirmation of the transposon based transgenesis further increases the value of Macrostomum lignano as a

powerful flatworm model organism.

METHODS

M. lignano lines and cultures

The NL10 line was previously described [18]. Animals were cultured under laboratory conditions in plastic Petri dishes (Greiner), filled with nutrient-enriched artificial sea water (Guillard's f/2 medium). Worms were fed ad libitum on the unicellular diatom

Nitzschia curvilineata (Heterokontophyta, Bacillariophyceae) (SAG). Climate chamber

conditions were set on 20˚C with constant aeration, a 14/10 h day/night cycle. Cultures designated for microinjection experiments were prepared as previously described [18, 27].

Cloning

The PB ITRs containing plasmid wasmade by cloning the commercially synthesized ITR sequences (IDT) into the pGEM-T backbone (Promega). Dlg4::mScarlet::3'UTR was first cloned before the ITRs using NcoI. EFa::mNeonGreen::3'UTR fragment was then cloned between the ITRs using BamHI and XhoI.

mRNA preparation

oPB plasmid carrying the codon optimized version of the PB transposase was used as a PCR template. During the PCR the T7 promoter was added and the product was used as a template for in vitro transcription. The reaction was carried out using the HiScribe T7 ARCA mRNA

Kit with tailing (NEB) according to the manufacturer's instructions.

Microinjections

All microinjections were carried out following the previous instructions. Only fresh,

one-cell stage M. lignano embryos were used. All the micromanipulations were done using a

microinjection stage equipped with: AxioVert A1 inverted microscope (Carl Zeiss), PatchMan NP2, TransferMan NK2, FemtoJet express and PiezoXpert (Eppendorf).

PCR excision screen

The excision screen was carried out on single positive worms. The PCR was run using the single worm-PCR protocol in 10 µl - SWLB buffer (50 mM KCl, 10 mM Tris-HCl pH 8.3, 2.5 mM MgCl₂, 0.45% NP-40, 0.45% Tween-20) + proteinase K incubated in liquid nitrogen for 15' and then incubated at 65˚C for 1,5h. 0,5 µl of the SW-PCR was used for a PCR reaction using Q5 polymerase (NEB, USA) with 5'- CGTAAAGCACTAAATCGGAACCCT-3' and 5'- GGCTCGTATGTTGTGTGGAATTGTG-3' as primers. The PCR product was then run on a gel, isolated and sent for sequencing to an external company (GATC)

Establishing transgenic line

The hatchling positive for mNeonGreen and negative for mScarlet was selected (P0) and singled out. It was then crossed with a single-wild-type worm that was raised in the same conditions. The pair was transferred to fresh food every 2 weeks. The progeny was screened and the positive F1 animals were selected, put together on fresh food, transferred to a separate dish and allowed to generate F2 progeny. The population was selected bi-weekly and only the mNeonGreen worms were kept. After reaching 200 positive worms, half of the population was sacrificed for genomic DNA extraction needed for the Genome Walker protocol. The rest of the worms were kept and used to set up a stable culture.

Identification of the integration site

Genomic location of the inserted DNA fragment was obtained using the Universal Genome Walker 2.0 Kit (Clontech Laboratories) with SmaI as the restriction enzyme. Sequencing was done by an external company (GATC)

Microscopy. Images were taken using a Zeiss Axio Zoom V16 microscope with an HRm digital camera and Zeiss filter set 38HE (FITC).

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