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Keywords:

  • Caenorhabditis elegans ;
  • GLR-1;
  • heteroduplex mobility assay;
  • mechanosensory behavior;
  • TALEN

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Author contributions
  9. References

Targeted genome editing using transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems has recently emerged as a potentially powerful method for creating locus-specific mutations in Caenorhabditis elegans. Due to the low mutation frequencies, one of the crucial steps in using these technologies is screening animals that harbor a targeted mutation. In previous studies, identifying targeted mutations in C. elegans usually depended on observations of fluorescent markers such as a green fluorescent protein or visible phenotypes such as dumpy and uncoordinated phenotypes. However, this strategy is limited in practice because the phenotypes caused by targeted mutations such as defects in sensory behaviors are often apparently invisible. Here, we describe a versatile strategy for isolating C. elegans knockout mutants by TALEN-mediated genome editing and a heteroduplex mobility assay. We applied TALENs to engineer the locus of the neural gene glr-1, which is a C. elegans AMPA-type receptor orthologue that is known to have crucial roles in various sensory behaviors. Knockout mutations in the glr-1 locus, which caused defective mechanosensory behaviors, were efficiently identified by the heteroduplex mobility assay. Thus, we demonstrated the utility of a TALEN-based knockout strategy for creating C. elegans with mutations that cause invisible phenotypes.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Author contributions
  9. References

Genome editing technologies have become powerful tools for interrogating, perturbing, and engineering biological systems (Pauwels et al. 2013). Current technologies mainly comprise two methods: transcription activator-like effector (TALE) nucleases (TALENs) and RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 (CRISPR-associated) endonucleases (Wei et al. 2013). TALENs comprise a non-specific FokI nuclease domain that is fused to a customizable TALE domain, which recognizes a predictable DNA sequence (Mussolino & Cathomen 2012). Specific DNA recognition by TALE domains directs the nuclease to introduce DNA double-strand breaks at a target site. Erroneous repair by non-homologous end-joining often yields a mutagenic deletion or insertion (indel) at this breakpoint (Joung & Sander 2013). In a CRISPR/Cas9 system, a customizable single guide RNA (sgRNA) supports site-specific cleavage of target DNA by Cas9, with target specificity determined by base pairing between the sgRNA and the target DNA (Cong et al. 2013; Mali et al. 2013).

These two methods were recently used for a genetically tractable model animal, Caenorhabditis elegans (Wood et al. 2011; Chen et al. 2013; Cheng et al. 2013; Chiu et al. 2013; Dickinson et al. 2013; Friedland et al. 2013; Katic & Großhans 2013; Lo et al. 2013; Tzur et al. 2013; Waaijers et al. 2013). During C. elegans genome editing, one of the most important steps is screening animals that harbor a heterologous targeted mutation due to the low mutation frequency. Previous mutant isolation was largely based on knowledge-based screening, which relies on observations of fluorescent markers or easily identifiable phenotypes like dumpy (Dpy) and uncoordinated (Unc) phenotypes.

However, these screening strategies have been limited in practice, particularly when applying TALENs to research on behavioral neurobiology. This is because behavioral phenotypes caused by targeted mutations, such as defects in chemotaxis, thermotaxis, or mechanosensory behavior, are often apparently invisible. Alternatively, one group used a CEL-1 assay to screen C. elegans mutants (Wood et al. 2011), in which a CEL-1 surveyor nuclease specifically digested a heterologous mutation. However, this screening method requires large amounts of expensive enzymes. Thus, we hypothesized that it would be necessary to demonstrate the practicality of TALEN-mediated genome editing for use in research on behavioral neurobiology.

In this study, we used TALEN for the genome editing of a neural gene, glr-1, a C. elegans AMPA-type glutamate receptor orthologue. We also applied a heteroduplex mobility assay to identify knockout mutations. An isolated glr-1 mutant exhibited defective mechanosensory behavior similar to that of a previously isolated glr-1 null mutant. This indicated that our strategy could be applied to efficiently isolate mutants that exhibit apparently invisible but measurable behavioral phenotypes. Thus, we demonstrated the utility of a TALEN-based knockout strategy for investigating neural functions, such as sensory behavior.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Author contributions
  9. References

Strain preparation

All C. elegans strains used in this study were maintained and manipulated using standard methods (Brenner 1974; Sugi et al. 2011). We used the wild-type N2 Bristol and glr-1(ky176) strains.

Construction of TALEN vectors

TALEN plasmids for glr-1 were constructed using the two-step Golden Gate cloning method as described previously (Sakuma et al. 2013a), with various modifications (Sakuma et al. 2013b). TALE repeats were cloned into pBluescript SK and assembled into pCS2+-based destination vectors harboring the optimized 5′ and 3′ untranslated regions that were described previously (Wood et al. 2011). The N- and C-terminal domains of TALE and the FokI nuclease domain were obtained from pTALEN_v2 (Addgene) (Sanjana et al. 2012).

TALEN mRNA synthesis and injection

TALEN mRNA was synthesized using previously reported methods (Wood et al. 2011). A 7-methyl guanosine cap analogue [m7G(5′)ppp(5′)G] were added to the 5′ end of TALEN mRNA using an mMessage mMachine kit (Ambion #A1340), and poly(A) tails were added to the 3′ end of TALEN mRNA using a PolyA tailing kit (Ambion #AM1350). The obtained mRNAs were purified with a MegaClear kit (Ambion AM#1908).

Germline transformations were performed for the wild-type N2 Bristol strain as described (Mello et al. 1991). mRNA injections were conducted using a Zeiss Axio Observer.A1 microscope and a Narishige IM31 injector. The germline syncytia of two P0 animals were injected with TALENs at 750 ng/μL for each mRNA (1500 ng/μL total).

Procedures for mutant isolation

Injected P0 worms were initially cultivated on a 60-mm Petri dish containing nematode growth medium (NGM) agar and Escherichia coli OP50 bacteria for 4 h. These P0 worms were individually transferred onto new plates according to the time schedule shown in Figure 1. F1 progeny were grown on separate NGM plates.

image

Figure 1. Experimental strategy to generate and screen Caenorhabditis elegans with engineered genomes. Procedure to identify worms harboring engineered genomes. Two P0 adult worms were injected at the same time. The time course for individually transferring the injected worms to new plates is indicated (Top). The second screening was performed using aliquots from single worm lysates obtained just before the first screening.

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TALEN-induced mutations were detected by a heteroduplex mobility assay (HMA). Initially, genomic DNA was separately isolated from all F1 worms by digestion using proteinase K. Next, a 364-bp short fragment that included the target site was amplified from genomic DNA using a forward primer (5′-GACATGTGTTAATCCG) and a reverse primer (5′-TGGGACAAGCGAATATGTATGG). Three-step polymerase chain reaction (PCR) was performed using the following protocol: 35 cycles of 98°C for 10 s, 55°C for 30 s, and 68°C for 20 s. The resulting PCR products were subjected to annealing and electrophoresed on 6% polyacrylamide gels using a Novex Mini-Cell (Life Technologies) and a Bio-Rad powerpac (Bio-Rad). F2 worms that carried a targeted mutation were recovered and subjected to sequence analysis using the above primers.

Assays for mechanosensory behavior

Worms were cultivated in a 60-mm Petri dish containing 10 mL NGM with 2% agar, on which Escherichia coli OP50 bacteria were seeded. On the first day, eight worms were deposited on an NGM plate and cultivated at 20°C. After 3 h, deposited P0 worms were removed to segregate F1 progeny. The assays for mechanosensory behavior were performed as shown in a previous report (Swierczek et al. 2011). At 80 h after P0 removal, behavioral tests were performed by applying tap stimuli to each Petri dish. Behaviors were recorded for 60 s using a CCD camera and quantified with the custom-made image-processing software. A reversal was scored when a worm that was moving forward at the time of the stimulus moved backwards within 1 s of the stimulus. A reversal was considered to be complete when a worm paused or began forward motion that lasted for more than 1 s. Cases when worms were already reversing were not used for analysis.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Author contributions
  9. References

Targeted genome editing by TALEN

AMPA-type glutamate receptors play pivotal roles in excitatory neurotransmission to ensure various neural functions from development to behavioral plasticity (Kessels & Malinow 2009). A C. elegans AMPA-type glutamate receptor orthologue, GLR-1, has been one of the most intensively investigated neural genes (Hart et al. 1995; Maricq et al. 1995). Although previously isolated glr-1 mutants appeared to show superficial wild-type phenotypes, behavioral analyses indicated that these mutants exhibited severe defects in several sensory behaviors, such as mechanosensory behavior (Rose et al. 2003; Chalasani et al. 2007). Therefore, because glr-1 mutants do not exhibit visible phenotypes, we selected the glr-1 locus for introducing targeted mutations.

To induce frameshift mutations within the first exon of glr-1, we designed a TALEN pair (TALEN-glr-1) that targeted a sequence downstream of the start codon. We then synthesized poly(A)-tailed TALEN-glr-1-encoding mRNAs with 5′ and 3′ untranslated regions that were favorable for germline translation. These mRNAs were injected into the distal region of gonad arms of wild-type hermaphrodites. It is general that an injected DNA or mRNA including a TALEN-mRNA is initially incorporated into the nuclei of oocytes. The oocytes enter the spermatheca and can be fertilized by sperm. Upon self-fertilization, partial embryonic development occurs in the uterus, and eggs are eventually laid before hatching. Thus, injected materials can enter into oocytes before fertilization. Due to these unique injection techniques different from other model animals such as vertebrates and insects, F1 individuals could carry a genetically fixed mutant allele.

Two injected P0 worms were transferred onto new NGM plates using the time schedule shown in Figure 1. In pilot experiments, we noted that mutations could be detected only for the F1 progeny that were laid within 24 h after injection but not for F1 progeny that were laid after this time (data not shown). This was consistent with a previous report (Wood et al. 2011). Thus, we analyzed only those worms that were produced within 24 h after injection.

Screening for targeted mutations by heteroduplex mobility assay

For this screening, we used a heteroduplex mobility assay (HMA) to detect heterozygous mutations (Ota et al. 2013). An HMA is based on denaturing and annealing PCR-amplified nucleotide strands that are not completely complementary and, therefore, generate homoduplexes and heteroduplexes. Heteroduplexes can readily be distinguished from homoduplexes by polyacrylamide gel electrophoresis (PAGE) because heteroduplexes migrate more slowly due to an opened single-strand configuration surrounding the mismatched region. A previous report indicated that the sensitivity of this assay was sufficient to distinguish a 2-bp difference between wild-type and mutant nucleotides (Ota et al. 2013). Although this assay requires DNA polymerase for PCR amplification, detection can readily be performed without using enzymes.

After self-fertilization, individual F1 progeny were picked and placed on separate NGM plates. Next, single worm lysis was performed for each F1 progeny after they laid eggs (Fig. 1, step 1). In the pilot experiment, we could detect at least 5% of a mutant haplotype in a DNA pool, indicating that at least 10 individual samples can be pooled into a single tube. Considering that an injected P0 yields about 100–150 F1 worms within 24 h after injection (Table 1), we pooled aliquots of four or five worm lysates into single PCR tubes, and 10 PCR tubes were simultaneously used for the polyacrylamide electrophoresis in the first screening (Fig. 1, step 2). We used 10 well-polyacrylamide gel for electrophoresis. Therefore, our strategy enables us to complete the first screening at only one or two time electrophoresis and ensures the applicability of HMA to the high-throughput screening. Figure 2A shows representative results of the first HMA screening for F1 progeny produced by an injected P0 worm A (Table 1). Heteroduplex bands were detected in four of the 10 PCR tubes, which indicated that these four PCR tubes were positive by HMA. Thus, aliquots of single worm lysates for the positive PCR tubes were applied to a second HMA screening (Fig. 1, step 3). As shown in Figure 2B, the four single worm lysates, 3E, 4C, 5A, and 8A, were positive for the second screening, which indicated that the NGM plates corresponding to these single worm lysates included worms that had the targeted mutation. Thus, the F2 progeny from these positive single NGM plates were assayed to identify homozygous mutants.

Table 1. Summary of transcription activator-like effector nuclease (TALEN)-mediated isolation of glr-1 mutants
Injected P0No. F1 progeny with mutations/No. analyzed F1 progeny on the 0–4 h plate (%)No. F1 progeny with mutations/No. analyzed F1 progeny on the 4–15 h plate (%)No. F1 progeny with mutations/No. analyzed F1 progeny on the 15–24 h plate (%)Frequency (%)
  1. Frequency indicates all F1 worms with mutations/all analyzed F1 worms for each injected P0 worm.

Worm A0/3 (0)4/50 (8.0)0/31 (0)4/84 (4.8)
Worm B0/4 (0)7/74 (9.5)3/52 (5.8)10/130 (7.7)
image

Figure 2. Heteroduplex mobility assay (HMA) detection of transcription activator-like effector nuclease (TALEN)-mediated genome modifications in Caenorhabditis elegans. (A, B) HMA for detecting TALEN-mediated genome modifications in the glr-1 locus. Several heteroduplex bands (slow mobility) were observed with 6% polyacrylamide gel electrophoresis. An HMA gel image during the first HMA screening for F1 progeny on a 4–15 h plate is shown in (A). Each lane indicates the mobility of a polymerase chain reaction (PCR) amplicon obtained using aliquots pooled from five single worm lysates. The gel images during the second HMA screening for F1 progeny are indicated separately in (B). Four different HMA profiles were detected (*, ◊, □, and #). M, 100 bp DNA ladder marker. (C) Sequence analysis of the F1 progeny from glr-1-targeting experiments. TALEN target regions were amplified from the genomic DNA of mutant progeny by PCR. PCR amplicons were subcloned into pBluescript vectors, and individual inserts were sequenced. The wild-type sequence is indicated at the top. TALEN target sequences are shown by blue letters. Deleted and inserted nucleotides in the DNA sequences are indicated by red dashes and red letters, respectively. Numbers to the right of the sequences indicate the net loss of bases for each allele.

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The results for mutant screening are summarized in Table 1. From two P0 worms that were injected with poly(A)-tailed TALEN-glr-1-encoding mRNAs, 214 F1 progeny were screened, among which 14 mutations (frequency of 6.5%) were identified. Both injected worms produced mutants, and a single worm yielded numerous mutant progeny. In particular, we efficiently obtained mutants from the 4–15 h post-injection plate.

These four resulting homozygous mutants were subjected to sequence analysis. The genomes of all four mutants had unique indels located within the expected target sequences (Fig. 2C). Three of four mutations successfully induced a frameshift mutation within the first exon of glr-1. These frameshifts would result in a loss-of-function of GLR-1. For all four alleles, the allele found in the F1 generation was efficiently transmitted to the F2 generation. Thus, TALEN-mediated gene modification produced a variety of heritable knockout mutations in the glr-1 locus.

Mechanosensory behaviors of the new glr-1 mutants

Caenorhabditis elegans usually responds to non-localized vibrations, such as tap mechanical stimulation to a Petri dish, and subsequently exhibits an escape response (reversal). A previous study showed that a glr-1(ky176) mutant (Fig. 3A), which has a deletion and a premature stop codon in the glr-1 locus, exhibited almost no reversal responses to moderate mechanosensory tap stimuli but did respond to a stronger stimulus such as a train of taps; the train comprised six taps within 600 ms (Rose et al. 2003). We quantified the behavioral responses of the glr-1(ky176) mutant and the newly isolated mutants to a strong single tap stimulus.

image

Figure 3. Comparison of mechanosensory behaviors of a newly isolated glr-1 mutant with a previously isolated glr-1(ky176) mutant. (A) Predicted protein product. In the st1 allele, the missense mutation and deletion were occurred at Thr16 and Val17, respectively. In the st2-4 alleles, the frameshifts were caused at the positions marked by the asterisk, resulting in the premature stop codon. TM means transmembrane resion. (B) Behavioral defects were observed for the glr-1(st4) mutant but not for the glr-1(st1) mutant as compared with wild-type worms. Assays for each strain were carried out at least three times. Error bars indicate standard errors of the mean (SEMs). Statistical comparisons were made by t-tests. Asterisk indicates < 0.01. NS, not significant.

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As shown in Figure 3B, glr-1(ky176) mutants exhibited reversal responses to these stimuli, although their reversal distances were less than those of wild-type worms. This indicated that although we did not apply a train of taps, the intensity of a single stimulus was sufficiently strong to induce a mechanosensory response by glr-1(ky176) mutants. Interestingly, the glr-1(st4) mutant, which harbored a frameshift mutation in the glr-1 locus, exhibited a behavioral defect similar in extent to that of the glr-1(ky176) mutant (Fig. 3B), unlike the glr-1(st1) mutant, which had a mutation that caused no frameshift in this locus. These behavioral analyses indicated that our TALEN-mediated knockout strategy could be used to identify those mutations that did not cause visible phenotypes.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Author contributions
  9. References

Currently available methods to generate mutations in the genome of C. elegans, such as chemical mutagenesis and imprecise excision of transposons, depend on procedures that take at least 1 month to conduct large-scale mutagenesis screening. Recently, to overcome the lack of effective reverse genetic tools, genome editing approaches using TALEN and CRISPR/Cas9 systems were applied to C. elegans. However, although the potential of these technologies has been demonstrated, their practical use requires improving their efficiency to obtain desired mutants, particularly by increasing the mutation frequency and accelerating the screening process.

With regard to mutation frequency, we obtained 11 glr-1 mutants from among 124 F1 progeny laid on 4–15 h plate, which indicated an 8.9% mutation frequency at this peak time (Table 1). In contrast, a previous study found that only 3.5% of the progeny during a 4-h peak window were mutants (Wood et al. 2011). Although it is difficult to directly compare the mutation frequency rates between both studies, the efficiency of genome editing in our study appears to be slightly higher than that in the previous study. We assume that this difference might arise from differences in the chromosomal structure around a targeted locus or in the architecture of TALEN itself (Sakuma et al. 2013).

Regarding the screening procedure, we used HMA analysis to identify mutations in the glr-1 locus. With our strategy, a procedure from injection to screening can be completed within only 1 week, and a desirable mutant without observable phenotypic selection or expensive enzymes for mutation detection can be produced. Considering that previous screening procedures were based on visible phenotypic selection or a CEL-1 assay, we propose that our strategy is more versatile for practically obtaining desired mutants.

Thus, we have described a versatile strategy for isolating TALEN-mediated knockout mutants in C. elegans. Indeed, we could create a mutation in the C. elegans AMPA-type receptor that caused defective sensory behavior like that of previously isolated glr-1 mutants. This versatile strategy allows for rapid interrogation of biological systems and facilitates functional annotation of a target genomic locus in C. elegans.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Author contributions
  9. References

We thank A. V. Maricq for sharing the glr-1(ky176) mutants, N. Ohnishi for fruitful discussions, and T. Kimata for a critical review of our manuscript. This research was supported by the Japan Science and Technology Agency under Precursory Research for Embryonic Science and Technology (PRESTO; to T. Sugi), Grants-in-Aid for Scientific Research (Ministry of Education, Culture, Sports, Science and Technology; to T. Sugi).

Author contributions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Author contributions
  9. References

T. Sugi, T. Sakuma, and T. Yamamoto conceived the project. T. Sakuma and T. Yamamoto created the TALEN-glr-1 constructs. T. Sugi created the poly(A)-tailed TALEN-glr-1 mRNA, performed germline transformations to introduce mRNA into C. elegans, and conducted behavior assays and data analysis. Y. Ohtani and T. Sugi performed HMAs. T. Sugi, T. Sakuma, and T. Yamamoto wrote the paper.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Author contributions
  9. References
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