Genomic Access to Monarch Migration Using TALEN and CRISPR/Cas9-Mediated Targeted Mutagenesis

Abstract

The eastern North American monarch butterfly, Danaus plexippus, is an emerging model system to study the neural, molecular, and genetic basis of animal long-distance migration and animal clockwork mechanisms. While genomic studies have provided new insight into migration-associated and circadian clock genes, the general lack of simple and versatile reverse-genetic methods has limited in vivo functional analysis of candidate genes in this species. Here, we report the establishment of highly efficient and heritable gene mutagenesis methods in the monarch butterfly using transcriptional activator-like effector nucleases (TALENs) and CRISPR-associated RNA-guided nuclease Cas9 (CRISPR/Cas9). Using two clock gene loci, cryptochrome 2 and clock (clk), as candidates, we show that both TALENs and CRISPR/Cas9 generate high-frequency nonhomologous end-joining (NHEJ)-mediated mutations at targeted sites (up to 100%), and that injecting fewer than 100 eggs is sufficient to recover mutant progeny and generate monarch knockout lines in about 3 months. Our study also genetically defines monarch CLK as an essential component of the transcriptional activation complex of the circadian clock. The methods presented should not only greatly accelerate functional analyses of many aspects of monarch biology, but are also anticipated to facilitate the development of these tools in other nontraditional insect species as well as the development of homology-directed knock-ins.

Keywords: CRISPR; TALENs; clock genes; germline targeting; insect.

Figure 1

Targeted mutagenesis induced by microinjection of cry2 TALEN mRNAs into monarch butterfly embryos. (A) Schematic diagram of the TALEN targeting sites. The black line depicts the genomic locus of monarch cry2, and the numbered black boxes represent the 9 exons of cry2. The blue region in exon 2 is expanded to provide the sequence that includes the TALEN binding sites (shown in boxes) and the previously described ZFN binding sites (underlined, Merlin et al. 2013). The restriction site (EagI) in the spacer is shown in blue. Arrows on top represent the position of the primers used to amplify the 478 bp targeted region for analysis of mutagenic lesions in B (cry2 F1, cry2 R1 in Table S1). (B) Targeted mutagenesis in representative founder G₀ butterflies validated by PCR and EagI digestion. For each founder, the PCR product was either not subjected (–) or subjected to EagI (+). Genomic amplicons carrying nonhomologous end-joining (NHEJ)-induced mutations (mut; red arrow) leading to the loss of the EagI site are resistant to restriction enzyme digestion. The frequency of NHEJ-mediated indels provided under each founder was estimated by relative quantification of the resistant and digested products. Red stars, founders crossed to determine germline targeting rates. (C) TALEN-induced mutations in somatic cells of larvae surviving the injection. TALEN binding sites are boxed on the wild-type sequence. EagI site is in blue, and the positions of deletion and insertions are in red (dashes and letters, respectively). TALEN, transcriptional activator-like effector nucleases; ZFN, zinc-finger nuclease.

Figure 2

TALEN-mediated targeted mutagenesis in somatic and germline cells of monarch butterfly clock. (A) Schematic diagram of the monarch clock genomic locus and the TALEN targeting sites. The red region in exon 3 is expanded to provide the sequence that includes the TALEN binding sites (shown in boxes). The restriction site (NgoMIV) in the spacer is shown in green. Arrows on top represent the position of the primers used to amplify the 417 bp targeted region for analysis of mutagenic lesions in B (clock F1, clock R1 in Table S1). (B) Targeted mutagenesis in representative founder G₀ butterflies validated by PCR and NgoMIV digestion. For each founder, the PCR product was either not subjected (–) or subjected (+) to NgoMIV. Genomic amplicons carrying NHEJ-induced mutations (mut; red arrow) leading to the loss of the NgoMIV site are resistant to restriction enzyme digestion. Estimation of the frequency of NHEJ-mediated indels is provided under each founder. Red stars, founders crossed to determine germline targeting rates. Black star, highly targeted G₀ butterfly that did not survive to adulthood. (C) TALEN-induced mutations in somatic cells of larvae surviving the injection. TALEN binding sites are boxed on the wild-type sequence. NgoMIV site is in green, and the positions of deletions and insertions are in red (dashes and letters, respectively). (D) The clock gene is located on the Z chromosome. Crosses between heterozygote clock mutant (G₁) and wild-type butterflies produce progeny that include heterozygote males (M) and homozygote females (F) for the mutated allele (mut; red arrow). (E) TALEN-induced mutations in the germline of founders 3, 8, 9, and 10. Red arrowhead, butterfly selected to establish a clock mutant line. (F) Relative expression level of clock in brains of wild-type monarch females (WT F) and males (WT M). Values are mean ± SEM of 18 animals, collected every 3 hr in triplicates over 24 hr. t-test: P = 0.37. PCR, polymerase chain reaction; SEM, standard error of the mean; TALEN, transcriptional activator-like effector nuclease; WT, wild-type monarchs.

Figure 3

Heritable CRISPR/Cas9-mediated targeted mutagenesis of monarch butterfly clock. (A) Schematic of part of the monarch clock genomic locus containing the CRISPR/Cas9 target site. The purple region in exon 2 is expanded to provide the sequence targeted for genome editing by a single guide RNA (sgRNA, purple letters). The protospacer adjacent motif (PAM site, 5′-NGG-3′) 3′ of the target sequence is highlighted in gray. Arrows on top and bottom represent the positions of the primers used to amplify the 839 bp (clock F2, clock R1; Table S1) and 756 bp (clock F2, clock R2; Table S1) targeted regions for analysis of mutagenic lesions in C and E, respectively. (B) Diagram of the in vitro cleavage assay used to detect mutagenic lesions showing a PCR amplicon subjected to the sgRNA and the Cas9 protein. Blue and red lines correspond to a wild-type (WT) genomic fragment cleaved by Cas9. Fragments with mutations induced by NHEJ at the site targeted by the sgRNA (red boxes) are uncleaved (black line). (C) Detection of mutagenic lesions at the clock locus in somatic cells of founder G₀ butterflies. For each founder, a PCR fragment was subjected to a Cas9-based in vitro cleavage assay. Blue and red arrows, WT fragments. Black arrow, amplicons carrying mutations (mut) at the site targeted by the sgRNA. Estimation of the frequency of NHEJ-mediated indels is provided under each founder. Black stars, founders selected for crosses to determine germline targeting rates. (D) CRISPR/Cas9-induced mutations in somatic and germline cells of founders 17 and 18. The sgRNA binding site is underlined on the wild-type sequence. Red dashes and red letters, deletion and insertions, respectively. (E) Genotyping of G₁ butterflies from founders 17 and 18 backcrossed to WT using the Cas9 in vitro cleavage assay. Heterozygote butterflies carrying the mutated allele are robustly discriminated by the presence of an additional uncleaved PCR fragment. CRISPR, clustered regularly interspaced short palindromic repeats; NHEJ, nonhomologous end-joining; PCR, polymerase chain reaction.

Figure 4

Highly efficient CRISPR/Cas9-mediated targeted mutations and deletions of monarch butterfly cry2 using dual sgRNAs. (A) Schematic of part of the monarch cry2 genomic locus containing the two CRISPR/Cas9 target sites. The orange and green regions in exons 2 and 3 are expanded to provide the sequences targeted for genome editing by each single guide RNA (sgRNA). Protospacer adjacent motifs (PAM site, 5′-NGG-3′) are highlighted in gray. Arrows on top represent the positions of the primers used to amplify the 1403 bp targeted region for analysis of mutagenic lesions in B (cry2 F2, cry2 R2 in Table S1). (B) Detection of mutagenic lesions at the cry2 loci targeted by sgRNA1 (left) and sgRNA2 (right) in somatic cells of founder G₀ butterflies (9 out of 13 are shown). For each founder, a PCR fragment was subjected to a Cas9-based in vitro cleavage assay with each of the sgRNAs. Black arrowhead, amplicons carrying mutations (mut). Estimation of the frequency of NHEJ-mediated indels is provided under each founder. Red arrowhead, amplicons carrying genomic deletions (del) at the sites targeted by the sgRNAs. Blue and gray arrows, cleaved wild-type (WT) PCR fragments. Black stars, founders selected for crosses to determine germline targeting rates. (C) Left, CRISPR/Cas9-induced mutations in somatic and germline cells with sgRNA1. Guide RNA binding site is underlined on the wild-type sequence. Red dashes and red letters, deletion and insertions, respectively. Right, Dual sgRNA-mediated genomic deletion in somatic cells. Positions of sgRNA1 and sgRNA2 are underlined on the sequence and represented by colored boxes on a schematic representation. CRISPR, clustered regularly interspaced short palindromic repeats; NHEJ, nonhomologous end-joining; PCR, polymerase chain reaction.

Figure 5

CLOCK deficiency disrupts circadian behavior and the molecular clockwork. (A) Profiles of adult eclosion in constant darkness (DD) of wild-type (^+/+, black), heterozygous (^+/−, gray) and homozygous mutant (^out, green) siblings of a clock (clk) mutant line carrying a 4 bp deletion in exon 3, entrained to LD (15 hr light, 9 hr dark) throughout their larval and pupal stages. Eclosion occurred on the first and second d of DD. Data from both d are pooled and binned in 1 hr intervals. Effect of genotype on eclosion time, one-way ANOVA: P < 0.0002; Tukey posthoc test: clk^+/+ vs. clk^+/−, P > 0.05; clk^+/+ vs. clk^out, P < 0.01; clk^+/− vs. clk^out, P < 0.01. Black horizontal bars, subjective night; gray horizontal bars, subjective day. (B) Circadian expression of period and timeless in brains of wild-type (black lines) and homozygous mutant (dashed green lines) siblings of the clk mutant line entrained to LD throughout their larval and pupal stages. Values are mean ± SEM of three animals, except for circadian time 0 (CT0) in clk^out, which represents the mean of two animals. Interaction genotype × time, two-way ANOVA: per, P < 0.0001; tim, P < 0.01. Box shading: gray, subjective day; black, subjective night.