TALENs mediate efficient and heritable mutation of endogenous genes in the marine annelid Platynereis dumerilii

Abstract

Platynereis dumerilii is a marine polychaete and an established model system for studies of evolution and development. Platynereis is also a re-emerging model for studying the molecular basis of circalunar reproductive timing: a biological phenomenon observed in many marine species. While gene expression studies have provided new insight into patterns of gene regulation, a lack of reverse genetic tools has so far limited the depth of functional analyses in this species. To address this need, we established customized transcriptional activator-like effector nucleases (TALENs) as a tool to engineer targeted modifications in Platynereis genes. By adapting a workflow of TALEN construction protocols and mutation screening approaches for use in Platynereis, we engineered frameshift mutations in three endogenous Platynereis genes. We confirmed that such mutations are heritable, demonstrating that TALENs can be used to generate homozygous knockout lines in P. dumerilii. This is the first use of TALENs for generating genetic knockout mutations in an annelid model. These tools not only open the door for detailed in vivo functional analyses, but also can facilitate further technical development, such as targeted genome editing.

Keywords: chronobiology; evolution; genome editing; invertebrate; marine.

Figure 1

Evidence for TALEN-induced mutation of Platynereis vasotocin-neurophysin. (A) Schematic of the vtn locus showing TALEN target site (yellow) in exon 2 (L, left TALEN; R, right TALEN). Blue arrows (F1/R3) indicate primers used for mutation screening PCR [size of amplicon (bp) indicated by double-ended arrows]. Green block indicates position of sequence from intron 3 detected as an insertion (sample 72). The mature peptide (highlighted in purple) is included by the majority of the TALEN spacer and includes the MfeI-screening site. (B) Restriction digest screening of 24-hpf larvae injected with vtnEx2_L and vtnEx2_R TALEN mRNA at concentration of 200 ng/µl/TALEN. Samples contain pools of four larvae. Black arrow indicates size of uncut PCR product; red arrow indicates 109-bp insertion detected in sample 72. (C) Results of MfeI digest of PCR products from adult worms: uncut PCR product (black arrowhead) cloned from adult injected worm vtn+13, representing 9-bp deletion. (B and C) PCR, un-digested PCR product; NI, non-injected; asterisk (*) indicates samples digested with MfeI. (D) Results of sequence analysis of uncut and insertion bands from samples in B and C. Length of mutations indicated by ∆ symbol with “−” indicating deletions and “+” indicating insertions. Restriction site is shown in boldface type; asterisks indicate frameshift mutations. Shading key: yellow, TALEN-binding sites; gray, spacer; blue, nucleotides differing from wild type; green, inserted nucleotides.

Figure 2

TALEN-induced mutations in the Platynereis estrogen receptor. (A) Schematic of the er locus showing target exons 2 and 3 with TALEN target sites (yellow). Blue arrows, primer positions; red double-ended arrow, region of sequence deleted in E; green, DNA-binding domain. Primer combinations used for screening are shown above in B–E. (B–E) PCR, undigested PCR product; NI, non-injected. (B) Restriction digest screening of larvae injected with erEx3_L2/R2 TALENs (mRNA concentration: 267 ng/µl/TALEN mRNA). Arrowhead indicates uncut PCR product following AflIII digestion (asterisk). (C) Mutation evidence at exon 2 site: uncut band adult worm +3 vs. fully digested product from mutation-negative (−) TALEN-injected worm. (D) Adult worms er+31 and er+37 with mutations at exon 3 site. (E) Deletions (red arrow) detected in larvae and adult worms resulting from simultaneous cleavage at exons 2 and 3 using 300 ng/µl/TALEN mRNA: deletion positive (+); deletion negative (−). Please note different primer pairs used for larval vs. adult samples. (F) Mutant sequences obtained from digest screening for exons 1, 2, and long deletions. Numbers in brackets indicate the sample or worm from which the sequence was obtained; all other sequences are from injected larvae shown in B. Length of mutations are indicated by ∆ with “−” indicating deletions and “+” indicating insertions. Restriction site is shown in boldface type; asterisks indicate frameshift mutations. Shading key: yellow, TALEN binding sites; gray, spacer; blue, nucleotides differing from wild type; green, inserted nucleotides.

Figure 3

Evidence of TALEN-induced mutations at l-cry locus. (A) Schematic of the l-cry genomic locus. Gray, exons; red, photolyase domain; yellow, TALEN sites; blue arrows, position of primers. Size of PCR amplicons and distance between TALEN sites indicated by double-ended arrows. Mutation screening results from larvae (B) and adult worms (D) injected with TALENn mRNA (l-cryEx2_L/R and l-cryEx3_L/R: 300 ng/µl/TALEN), showing uncut PCR bands following AvaII digestion (black arrow) and/or long deletions (red arrow). (D) Worm l-cry+45 shows completely undigested band suggesting biallelic mutation. Mutant sequences obtained from injected larvae (C) and adult worms (E) following AvaII digestion (asterisks). Length of mutations indicated by ∆ with “−” indicating deletions and “+” indicating insertions. Restriction site is shown in boldface type; asterisks indicate frameshift mutations. Shading key: yellow, TALEN0binding sites; gray, spacer; blue, nucleotides differing from wild type; green, inserted nucleotides. (B and D) PCR, undigested PCR product; NI, non-injected.

Figure 4

Evidence for germline transmission of TALEN-mediated mutations. (A) Mutation screening results: G1 from male worm (l-cry+36) injected with l-cry TALENs. Pools of four G1 larvae were digested using proteinase K buffer and analyzed by PCR and AvaII digestion. Aliquots of undigested PCR products (PCR) are run next to a digested aliquot (*) for each sample. Sample G1_5 shows both uncut (black arrowhead) and deletion (red arrowhead) bands. (B) Sequence of uncut band in sample G1_5 in A. (C and D) Evidence of long-range deletions (red arrows) in pooled G1 larvae from er TALEN-injected worms er+32 and er+59 (male symbols indicate PCR products from these male worms). (D) Sequenced deletions from G1 samples: G1_2 and G1_7 shown in C. Asterisks indicate frameshift mutations.