Cas9-mediated gene editing in the black-legged tick, Ixodes scapularis, by embryo injection and ReMOT Control

Abstract

Despite their capacity to acquire and pass on an array of debilitating pathogens, research on ticks has lagged behind other arthropod vectors, such as mosquitoes, largely because of challenges in applying available genetic and molecular tools. CRISPR-Cas9 is transforming non-model organism research; however, successful gene editing has not yet been reported in ticks. Technical challenges for injecting tick embryos to attempt gene editing have further slowed research progress. Currently, no embryo injection protocol exists for any chelicerate species, including ticks. Herein, we report a successful embryo injection protocol for the black-legged tick, Ixodes scapularis, and the use of this protocol for genome editing with CRISPR-Cas9. We also demonstrate that the ReMOT Control technique could be successfully used to generate genome mutations outside Insecta. Our results provide innovative tools to the tick research community that are essential for advancing our understanding of the molecular mechanisms governing pathogen transmission by tick vectors and the underlying biology of host-vector-pathogen interactions.

Keywords: Biological sciences; Biological sciences research methodologies; Biology experimental methods; Biotechnology; Genetic engineering.

Graphical abstract

Figure 1

Gene editing by embryo injections (A) Gene maps showing exons (gray boxes) and introns (wavy lines) and designed sgRNAs targeting the first exon for Proboscipedia (ISCW021086), and the third exon for Chitinase (ISCW003950) genes used for embryo injections in I. scapularis. (B) Workflow schematic for embryo injections against target genes.

Figure 2

Sequences of Proboscipedia G₀ larvae edited by embryo injection aligned to wildtype (WT) (A) Sequences and chromatograms of wildtype (WT), heterozygous deletion (L1), and homozygous deletion (L2). GC-rich regions flanking the homozygous deletions are underlined in blue. Black squiggly lines represent an additional deleted sequence in that region. (B) Control and mutant larva (L1) showing extended hypostome phenotype. (C) Illustration of tick mouthparts and measurements (top), a graph showing hypostome length in wildtype (control) and injected larvae (bottom).Data are presented as mean ±SEM. An unpaired Student’s t test was used to calculate significance ∗ = p> 0.05

Figure 3

Sequences of Chitinase G₀ larvae edited by embryo injection aligned to wildtype (WT) (A) Summary of deep sequencing data of animals injected with sgRNA 1. Indel percentage is on the Y axis. The X axis depicts predicted Cas9 cut site (position 0) and sequences up- and downstream of the cut site. (B) Representative sequences containing deletions corresponding to sgRNA 1. The predicted cut site is shown by a black arrow and dotted line. PAM site is indicated by a red rectangle outline. Alignments were generated using Snapgene. (C) Forward (F) and Reverse (R) chromatograms of WT, CH5, and CH6 larvae. Additional sequences, chromatograms, and indel/substitution percentages calculated by deep sequencing are shown in Figure S5 and Table S4.

Figure 4

Delivery of injection components to ovaries via ReMOT Control (A) Visualization of delivery of injection components to ovaries via fluorescent imaging following injection of P2C-GFP. (B) Workflow schematic of blood-fed adult female injections with P2C-Cas9 protein, saponin, and pooled sgRNAs. (C) Measurements of survival: egg-laying and larval hatching following injections with varying concentrations of saponin and sgRNAs. Vehicle control = dialysis buffer + saponin (36 μM). Data are presented as mean ±SEM.

Figure 5

Sequences of Proboscipedia G₀ larvae CRISPR edited by ReMOT Control aligned to wildtype (WT) (A) Map depicting aligned homozygous and heterozygous deletions. (B) Representative chromatogram alignment of WT and two homozygous mutants (R4 and R6). GC-rich flanking regions are underlined in blue. PAM sites are indicated by a red rectangle. The approximate cut sites are shown by black arrows and dotted lines. PAM sites are indicated by a red rectangle. The approximate cut sites are shown by black arrows. Alignments were generated using Snapgene.