CRISPR-Cas9-Guided Genome Engineering in Caenorhabditis elegans

Abstract

The CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein) system is being used successfully for efficient and targeted genome editing in various organisms, including the nematode Caenorhabditis elegans. Recent studies have developed a variety of CRISPR-Cas9 approaches to enhance genome engineering via two major DNA double-strand break repair pathways: nonhomologous end joining and homologous recombination. Here, we describe a protocol for Cas9-mediated C. elegans genome editing together with single guide RNA (sgRNA) and repair template cloning (canonical marker-free and cassette selection methods), as well as injection methods required for delivering Cas9, sgRNAs, and repair template DNA into the germline. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Guide RNA preparation Alternate Protocol 1: sgRNA cloning using fusion PCR Basic Protocol 2: Preparation of a repair template for homologous recombination Alternate Protocol 2: Preparation of repair template donors for the cassette selection method Basic Protocol 3: Injecting animals Basic Protocol 4: Screening transgenic worms with marker-free method Alternate Protocol 3: Screening transgenic worms with cassette selection method.

Keywords: C. elegans; CRISPR; CRISPR-Cas; Cas9; genome editing; genome engineering.

Figure 1.

Schematic representation of the CRISPR-Cas9 genome editing approach in C. elegans. Young adult hermaphrodites are injected with the CRISPR-Cas9-containing DNA mixture. A DSB generated by Cas9 is repaired via error-prone NHEJ or error-free HR. Yellow box represents small nucleotide insertion and green box represents insertion of GFP tag.

Figure 2.

Outline of building genome engineered C. elegans in this protocol. Note that we present two alternative protocols for guide RNA and repair template. Each requires its own screening strategy.

Figure 3.

Schematic representation of the sgRNA cloning by two alternative methods. A. sgRNA cloning using an empty sgRNA expression vector. The sgRNA comprised of a pair of annealed oligos specifically targeting ztf-8 is cloned into the NotI and BamHI digested empty sgRNA vector (left) using Gibson assembly. B. sgRNA cloning using fusion PCR. To generate an sgRNA containing the EcoRI-HindIII fragment, two amplicons are stitched by PCR (right). Both the fused PCR fragment and the empty sgRNA vector are digested with EcoRI and HindIII and ligated to create the sgRNA expression vector.

Figure 4.

Schematic representation of the construction of the repair template. A. Diagrams illustrate the N- and C-terminal GFP tagging of a gene of interest. Note the position of GFP as well as the Start and Stop codons of the protein. Flag represents start codon. B. Schematic representation of the repair template cloning for C-terminal GFP tagging. Three PCR amplicons are assembled into a KpnI and SalI digested pUC19 vector to create a repair template (donor plasmid).

Figure 5.

A. Introduction of silent mutations in S (serine) and R (arginine) with the primer ztf-8 UP-R to avoid cutting repair template. The original wild type and designed repair template sequences are compared. B. Primer sequences for amplifying homology arms for cassette selection method. Identical sequences are highlighted with the same colors. For example, all constructs share the downstream R primer (cyan color) in common. The reverse complement (RC) is underlined. N: ~20bp sense complementary sequence to your gene of interest for a PCR reaction. N-RC: ~20bp reverse complementary sequence to your gene of interest for a PCR reaction (underlined).

Figure 6.

Schematic representation of the construction of the repair template with a selection cassette toolkit. Top left: Diagrams illustrate the PCR amplification of sequences up and downstream of the target gene. Top right: Vector containing backbone and selection cassette is digested with restriction enzyme (RE) and stitched together with up and downstream homology arms through a 4 fragment Gibson assembly reaction. Note that 5 types of vectors (disruption/deletion, GFP, RFP, 3xHA and GFP::3xHA) are available for generating distuption/deletion or tagging template. The cassette in tagging vectors contains selection markers (GFP + neomycin) and DNA sequences for tagging while the cassette in disruption/deletion vector contains only selection markers. Color code denotes homology containing DNA sequences.

Figure 7.

Workflow and timeline for CRISPR-Cas9-guided genome editing in C. elegans. The expected timelines (14 days and 27 days for completion for the marker-free and cassette selection methods, respectively) assume that each step is not delayed. See Table 6 for details.