Genome editing technologies have transformed biomedical research, enabling precise alterations to DNA in C. elegans. These tools allow for gene disruption, insertion, correction, or replacement, with wide applications in functional genomics, disease modeling, and therapeutic development.

Major Genome Editing Mechanisms

NHEJ – Non-Homologous End Joining

NHEJ disrupts gene expression by creating some bases insertion or deletion, called indel mutations. One DSB repaired by NHEJ induce frame shift, and two DSB repaired by NHEJ produce a fragment deletion. For screening convenience, we involved any visible marker carried by transposable elements in two DSB CRISPR.

• Mechanism: DNA double-strand breaks (DSBs) are repaired by directly joining broken ends without a template.
• Outcome: Often introduces insertions/deletions (indels), leading to gene disruption.
• Use Case: Efficient for gene knockout.

HDR – Homology-Directed Repair

HDR functions as a versatile knock-in tool to bring a fragment in the gene-of-interested to create premature stop, to delete large gene region and replace it with an attP-landing site for further modifications, to bring in RMCE cassette for further gene exchange or reporter (such as Gals4) exchange, to replace some amino acids, and to fuse a fluorescence protein for trace. We accept customized designs for your research needs.

• Mechanism: Uses a DNA repair template with homology arms to introduce precise changes at the break site.
• Outcome: Allows for exact insertion, correction, or replacement of DNA sequences.
• Use Case: Ideal for gene knock-in or precise editing, but less efficient and restricted to dividing cells.

TALEN – Transcription Activator-Like Effector Nucleases

The target site of TALEN is composed of over 20 base nucleotides, usually 30~40 bases. Each nucleotide is recognized by a tandem array of 33~35 amino acid repeats. Specifically, the specificity of each repeat domain is determined by the 12th and 13th amino acids, which are called RVD (repeat variable di-residues). However, TALEN cloning is a time-, effort- and money-consuming step since over 20 35-amino-acid-repeats need to be put on the same construct.

• Mechanism: Engineered proteins (TALENs) bind specific DNA sequences and induce DSBs via FokI nuclease domains.
• Outcome: DSBs repaired via NHEJ or HDR.
• Use Case: Custom gene editing with high specificity but labor-intensive to design.

CRISPR/Cas9 – Clustered Regularly Interspaced Short Palindromic Repeats

CRISPR make genome double stand break easy and specific. CRISPR function as a RNA-guided engineered nuclease, and is composed of crRNA, tracRNA and Cas9 nuclease. crRNA is processed from pre-crRNA under the contribution of tracRNA, and the 20-base sequence on crRNA is responsible for the specificity of target site. DualRNA-Cas9 then form an active DNA endonuclease to recognize 20 bases + NGG sequence on genome and undergo endonuclease activity. This makes CRISPR an easier, faster, cheaper way to edit genome.

• Mechanism: Cas9 endonuclease guided by RNA (gRNA) to target DNA sequences for cleavage.
• Outcome: DSBs repaired by NHEJ or HDR pathways.
• Use Case: Widely used for gene knockout, knock-in, base editing, and multiplex genome editing due to simplicity and scalability.

Figure 1. CRISPR-Cas9 genome editing approach in C. elegans. (Kim HM, et al. 2019)

Comparison of Genome Editing Technologies

Feature	NHEJ	HDR	TALEN	CRISPR/Cas9
Editing Precision	Low	High	High	High
Repair Mechanism	Error-prone DSB joining	Template-guided DNA repair	DSB via protein-DNA recognition	DSB via RNA-guided Cas9
Indels / Knockout	✅ Highly efficient	❌ Rare	✅ Moderate	✅ Highly efficient
Knock-in / Point Mutation	❌ Not suitable	✅ Precise but low efficiency	✅ Moderate (with HDR)	✅ Widely used with HDR
Targeting Specificity	N/A	N/A	✅ High	✅ Moderate to high (with improved gRNA design)
Multiplexing Capability	❌	❌	❌	✅ Easily multiplexed
Ease of Design & Use	N/A	❌ Needs donor template	❌ Complex protein engineering	✅ Simple RNA design
Cost & Scalability	N/A	❌ Higher	❌ Expensive and slow	✅ Low-cost and scalable
Cell Cycle Dependence	❌ Cell cycle–independent	✅ Requires S/G2 phase	❌ Mostly cell cycle–independent	❌/✅ Depending on pathway used

Key Insights

• NHEJ is best suited for quick and efficient gene disruption.
• HDR allows for precise gene editing, but is limited to dividing cells and has lower efficiency.
• TALENs offer high specificity but are labor-intensive to produce and modify.
• CRISPR/Cas9 has become the go-to technology due to its simplicity, flexibility, and adaptability to various editing contexts.

Reference

1. Kim HM, Colaiácovo MP. CRISPR-Cas9-Guided Genome Engineering in Caenorhabditis elegans. Curr Protoc Mol Biol. 2019 Dec;129(1):e106.