CRISPR/Cas9 Delivery in Rat Zygotes for Germline Transgenesis: Microinjection vs. Electroporation

The generation of genetically modified rats through CRISPR/Cas9-based genome editing has been significantly advanced by the development of efficient delivery techniques targeting one-cell zygotes. The two principal methods employed for the introduction of CRISPR/Cas9 components—microinjection and electroporation—each offer distinct advantages and trade-offs in terms of editing efficiency, technical complexity, and embryo viability. Microinjection involves the direct injection of Cas9 mRNA or protein and single-guide RNA (sgRNA) into either the cytoplasm or pronucleus of fertilized rat zygotes. While pronuclear injection has traditionally yielded higher integration rates, cytoplasmic injection reduces the risk of damage to the nucleus and can be easier to perform consistently. This method requires skilled micromanipulation under a microscope and is labor-intensive, limiting throughput.

Electroporation, by contrast, allows for the simultaneous delivery of CRISPR/Cas9 ribonucleoprotein complexes into batches of zygotes using short electrical pulses to transiently permeabilize the cell membrane. This technique significantly increases the number of embryos that can be processed in a single session and has demonstrated comparable gene editing efficiencies to microinjection when optimized appropriately. Key electroporation parameters include voltage amplitude, pulse width, number of pulses, and the composition of the electroporation buffer. Rat zygotes are particularly sensitive to physical and osmotic stress, so the use of low-conductivity, isotonic buffers such as Opti-MEM is critical to preserving embryo viability during electroporation.

An important consideration in both methods is the timing of delivery relative to fertilization, as the zygotic cell cycle stage impacts the integration efficiency and frequency of mosaicism in the resulting embryos. Microinjection or electroporation should ideally occur within a narrow post-fertilization window to ensure editing occurs prior to the first round of DNA replication. Mosaicism—when some cells in the resulting organism carry different genotypes—can complicate phenotypic analysis and requires breeding to isolate homozygous mutant lines. Validation of genome editing is typically performed through PCR-based genotyping, T7 endonuclease assays, or sequencing of target loci in the resulting pups.

Off-target effects remain a concern in germline editing, especially in a therapeutic or regulatory context. High-fidelity variants of Cas9 and careful in silico sgRNA design can mitigate these risks, along with whole-genome sequencing of founder animals when required. In conclusion, both microinjection and electroporation represent powerful strategies for CRISPR/Cas9-mediated germline modification in rats. While microinjection remains the gold standard for precise genome engineering, electroporation offers scalable, high-throughput delivery with less labor and equipment intensity, making it increasingly attractive for laboratories generating transgenic rat models.