Knock-in genome editing allows researchers to investigate the function of specific gene variants, study genome regulation, and introduce reporter genes (e.g., green fluorescent protein) for monitoring gene expression. It also promises to be an effective therapeutic strategy where it is used to correct a mutation or replace a faulty gene. Knock-in mouse models are essential tools for such projects as they mimic the human condition from a genetic standpoint. However, developing mouse models with large genomic modifications has proven to be a challenging task. This article explains how a method known as recombinase-mediated cassette exchange (RMCE) can provide mouse models with knock-ins as large as 300 kb. It also reports on the use of two gene-editing technologies based on RMCE (TurboKnockout® and CRISPR-Cas9) for producing large fragment knock-ins (LFKIs).
Knock-in mouse models are widely used to mimic human genetic disorders, investigate the role of specific genes during embryonic development, and evaluate potential drug candidates for their efficacy in treating disease. They are typically produced using site-specific recombinases (SSRs) to exchange DNA between a vector and a recombination site in the genome of fertilized mouse eggs; these are then implanted into surrogate mothers, with the resultant offspring being screened by PCR to reveal any insertions. Critically, the vector and the recombination site must share a certain degree of sequence homology, with the recombination site comprising two motifs with a partial inverted-repeat symmetry; the relative orientation and molecular location of these motifs dictates whether genes are inverted, excised, or inserted. While various SSR-based strategies have been developed, RMCE offers several important advantages.
A major advantage of RMCE over other methods used for generating knock-in mouse models is that it enables knock-ins of up to 300 kb, which includes many whole genes. To achieve this, the gene of interest (GOI) is integrated into the mouse genome by exchanging a pre-existing gene cassette for an analogous cassette containing the GOI. For proper insertion, the wild-type gene and the vector-delivered gene must each be flanked by the same two (unique) recognition sites, with a recombinase (e.g., Cre) being used to perform the exchange. The capacity to introduce large fragments has led RMCE to be used for many different applications, including the generation of conditional, reporter, and transgenic mouse models; it has also been adapted to produce humanized mouse models that replicate human gene expression patterns in vivo.
Another important application of RMCE is to generate mutant models for large-scale mutation screens, characterization of domain-specific protein functions, and other comparative studies. In this scenario, RMCE is used to insert a mutation (either a point mutation, large gene mutation, or a gene replacement) directly into a parental embryonic stem (ES) cell clone, with different targeting vectors being used to produce a broad range of unique mutant cell lines. Since just one ES clonal line is required to produce multiple mutants, RMCE provides an efficient and cost-effective approach to performing large-scale genomic studies.
In addition to being a stand-alone technology for developing large fragment knock-in models, RMCE can enhance other gene-editing strategies. One such example is CRISPR-Cas9, which uses a guide RNA (gRNA) to target the Cas9 endonuclease to a gene of interest and cause a double-strand break. When CRISPR-Cas9 is used for gene knockout, any insertions or deletions introduced at the cut site via cellular repair mechanisms are inconsequential. Yet, when CRISPR-Cas9 is used for gene knock-in, it is vital that the break be repaired perfectly. Harnessing CRISPR-Cas9 with RMCE for targeted gene insertion into the ROSA26 locus—often referred to as a ‘safe harbor’ locus—helps ensure minimal insertion site side effects and the stable ubiquitous expression of inserted genes to achieve accurate modification of higher eukaryotic genomes. Another example is TurboKnockout®, a gene targeting method that uses a super competent ES cell line to eliminate the unpredictability of germline transmission, generating 100% ES cell-derived founder mice and circumventing the chimera stage. When combined with RMCE, TurboKnockout® can provide mouse models with knock-ins upwards of 300 kb, guaranteed genotype, and no off-target effects.
Cyagen offers a broad portfolio of custom genetically engineered models, including knock-in mouse models generated using RMCE. To learn more, visit cyagen.com
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Large Fragment Knock-In (LFKI) Mouse Models Using Recombinase-Mediated Cassette Exchange (RMCE)
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