Twenty years ago, Gu et al. (1) published the first paper using the Cre-loxP system to study the immune response, entitled “Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting.” That experimental strategy has enabled construction of clean mutations in essentially any gene of interest, in almost any cell type, and it has been fundamental to our current detailed understanding of immunity and development. It is especially interesting to look back at this achievement in light of the rapid recent progress in the use of customized nucleases for targeted gene correction and gene disruption.

Gene disruptions, in contrast to deletions, had become common following publication of the classic homologous gene targeting paper by Thomas and Capecchi in 1987 (2). However, targeting constructs typically bore a selectable neomycin expression cassette that remained embedded in the engineered locus. This cassette in principle could (and occasionally did) corrupt expression of the targeted gene or the function of its product. The paper by Gu et al. (1) posed the question of whether cis-elements regulate class switch recombination (CSR). Rigorous analysis required a deletion mutation, because persistence of the promoter and enhancer driving neomycin transcription had clear potential to confound identification of cis-regulatory elements at IgH.

Enter Cre-loxP. Rajewsky’s laboratory (1) targeted the region of IgH spanning JH–Eμ for deletion using a targeting construct very much like those used for gene ablations, but distinguished by the presence of selectable markers and loxP sites flanking the region targeted for deletion. Expression of Cre was predicted to cause deletion of the region bound by the loxP sites, and this did indeed occur, at a frequency of 2–4%. Three lines produced by deletional recombination were successfully used to generate mutant founder mice that transmitted the JH–Eμ deletion through the germline, whereas a fourth gave rise to a single male chimeric mouse, which was sterile. Finally, the JH–Eμ deletion had a clear effect on both CSR and B cell development. In heterozygous mice bearing the JH–Eμ deletion on a single chromosome, VDJ recombination and efficient CSR at μ occurred on the wild-type chromosome but not on the chromosome bearing the deletion. Homozygous mutants lacked all B cells, reflecting failed VDJ recombination in the absence of Eμ.

Genome engineering can be accompanied by collateral damage, and the paper offered reassurance on this point. Cre expression appeared not to cause rampant genome-wide instability, based on the observation that three of four embryonic stem cell lines tested were capable of germline transmission. Additionally, the excised DNA fragment appeared not to reintegrate elsewhere in the genome, as assayed by Southern blotting. Neither concern was without foundation. In subsequent experiments designed specifically to assay toxicity, high-level Cre expression in spermatids was shown to abolish fertility (3). Additionally, later experiments found that reintegration of the fragment deleted by RAG1/2-mediated V(D)J recombination occurs with a frequency of 1 in 50,000 events (4).

Cre is one member of a family of site-specific recombinases that cleave 34-bp target sequences (5). Cre derives from the coliphage P1, and its temperature optimum of 37°C is perfectly suited for genome engineering in murine cells. A related system, Flp-FRT, derived from Saccharomyces cerevisiae, had also shown initial promise for genome engineering. Flp-FRT was used by Jung et al. (6) to generate a targeted deletion 5′ of Sγ1, reported a few months before the Gu et al. paper came out. However, Flp has a 30°C optimum, making it less effective than Cre in mouse cells, and Cre came rapidly to dominate murine genetics (7). Flp-FRT has been improved over the years, and it now matches Cre-loxP in efficiency (5). These two site-specific recombination systems are now readily combined for sophisticated genome engineering applications.

Much of the power of the Cre-loxP system derives from the potential to generate conditional mutants. Conditional deletions can overcome the barrier that embryonic lethality otherwise poses to studying essential genes, as first shown by Rajewsky’s laboratory in experiments that analyzed T cell–restricted deletion of the repair polymerase, pol β (8). Conditional deletions have since been key to a vast number of studies that have elucidated the functions of specific genes and regulatory elements in the immune response and development. Inspired by the many successes, the International Knockout Mouse Consortium developed a repository of murine embryonic stem cell lines in which essentially every gene is flanked by loxP sites (or “floxed”) to enable studies of individual gene functions in specific tissues (9). However, conditional deletion of every gene in every cell type may not become a reality. Conditional deletions are only as good as the promoters that regulate Cre expression, and transgenic Cre driver lines have proven to be somewhat problematic, exhibiting expression outside the target tissue as well as inefficient cleavage leading to mosaicism (10).

Will Cre-loxP and related systems continue to dominate? Alternatively, has genome engineering entered a new era, now that TALEN or CRISPR/Cas-based nucleases can be readily customized to cleave at essentially any genomic site (11, 12)? These new approaches are enabling genome engineering in organisms that were recalcitrant to Cre-loxP, such as zebrafish (13), where it now takes only 2 wk to generate a targeting TALEN and use it to create a modified embryo (14). TALEN and CRISPR/Cas-based approaches must surmount some of the same hurdles initially confronted by Cre-loxP or Flp-FRT, such as optimizing efficiency and minimizing collateral damage. Current technologies such as high-throughput screens and deep sequencing will undoubtedly accelerate optimization of TALENs and CRISPR/Cas-based systems. Nonetheless, in the end these new approaches will be judged not by how rapidly the genome was engineered but by the quality of the mutants generated. The Cre-loxP system has set a very high bar.

Abbreviation used in this article:

CSR

class switch recombination.

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The author has no financial conflicts of interest.