CRISPR meets Cre: conditional gene inactivation just got easy

In 1960, the U.S Navy introduced the KISS principle (Keep It Simple, Stupid), proposing that the best way to make a system working at its best, is to keep it simple.

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I do not know much more about the U.S. Navy but I would argue, that this is particularly good advice for daily life as well. And indeed, why would you choose a complicated solution if there is an easy one? The KISS principle can also be applied to many aspects in life science and now even to the method of conditional gene inactivation. But what does this actually mean and how can such an approach actually spark joy when it comes to genome editing? When you deal with an adult stem cell population, you are interested in several questions. For one, you want to know the potential of the stem cell; meaning what does it gives rise to. You also want to know if the fate of the progeny can be changed, for example by the misexpression of another factor. And finally, you want to know which factors are in control of the stem cell fate and its progeny. To address all of these questions, scientists depend on genetic tools that allow them to manipulate the stem cell population at adult stages. Of all the systems available, Cre/lox is outstanding and superior. The Cre/lox system allows genetic lineage tracing, temporal and spatial misexpression and conditional gene inactivation studies. In its native form, Cre/lox is very simple. It relies on the recombinase Cre and certain sequences in the DNA, called lox. There are a couple of different lox sites, which only differ slightly in their sequences, with loxP being the most famous one (figure 1A). When Cre encounters two lox sites in the same orientation, it deletes the intervening DNA sequence. This enables lineage tracing, misexpression or conditional gene inactivation (figure 1B). To control the activity of Cre at later or even adult stages, one can use ligand-inducible Cre variants. Here, Cre is fused to a modified estrogen receptor (e.g. CreERT2) allowing to turn on Cre activity by the application of a ligand. This way, a single CreERT2 source, expressed in a given stem cell population, allows to address all questions mentioned above. One simply combines the CreERT2 source with any Cre effector which can be easily generated via transgenesis. Both approaches have been performed in many different model organisms like mouse, zebrafish or fruit fly. In stark contrast, conditional gene inactivation requires sophisticated genome engineering to equip the gene-of-interest with two lox sites. For years and even decades, mammals were the only organisms in which this could be achieved, setting the gold standard. Only embryonic stem cell cultures allowed the introduction of two lox sites into the gene-of-interest. Subsequently, the engineered gene-of-interest was combined with a Cre or CreERT2 source to perform conditional gene inactivation studies. The advent of CRISPR was a game changer and provided a quantum leap for all other model organisms, opening the door for conditional gene inactivation studies also in zebrafish. Following the same logic, scientists use CRISPR to equip their gene-of-interest with two lox sites and combine the engineered gene-of-interest with a Cre or CreERT2 source to perform conditional gene inactivation studies. However, genome engineering and generation of animals carrying the desired genetic composition is time and labor consuming. Moreover, the overall success of the experiment is by far not guaranteed which leaves plenty of room for frustration after spending so much time and energy into a project. Hence, an alternative approach following the KISS principle was needed. This is where Cre-Controlled CRISPR comes into play; an easy and straightforward system that allows conditional gene inactivation in a Cre-dependent manner (figure 1C). Instead of using CRISPR and Cre/lox technology sequentially, Cre-Controlled CRISPR exploits the best of both worlds and combines the benefits of Cre/lox and the ease of gene editing of CRISPR into a win-win situation.  Cre-Controlled CRISPR has just recently been published in Nature Communications (https://www.nature.com/articles/s41467-021-21427-6).

Figure 1: (A) The Cre/lox system relies on the Cre recombinase and its target sequence called lox. (B) Rationale of the mostly applied approaches for Cre/lox: genetic lineage tracings, temporal and spatial misexpressions and conditional gene inactivations. For genetic lineage tracing and temporal and spatial misexpression the construct relies on the expression of a marker like GFP or the expression of a gene-of-interest (GOI) following a Cre-dependent deletion event. For conditional gene inactivation, the gene-of-interest needs to be flanked by two lox sites resulting in a deletion of the gene-of-interest following Cre-mediated recombination. (C) Rationale of Cre-Controlled CRISPR. A Cre effector construct controls the expression of a floxed Stop cassette upstream of the sequence encoding a fusion protein of Cas9 and GFP. In addition, an U6a promoter drives the constitutive expression of a gRNA targeting a gene-of-interest (GOI). Cre-mediated recombination results in the expression of Cas9-GFP. Combined with the gRNA a functional CRISPR complex is formed and mutates the gene of interest.

Dr. Stefan Hans

Subgroup leader, CRTD, Technische Universität Dresden