What is Cre-Lox Recombination?

Keywords

Genetic manipulation, gene expression, Cre recombinase, recognition site, P1 bacteriophage, genomes, recombination.

Introduction

Cre-Lox is a powerful tool for genetic manipulation in vivo because it allows for excellent spatial and temporal control of gene expression. This is invaluable when working with animal models, where unchecked gene expression or complete gene knockout may be detrimental or lethal.

As the name suggests, the Cre-Lox system relies on two components to function: a Cre recombinase, and its recognition site, loxP. These components have been adapted from the P1 bacteriophage for use in genetic manipulation. LoxP sites are directional 34 bp sequences made up of two 13 bp recognition sites separated by an 8 bp spacer region.

The sequences don’t occur naturally in any known genomes other than the P1 bacteriophage and are long enough that they are unlikely to occur by chance. For this reason, they can be used for specific genetic manipulations without unintended side effects. LoxP sites are always used in pairs, often flanking a gene or interest or reporter. The orientation of the loxP sites will determine the outcome of recombination.

Mechanism of Cre-Lox Recombination

First, two Cre proteins recognize a loxP site and bind to it, forming a dimer. Two Cre-loxP dimers come together to form a tetramer, bringing the two loxP sites together with opposing directionality. Finally, dsDNA cleavage occurs in the center of the loxP site, and a crossover event occurs. Based on the orientation of the loxP sites, there are three outcomes that can result from the recombination:

Excision

If lox sites are in the same orientation flanking a sequence, recombination will result in the excision of the sequence, deleting it from its original locus. This process is essentially irreversible.

Inversion

If lox sites are in opposite orientations flanking a sequence, recombination will result in the inversion of that sequence. Since the lox sites remain unchanged, this process is reversible, and the gene can “flip” back and forth between both orientations indefinitely.

Translocation

If lox sites reside on separate pieces of DNA, recombination will result in a reversible translocation event. Lox-flanked regions of DNA are often said to be “floxed”.

DIO and DO Switches

The inverted version of a gene will not be expressed, so DIO and DO vectors can be used to control gene expression. DIO and DO vectors have the sequence of interest flanked by two sets of different lox sites. These sequences can be combined to make DIO (Double-floxed Inverse Orientation) or DO (Double-floxed Orientation) switches.

A DIO vector will have the gene of interest cloned in the inverted orientation and will become correctly oriented in the presence of Cre; thus, DIO vectors are “Cre-On”. A DO vector will have the gene of interest present in the correct orientation and become inverted in the presence of Cre; therefore, they are “Cre-Off”.

Applications

The basic Cre-loxP recombination event is most useful for excision of genetic sequences, due to the irreversible nature of this event.

Cre-dependent Sequence Knockout

If a sequence is flanked with two loxP sites in the same orientation, the sequence will be excised when Cre is present. This can be useful when performing gene editing experiments; successfully edited clones may be found using a selection marker, which can later be removed using Cre-Lox. However, a complete gene knockout isn’t a good choice for studying essential genes, since this would result in a lethal phenotype.

Cre-dependent Gene Expression

A ‘lox-stop-lox’ cassette can be placed upstream of a gene. Without Cre, the stop cassette prevents the translational expression of the gene. In the presence of Cre, the stop cassette is deleted and gene expression proceeds. However, using the ‘lox-stop-lox’ approach to control gene expression has some disadvantages. This strategy has noticeable levels of background expression when Cre is absent. Also, stop cassettes are large, which makes them difficult to package into small viruses, like adeno-associated virus (AAV).

Advanced Applications

Optogenetics

A particularly popular use for DIO vectors is optogenetic experiments. An opsin can be expressed from a DIO AAV that has been injected into a mouse brain. The mouse will express Cre from a cell type-specific promoter, so that the opsin will only be expressed in certain neurons. Behavioral changes can be observed when the opsin is activated via light in those neurons.

Fate-mapping Experiments

DIO switches can also be used for fate-mapping experiments, used to determine how certain progenitor cells will replicate as an organism grows. Cre expression isn’t necessary for reporter expression in descendent cells, since the Cre-On genetic modification is irreversible and inheritable.

Co-expression of Multiple Reporters

DIO vectors are also extremely useful for controlling co-expression of multiple reporters. Reporter vectors can be co-transfected at high molar ratios, while a limiting amount of Cre-expressing vector is added. This is useful for mapping certain cell types, such as neurons and their synapses, which are difficult to image at high levels of reporter expression.

Studying Functional Mutations

Placing the lox sites in a different position can cause the activation of one gene and simultaneous excision of another. This system can be used to easily replace the wildtype version of a gene with a mutated version. This is an excellent way to study essential genes since traditional knockout studies would be lethal.

Conclusion

Technology using Cre-loxp system provides the sophistication to study the gene functions. Recently, Cre-loxp system for more precise control has been continuously developed. In addition, Cre-loxp system approaches have been continually being developed using Crispr/Cas9 technology and viral system.

Using more sophisticated control techniques, researchers will be able to understand more precise gene functions by studying the function of specific genes at desired time (temporal) and tissue (spatial). The useful Cre-related portal sites and databases will enhance the efficiency of research by allowing researchers to find and obtain the suitable Cre-driver lines for research.