GENETIC MANIPULATION WITH

CRE-LOXP
Cre-Lox Recombination

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.
Introduction to the Cre-Lox System

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.
 Sequence of a loxP site, made up of two flanking palindromic
recognition regions and a spacer region which gives it
directionality.

13 bp Recognition 13 bp Recognition
Region 8 bp Spacer Region
ATAACTTCGTATA ATGTATGC TATACGAAGTTAT

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
 Four Cre proteins form a tetramer. Tyrosines at position 324 cleave the DNA
backbone within the loxP site, and the released hydroxyl groups attack the
partner strand to form a Holliday intermediate. The remaining strands of DNA
are then cleaved and strand exchange occurs. The resulting sequences are also
lox sites which can undergo subsequent rounds of recombination
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”.

The directionality of the lox sites determines the outcome of the recombination.
If lox sites are in the same orientation, the floxed region will be excised. If the lox
sites are in opposing orientations, the floxed region will be inverted. If the lox
sites are on separate pieces of DNA, recombination results in a translocation
event
Basic Applications

The basic Cre-loxP recombination event is most useful for excision of genetic
sequences, due to the irreversible nature of this event. It has been adapted for two
main purposes:

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. As well, stop cassettes are large (~1.8 kb), which makes them difficult to
package into small viruses, like adeno-associated virus (AAV)
DIO and DO Switches

Scientists have discovered that if the spacer region of the lox site is altered, Cre
doesn’t cause recombination between the wildtype and mutant lox sites.

However, two of the same mutant lox site can recombine with each other.

Many variations of lox site have now been developed, which are not cross-
compatible:
Name Left Recognition Spacer Right Recognition Region
Region

loxP ATAACTTCGTATA ATGTATGC TATACGAAGTTAT

lox511 ATAACTTCGTATA ATGTATAC TATACGAAGTTAT

lox2272 ATAACTTCGTATA AAGTATCC TATACGAAGTTAT

lox5171 ATAACTTCGTATA ATGTGTAC TATACGAAGTTAT

m2 ATAACTTCGTATA AGAAACCA TATACGAAGTTAT

m3 ATAACTTCGTATA TAATACCA TATACGAAGTTAT

m7 ATAACTTCGTATA AGATAGAA TATACGAAGTTAT

These sequences can be combined to make DIO (Double-
floxed Inverse Orientation) or DO (Double-floxed Orientation) switches.

These are also sometimes known as FLEx (or Flip Excision) switches.

DIO and DO vectors have the sequence of interest flanked by two sets of
different lox sites.

Two recombination steps occur – the first is an inversion of the flanked

sequence using one set of lox sites.

This leaves two identical sites with the same orientation on one side of the
gene.

The second recombination causes the excision of the intervening sequence.

The order in which the lox sites undergo recombination is random, but the
final product will be the same in both cases.
The inverted version of a gene will not be expressed, so DIO and DO
vectors can be used to control gene expression.

Unlike the large lox-stop-lox cassette, two sets of lox sites only take up
~5% of an AAV’s packaging capacity, leaving more room for other genetic
elements.

A DIO vector will have the gene of interest cloned in the inverted
orientation (eg. 3’-> 5’), 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”.
 A DIO vector, which has a Double-floxed gene
with Inverted Orientation. Inversion occurs via either loxP or lox2272,
followed by the excision of two lox sites. The DIO vector turns on gene
expression only in the presence of Cre
Cre-Lox for in vivo Research

Cre-lox recombination is ideal for performing genetic manipulation in vivo,

such as in mouse models.

When working in vivo, one must take into account some unique concerns:

1. Breeding mice is a lengthy and laborious process.

2. Mimicking a disease or mapping an organ may require a gene to be

overexpressed only in certain tissues.

3. Preventing lethality may require a gene to be overexpressed only after

embryonic development.
Despite these difficulties, mouse models are invaluable as a research tool due to
their similarity to humans.

Cre-Lox recombination addresses these concerns:

1. Cre-Lox is commonly used in mice, so there are many commercial Cre-

expressing mouse lines available for purchase. As well, one Cre-expressing
mouse line can be re-used for many different experiments, simply by
changing which floxed gene is expressed from a vector.

2. Tissue-specific expression in mouse models is easy to achieve with a DIO

switch. A tissue-specific promoter can be used to express Cre, which will then
control where the floxed gene of interest is expressed.

3. Cre expression can be controlled using an inducible promoter or regulator.

Cre won’t be expressed until the inducer is added, at which point the DIO/DO
switch will turn on or off.
Advanced Applications

Optogenetics

A particularly popular use for DIO vectors are optogenetic experiments. In

these experiments, an opsin (a neuronal channel or pump that is activated by
light) 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.

Then, the mouse can be fitted with a fiberoptic cannula and awoken.

Behavioural changes can be observed when the opsin is activated via light in
those particular 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.

For these experiments, two mouse lines are crossed together: a recombinase-
expressing mouse, and an indicator mouse.

The recombinase mouse has Cre expressed via a promoter specific for the
progenitor cell of interest.

The indicator mouse will ubiquitously express a reporter such as β-galactosidase

controlled by a Cre-On switch.

In crossed embryos harbouring both genes, the recombinase will irreversibly turn
‘on’ expression of the indicator only in the progenitor cell of interest.

All cells that originate from the progenitor cell will also express the indicator.

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 helps ensure that all cells to be labelled have been co-transfected with
both reporter vectors, but that the level of expression is not too high.

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.
 Cre-Lox for Gene Knock-In

A dual lox system can also be used to make gene knock-ins easier by using
Recombinase Mediated Cassette Exchange (RMCE).

In this approach, a selection marker would be present in the genome flanked by

different lox sites.

By introducing a vector carrying a gene of interest flanked by those same lox

sites, the gene of interest will be swapped into the genome 50% of the time.

Cells in which the swap has occurred can be enriched for or screened using the
absence of the selection marker.

This is a powerful tool for in vivo knock-in, since the same mouse line can be used
to generate many different knock-in strains; you simply need to change the gene
in your vector.
Recombinase-mediated cassette exchange
using Cre-Lox
An alternate method uses inverted-repeat variants, which have been mutated
in the left or right inverted repeat (called LE mutant sites or RE mutant sites,
respectively.

Recombination will occur between a LE site and a RE site, but the result will be
one wildtype loxP site and one LE/RE double mutant site.

The LE/RE site is not able to undergo further recombination, so the integration
is permanent.
Gene insertion using mutant lox sites. Recombination between a LE
mutant lox site and RE mutant lox site results in one LE/RE mutant
site and one wildtype loxP site. The LE/RE site is incompatible with
the loxP site, so no further recombination events occur.
 Other Site-Specific Recombinases

The use of other site-specific recombination systems in combination with Cre-Lox

allows for even greater flexibility and control of genetic experiments.

Another popular site-specific recombination system is Flp-FRT, which uses a Flp

recombinase to recombine FRT sites.

However, other recombinases have also been used, including R4, lambda, phi31,
HK022 and TP901-1.

The use of two recombinases allows for the cloning of complex genetic switches.

For example, one can target tissue-specific gene expression with even more
precision by using two tissue-specific promoters – one to express Cre, and the other
to express FLP.

This approach can be used to express any gene of interest only at the intersection
(in space or time) where Cre and FLP co-exist