Download Applications of site-specific recombination As can be

Applications of site-specific recombination
As can be seen from the examples discussed above, the same mechanism of DNA recombination
can be utilized in different biological contexts to bring about integration, excision (deletion) and
inversion of DNA segments. In principle, then, one should be able to adapt site-specific
recombination systems to direct one or more of these types of DNA rearrangements in selected
regions of a genome of interest. This expectation has been fully validated. Site-specific
recombination has been utilized in promoting genetic alterations for answering fundamental
questions in biology and for developing biotechnological tools.
Since Cre and Flp have extremely simple reaction requirements, these recombination
systems have been reconstituted in a variety of organisms-bacteria, fungi, plants, nematodes,
flies and animals. Cre and Flp can be placed under regulatable promoters for conditional or tissuespecific expression.
An important first step in applying site-specific recombination in a genetic context of
interest is the introduction of the target site or sites at the desired locale(s). Once this has been
accomplished, the rest of the experimental steps are quite straightforward.
Tracking cell lineage during development
One of the most useful applications of site-specific recombination in basic biology has been in
tracking the lineage of cells during development. The early work was done in Drosophila using
the Flp-FRT system. The method has been extended to a variety of developmental systems,
helped, in particular, by important technological advances in fluorescence microscopy and the
development of multi-color reporter constructs.
The principle of the method is illustrated in the figure below. Here site-specific
recombination is used to bring about mitotic recombination between homologous chromosomes.
In an organism such as Drosophila, the reporter constructs and the recombination sites can be
appropriately placed in the chromosomes to be manipulated.
The FRT sites are placed at identical positions close to the centromeres of a pair of
homologous chromosomes. In one chromosome a GFP (green fluorescent protein) reporter gene,
lacking a promoter, is placed adjacent to the FRT site as shown in the figure below. In the
homologue, a promoter sequence is placed next to the FRT site. At a certain stage in
development, as demanded by the experimental objective, the Flp protein is expressed by turning
on a conditional promoter that controls the FLP gene. Consider the state of a cell in which DNA
replication has been completed. There are two sister copies of each of the two chromosomes that
we are interested in. Each pair of sister chromatids is held together, so that they can be
segregated to the daughter cells in a one-to-one fashion. When recombination occurs between
FRT sites as diagrammed, one chromosome acquires the promoter from its partner, and becomes
competent to express GFP. When the cell divides, only one of the two daughter cells will acquire
this chromosome; therefore only this cell will express GFP, and appear as green under the
fluorescence microscope. The other will lack fluorescence. All the cells resulting from the division
of the fluorescent progenitor cell will also be green. These clones will form a mosaic against the
majority of cells that are non-fluorescent. Thus, by tracking fluorescence, cell lineage can be
followed during development.
Ablating a gene function during development
One can also delete a particular gene at a given stage of development, and follow the
consequence of the deletion during further development. Here the locus of interest is flanked by
two copies of FRT in a direct (or headͲtoͲtai) orientation (see the figure above). The Flp protein is
induced from a regulated promoter. If one expresses the Flp gene from a tissue specific promoter,
the deletion will occur only in that particular tissue. The effects of removing the gene function in a
given tissue or a set of tissues can be followed.
Inducing the expression of a gene at a specific time in development
One can induce the expression of a gene at a desired point in development via site-specific
recombination. For example, imagine a gene X engineered in such a way that its promoter is
oriented in the nonͲfunctional direction (see the figure below). The promoter is flanked by two FRT
sites in the direct or headͲtoͲhead orientation. When the Flp protein is induced at the appropriate
time, recombination by Flp will invert the DNA segment containing the promoter. It is now turned
around, and acquires the functional orientation, thus turning on the gene X.
+
Alternatively, one can introduce a transcription terminator site between the promoter and
the gene of interest (see the figure below). The terminator is flanked by a recombination target
site at either end, with the sites arranged as direct repeats. With the terminator present,
transcription initiated at the promoter cannot get past the terminator and enter the gene. However,
when the terminator is removed by recombination, transcription proceeds into the gene, thus
activating its function.
Site-specific recombination in biotechnological applications
Once the target site has been inserted into the genomic locus of interest, one can perform
integration of an exogenous DNA sequence into this target site. Since the inserted sequence is
flanked by two directly repeated (head-to-tail) target sites, it can also be excised from the genome
by recombination. These operations can be performed in a regulated fashion by inducible
expression of the recombinase. Similarly, if two target sites are inserted on either side of a certain
genetic locus, recombination can be used to excise the locus. Removal of integrated harmful viral
sequences would be a potential beneficial application of site-specific recombination.
A more recent application of site specific recombination in biotechnology is described as
recombination mediated cassette exchange (RMCE; see Figure below).
This method uses two recombination events to replace the endogenous chromosomal locus by a
new one. Originally, a single recombinase was used for RMCE (shown in A). RTa and RTa* are
slightly altered sites, so that Rta x Rta* recombination cannot occur. However, the recombinase
can act on both RTa and RTa* with nearly equal efficiencies. [I shall explain in class how this can
be done.] In later versions of RMCE, two recombinases and their respective target sites were
employed. The two cross-over events diagrammed cleanly splices out the pre-existing locus, and
exchanges it for the incoming locus.
Expanding the utility of site-specific recombination in biotechnology
As we already saw, one of the limitations in the application of site-specific recombination in
biotechnology is the introduction of the target site being a pre-requisite for manipulating a genome
of interest. The other is the small number of recombinases that can be easily manipulated for
genome engineering purposes. For all practical purposes, the number is two, Cre and Flp. For
each recombinase, one can generate a small set of variant target sites that are not cross reactive
with each other, but are normal or nearly normal in self-reactions. These target sites are created
by changing the sequence of the short strand exchange region without changing the binding sites
for the recombinase. The absolute sequence of the strand exchange region is not terribly
important for recombination (although some sequences work poorly). However, there has to be
perfect homology between the strand exchange regions for recombination to occur between two
target sites. In other words, an altered site is reactive with a second copy of itself, but non-reactive
with the native site or with another altered site containing a different substitution.
A potentially useful approach to expand the utility of site-specific recombination is to
generate recombinases with altered binding (DNA recognition) specificities. That is, change the
sequence of the binding elements, and then produce active recombinase variants that have
acquired the corresponding new recognition capabilities. Although this idea would seem
reasonably straightforward, this is quite difficult to accomplish in practice. Note that the present
day Flp or Cre represent the optimization of DNA-protein recognition and catalysis over
evolutionary time. It would be quite difficult to redesign that optimization within the time frame of
laboratory experiments. Nevertheless, what one tries to do is mimic the process evolution in the
test tube. The approach is called directed in vitro evolution, and is briefly described below.
On can generate a large pool of random mutants of the recombinase gene, preferably by
PCR- based mutagenesis. Among the 107 or 108 of mutants generated, there might be one or a
tiny number of recombinase variants that might have acquired specificity for the new target
sequence that we designed. The predominant majority will consist of recombinases that have lost
function because of acquired mutations or have not changed their DNA recognition. These are
not of interest to us. The problem is how to find the needle in the hay stack. For this one has to
have a simple and quick genetic or physical screens to track down the clones of interest to us.
The way the directed evolution protocol is carried out is as follows. First, we transform the
mutant library into an E. coli host, and collect the transformants, say, 107 independent
transformants. To screen just one equivalent of this library by standard genetic assays, one would
require about 104 large petri dishes (about 1000-1500 colonies can be plated out on a dish without
overcrowding). Many times more colonies can be analyzed by physical screen using fluorescence
reporters and high throughput cell sorting machines. 109 cells can be sorted in several hours using
state of the art sorters.
Changing the target specificity of Flp
Here is one example of a genetic screen. We want to identify Flp variants with their
specificity shifted away from the native FRT site and towards a mutant FRT site, called mFRT.
The two sites shown in the figure below differ in one key base pair of the Flp recognition sequencea change from a CG bp to GC bp. Two reporter plasmids are constructed, and the assay is carried
out in E. coli. The experimental design is to distinguish FRT x FRT recombination from mFRT x
mFRT recombination by a colony color assay (See the figure below). In one case, the LacZ
reporter is flanked by two direct copies of mFRT sites. In the other the gene for RFP
(red fluorescent protein) is flanked by two direct copies of FRT. Recombination between two FRT
sites will eliminate the RFP gene, and recombination between two FRT sites will eliminate the
LacZ gene. Either one or both the genes will be expressed only when the relevant recombination
event or events fail to occur. The library of Flp variants are expressed from the P-BAD promoter,
which is turned on only in the presence of arabinose. The color of the colonies after addition of
arabinose to the growth medium will indicate the occurrence or non-occurrence of the designed
recombination events.
Colonies expressing lacZ (absence of mFRT recombination) will be blue in the presence
of the indicator substrate X-gal. Colonies expressing both LacZ and RFP (absence of mFRT and
FRT recombination) will also appear as blue as the blue color is strong, and masks the red color.
Thus blue colonies declare a given Flp variant clone is either inactive on mFRT or inactive on
both FRT and mFRT. Colorless (white) colonies will indicate that a Flp variant is active on both
FRT and mFRT. Red colonies will denote mFRT recombination and the absence FRT
recombination by a Flp variant. One can collect the red colonies, isolate the Flp plasmids from
them, and identify the mutations responsible for the specificity switch. One can pool the variant
library, subject them to further random mutagenesis and screening to identify more robust variants
with strong mFRT recombination activity and high discrimination against FRT.
In panel A of the figure, the design of the Flp expression vector and of the two reporter
constructs is schematically shown. In Panel B, the expectations and outcomes are summarized.
Changing the target specificity of Cre
To facilitate the rapid screen of desired variants, in this set up, bacterial cells containing the library
of variants were screened by fluorescence-based cell sorting. The sequence of the native Cre
target site (called LoxP) and a mutant site M5 on which wild type Cre does not act are shown in
the figure below.
We wish to identify Cre variants that can recombine M5-LoxP sites.
The reporter genes here are GFP or YFP (yellow fluorescent protein). The LoxP sites, either
native or M5, are arranged in the reporter in inverted orientation (blue arrows). The promoter is
placed adjacent to the LoxP site (or M5 site) proximal to the YFP gene. The GFP gene in the
opposite orientation lacks a promoter. In the absence of Cre mediated recombination, the cells
will only express the YFP protein, and will display yellow fluorescence (left panel of the Figure
below). If recombination occurs between the sites, the DNA sequence between the LoxP (or M5)
sites will be inverted, and GFP will become in register with the promoter. Cells will now show both
green and yellow fluorescence (as shown in the right panel of the figure below. Cells with binary
fluorescence can be separated from the yellow fluorescent cells, and further enriched by growing
them to suitable cell density, and taking them through repeated rounds of recombinase, induction,
sorting etc.
The advantage of the sorting method over the genetic screen is that the former is much faster
and the cell populations covered are larger in number by two to three orders of magnitude.
Making the genetic screen more robust
The genetic method can be made more efficient by using a reporter gene that can be selected
against. For example the E. coli GalK gene is lethal when the strain carries a mutation in the GalE,
gene, which acts down stream of GalK. These genes code for enzymes involved in the utilization
of galactose. The product of GalK action, Gal-1-phopshate is toxic to the cell if it is not metabolized
further. Thus, a reporter carrying the GalK gene bordered by directly repeated recombination
target sites offers a means for selecting for cells that have eliminated GalK by recombination. The
cells carrying the reporter construct is grown in the presence of glucose (not galactose in the
medium, which would cause toxicity), and following the induction of recombinase, they are plated
on galactose plates. Here a million or more cells can be plated on a single plate as only the few
cells that successfully performed recombination will be able to grow. The rest of the ell population
will die due to the galactose toxicity.
Structure based mutagenesis
When detailed structural information on recombinase-DNA interaction are available, the directed
evolution procedure can be refined by randomizing a selected set of amino acid residues that
make direct contact with DNA bases or are located in close proximity to such residues. This
approach is much more efficient than targeting the entire protein to random mutagenesis. A library
of selective randomization will have a much more complete representation of different amino acids
at the relevant positions than a randomly mutagenized library, and is therefore likely to comprise
a significantly higher frequency of the desired recombinase variants.
Recombinases that utilize native genomic sequences as recombination target sites
The directed evolution strategy has been used with some degree of success to shift the specificity
of a recombinase through stepwise changes, leading ultimately to specificity for a sequence that
is already present within a genome. Achieving specificity to native sequences eliminates the
problem of having to introduce the natural target site of the recombinase into the genome to be
manipulated.
Computer algorithms can scan through the sequence of a genome, and identify sites that
resemble the recombinase site in sequence as well as organization. The degree of resemblance
can be ranked by a suitable scoring scheme. The high ranking sites are likely more amenable to
recognition by the recombinase (after directed evolution) than a lower ranking site. Once a
potential target site is chosen, the step-wise evolution can be performed. First, one or a few
changes corresponding to the native genomic sequence are incorporated into the target site, and
specificity is selected for the partially altered site. Additional substitutions are then introduced into
this altered site, and the evolution-selection schemes are repeated. Thus, through multiple rounds
of evolution, a recombinase variant is identified that can accept a genomic sequence as a
recombination target site. By subjecting Flp to this evolutionary scheme, it has been possible to
obtain a variant that can mediate recombination at a sequence contained within the human IL-10
gene. Similarly, a Cre variant capable of recombining a sequence within the LTR (long terminal
repeat) of a human immunodeficiency virus (HIV) has been evolved. Such recombinases can be
potentially harnessed for cleansing the genome of integrated retrovirus.