Improving recombinase temporal fidelity

Abstract

The technology as disclosed herein includes systems, methods, and compositions for improving the temporal fidelity of recombinases, including ligand-inducible recombinases. Implementations of the systems, methods, and compositions provided herein include and / or utilize and / or provide for reduced premature recombination (leakiness) of recombinases, including ligand-inducible recombinases. Implementations of the systems, methods, and compositions provided herein include and / or utilize and / or provide reduced premature recombination (leakiness) of inducible recombinases while retaining sufficient recombinogenic efficiency upon induction with ligand.

Description

IMPROVING RECOMBINASE TEMPORAL FIDELITYDESCRIPTIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present invention claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 675,706, filed July 25, 2024, the contents of which are incorporated herein by reference and made a part hereof.FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] N / AFIELD OF THE INVENTION

[0003] The present invention relates generally to systems, methods, and compositions for improving the temporal fidelity of recombinases, including ligand-inducible recombinases.BACKGROUND

[0004] Recombinase systems, methods, and compositions (including inducible recombinase systems) are important tools in biotechnology. For example, inducible recombinase systems allow for temporal control, upon ligand (e.g., tamoxifen) delivery, for when a specific recombination event occurs in a living cell both in vitro and in vivo. However, these tools often suffer from premature recombination of the targeted sequences in the absence of ligand.

[0005] Recombinase protein (including excess recombinase protein) in the cell can inadvertently enter the nucleus and induce recombination, and the odds of this happening increase with protein levels and time. Only relatively small amounts of the recombinase enzyme are actually required to facilitate a single one-time recombination event, after which the enzyme is no longer needed.

[0006] There are no viable alternative systems, methods, and compositions for providing the temporally stabilized expression of recombinases, including ligand induciblerecombinases. Thus, there is a need for new systems, methods, and compositions which can reduce premature recombination events. Thus, the systems, methods, and compositions provided herein provide such reduction of premature recombination events. Systems, methods, and compositions for temporally stabilized expression of recombinases are badly needed in this field more than ever and are provided herein.SUMMARY OF THE INVENTION

[0007] The technology as disclosed herein includes systems, methods, and compositions for improving the temporal fidelity of recombinases, including ligand-inducible recombinases.

[0008] Implementations of the systems, methods, and compositions provided herein include and / or utilize and / or provide for reduced premature recombination (leakiness) of recombinases, including ligand-inducible recombinases.

[0009] Implementations of the systems, methods, and compositions provided herein include and / or utilize and / or provide reduced premature recombination (leakiness) of inducible recombinases while retaining sufficient recombinogenic efficiency upon induction with ligand.

[0010] Implementations of the systems, methods, and compositions provided herein include and / or utilize and / or provide for selectively destabilized recombinases and / or selective reduction of recombinase levels, thus reducing premature recombination events.

[0011] The systems, methods, and compositions disclosed herein also provide for modification of non-inducible recombinase systems (for example the non-inducible Cre enzyme) to allow for improved efficacy of recombination targeting, including for example by providing for better tissue specific targeting of recombinase activity.

[0012] In accordance with one aspect of the invention, a composition is provided comprising (a) a recombinase, and (b) a first destabilization motif, a first nuclear export sequence (NES) motif, or a combination thereof.

[0013] The recombinase can be a ligand inducible recombinase or a Cre recombinase or a ligand inducible Cre recombinase. Moreover, the recombinase can include SEQ ID NO: 1 or 2.

[0014] The first destabilization motif can be an N-terminal to the recombinase or a C- terminal to the recombinase. Additionally, the composition of Implementation can include a second destabilization motif or a second NES motif.

[0015] The first destabilization motif can include a PEST domain, a non-cleavable ubiquitin (Ub) N-terminal moiety, or an RNA destabilizing element. The second destabilization motif can include a PEST domain, a non-cleavable ubiquitin (Ub) N-terminal moiety, or an RNA destabilizing element.

[0016] The first destabilization motif can be a PEST domain or a non-cleavable ubiquitin(Ub) N-terminal moiety or an RNA destabilizing element. The RNA destabilizing element can be a c-myc 3’-UTR, a c-fos 3’-UTR, or a SLDE domain from the G-CSF 3’-UTR.

[0017] The first destabilization motif can include SEQ ID NO:3, 5, or 6. Additionally, the first nuclear export sequence (NES) motif can include SEQ ID NO:4.

[0018] In accordance with another aspect of the invention, a protein comprises an amino acid sequence of SEQ ID NO:7, 8, 9, 10, 11, 12, 13, 14, 15, or 15.

[0019] In accordance with yet another aspect of the invention, a composition comprises a protein with an amino acid sequence that has at least 95% sequence identity with SEQ ID NO:7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0020] In accordance with another aspect of the invention, a method for genetic recombination comprises destabilization of a ligand inducible recombinase with a one or more destabilization motif, one or more nuclear export sequence (NES) motif, or any combination thereof.

[0021] In accordance with another aspect of the invention, a method comprises (a) genetic recombining with a recombinase, and (b) destabilizing the recombinase by fusing a a first destabilization motif, a first nuclear export sequence (NES) motif, or a combination thereof, thereto.

[0022] In the method the recombinase can be a ligand inducible recombinase or a Cre recombinase or a ligand inducible Cre recombinase. The recombinase can include SEQ ID NO:1 or 2.

[0023] In the method, the first destabilization motif can be N-terminal to the recombinase or a C-terminal to the recombinase.

[0024] The method can include destabilizing the recombinase by fusing a second destabilization motif thereto, or destabilizing the recombinase by fusing a second NES motif thereto.

[0025] In the method the first destabilization motif can be a PEST domain, a non- cleavable ubiquitin (Ub) N-terminal moiety, or an RNA destabilizing element. The second destabilization motif can be a PEST domain, a non-cleavable ubiquitin (Ub) N-terminal moiety, or an RNA destabilizing element.

[0026] In the method the RNA destabilizing element can be a c-myc 3’-UTR, a c-fos 3’- UTR, or a SLDE domain from the G-CSF 3’-UTR.

[0027] In the method the first destabilization motif can include SEQ ID NOG, 5, or 6. Additionally, the first nuclear export sequence (NES) motif can include SEQ ID NO:4.

[0028] In accordance with another aspect of the invention, a composition for selective destabil izalion of a ligand inducible recombinase comprises (a) a ligand inducible CRE recombinase, and (b)a PEST domain.

[0029] The composition of SEQ ID NO:7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. Additionally, a protein comprising SEQ ID NO:7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0030] In accordance with another aspect of the invention, a composition comprises the amino acid sequence:

[0031] An amino acid comprises the sequence encoded in SEQ ID NO:7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. Additionally, a nucleic acid encoding the sequence of SEQ ID NO:7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0032] In accordance with another aspect of the invention, a composition comprises at least one of: a recombinase, a ligand inducible recombinase, a CRE recombinase, or a ligand inducible CRE recombinase; and at least one of: a destabilizing motif, a PEST domain, or a modified PEST domain. A composition comprises a recombinase and a PEST domain43. A composition comprising a CRE recombinase and a destabilizing motif. A composition comprising a CRE recombinase and a PEST domain.

[0033] Other features and advantages of the invention will be apparent from the following specification taken in conjunction with the following Attachments and Figures.BRIEF DESCRIPTION OF THE DRAWINGS AND ATTACHMENTS

[0034] To understand the present invention, it will now be described by way of example, with reference to the accompanying drawings and attachments in which:

[0035] Figure 1 is a schematic diagram illustrating aspects of the present invention;

[0036] Figure 2 is a schematic diagram illustrating aspects of the present invention;

[0037] Figure 3 is a schematic diagram illustrating aspects of the present invention; and,

[0038] Figure 4 is a schematic diagram illustrating aspects of the present invention.

[0039] While the technology as disclosed is susceptible to various modifications and alternative forms, specific implementations thereof are shown by way of example in the drawings and will herein be described in detail. It will be understood, however, that the drawings and detailed description presented herein are not intended to limit the disclosure to the particular implementations as disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the scope of the present technology as disclosed and as defined by the appended claims.DETAILED DESCRIPTIONOverview

[0040] There is a need for new compositions and methods which can reduce premature recombination events in recombinase systems. Thus, the systems, methods, and compositionsprovided herein provide such reduction of premature recombination events. Compositions and methods for temporally stabilized expression of recombinases are badly needed in this field more than ever and are provided herein.Definitions

[0041] To the extent necessary to provide descriptive support, the subject matter and / or text of the appended claims is incorporated herein by reference in their entirety.

[0042] The headings provided herein are solely for ease of reference and are not limitations of the various aspects or aspects of the disclosure, which can be had by reference to the specification as a whole.

[0043] It will be understood by all readers of this written description that the exemplary implementations described and claimed herein may be suitably practiced in the absence of any recited feature, element or step that is, or is not, specifically disclosed herein.

[0044] It must be noted that as used herein and in the appended claims, the singular forms“a,” an,” and “the” include the plural reference unless the context clearly dictates otherwise; for example, “an clement” is a reference to one or more elements and includes equivalents thereof known to those skilled in the art. Similarly, for another example, a reference to “a step” or “a means” is a reference to one or more steps or means and can include sub-steps and subservient means. All conjunctions used are to be understood in the most inclusive sense possible. Thus, the word “or” should be understood as having the definition of a logical “or” rather than that of a logical “exclusive or” unless the context clearly necessitates otherwise. Structures described herein are to be understood also to refer to functional equivalents of such structures. Language that can be construed to express approximation should be so understood unless the context clearly dictates otherwise. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein. Singular words should be read as plural and vice versa and masculine as feminine and vice versa, where appropriate, and alternative implementations do not necessarily imply that the two arc mutually exclusive.

[0045] Furthermore, “and / or” where used herein is to be taken as specific disclosure of each of the specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B,and / or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone): B (alone); and C (alone).

[0046] It will be further understood that use of the word “can” and / or “may” will be understood to refer to the active, and enabling, dictionary meanings of “is able,” “be able”, “to know,” “be able to through acquired knowledge or skill,” “to know how to do something,” and / or “to have the ability to do something”; and not understood to intend a sense of “maybe” or permissiveness.

[0047] Reference in the specification to “one embodiment” or “an embodiment”; “one implementation” or “an implementation” means that a particular feature, structure, or characteristic described in connection with the embodiment or implementation is included in at least one embodiment or implementation of the present invention. The appearances of the phrase “in one embodiment,” or “in an embodiment,” or “in one implementation,” or “in an implementation” in various places in the specification are not necessarily all referring to the same embodiment or the same implementation, nor are separate or alternative embodiments or implementations mutually exclusive of other embodiments or implementations. The words “embodiments)” and “implementation(s)” can be used interchangeably based on context.

[0048] It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of and / or “consisting essentially of are also provided.

[0049] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. Although any methods, materials, features and / or components, similar or equivalent to those described herein can also be used in the practice or testing of various implementations of the present disclosure, exemplary methods and materials are now described.

[0050] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications arc cited.

[0051] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0052] Numeric ranges are inclusive of the numbers defining the range. Even when not explicitly identified by “and any range in between,” or the like, where a list of values is recited, e.g., 1, 2, 3, or 4, the disclosure specifically includes any range in between the values, e.g., 1 to 3, 1 to 4, 2 to 4, etc. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the present disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the present disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the present disclosure.

[0053] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual implementations described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several implementations without departing from the scope or spirit of the present disclosure.

[0054] It is also to be understood that the terminology used herein is for the purpose of describing particular implementations only, and is not intended to be limiting, of the scope of the present disclosure.

[0055] The terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0056] Implementation(s) of the present invention are discussed below with reference to the Figures. However, those skilled in the art will readily appreciate that the detailed description given herein with respect to these Figures is for explanatory purposes as the present invention extends beyond these limited implementations. For example, it will be appreciated that those skilled in the art will, in light of the teachings of the present invention, recognize a multiplicity of alternate and suitable approaches, depending upon the needs of the particular application, to implement the functionality of any given detail described herein, beyond the particular implementation choices described and shown. That is, there are modifications and variations of the present invention that are too numerous to be listed but that all fit within the scope of the present invention.

[0057] According to the implementation(s) of the present technology as disclosed, various views are illustrated in Figures throughout, and with like reference numerals being used consistently throughout to refer to like and corresponding parts of the technology for all of the various views and Figures of the drawing. Also, please note that the first digit(s) of the reference number for a given item or part of the technology should correspond to the Figure (Fig.) number in which the item or part is first identified.

[0058] As is evident from the foregoing and following description, certain aspects of the present implementation(s) are not limited by the particular details of the examples illustrated herein, and it is therefore understood that other modifications and applications, or equivalents thereof, will be apparent to those skilled in the art. It is accordingly intended that the claims shall cover all such modifications and applications that do not depart from the scope of the present implementation(s). Accordingly, the specification and drawings are to be regarded in an illustrative rather than a restrictive sense.

[0059] Other aspects, objects and advantages of the present technology as disclosed herein can be obtained from a study of the drawings, the disclosure and the appended claims.

[0060] Implementations of the disclosed invention are relevant to multiple and varied applications. For example, certain implementations are useful in, for example, transgenic models of gene function where a gene is intended to be turned on or off at a specific point in time. Certain implementations allow for the use of an inducible Cre expressed from immediate early gene promoters for marking memory engrams in transgenic animals. In certain implementations the disclosure helps improve experiments involving viral vector mediated delivery of an inducible recombinase. Improvements in experimental quality, therapeutic potential, and the derivation of conclusion related to the temporal fidelity of the recombinase system are all provided herein.

[0061] The compositions disclosed herein are useful in multiple overlapping manners as well, for example, in certain implementations, the recombinant protein disclosed in, for example, SEQ ID NO:7 provides utility useful in situations where nuclear export is compromised. In certain implementations, the recombinant protein disclosed in, for example, SEQ ID NO: 10 provides utility in conditions where proteasome function is impaired.

[0062] While the present invention is susceptible of embodiments in many different forms, there is shown in the drawings and will herein be described in detail preferredimplementations of the present invention with the understanding that the present disclosure is to be considered as an exemplification of the principles of the present invention and is not intended to limit the broad aspect of the present invention to the embodiments illustrated.

[0063] Many modifications and variations of the present invention are possible in light of the above teachings. It is, therefore, to be understood within the scope of the appended claims the invention may be protected otherwise than as specifically described.Example 1

[0064] For decades the Cre system and inducible Cre systems have been used and investigators have been plagued by non- specificity (leakiness) of recombination with no effective solution to this problem.

[0065] In order to provide solutions to these problems, the present invention alters the existing state of the art for the ligand inducible Cre system by taking an unorthodox approach: deliberately destabilizing the recombinase despite not knowing until directly tested whether this induced reduced stability of the recombinase would simply eliminate all effective recombinase activity or simply have no significant effect on leakiness of the system. Counterintuitively, implementations of the invention provided herein novelly combines two disparate molecular technologies (recombinase systems and destabilizing motifs) in a very unconventional way to specifically address the issue of recombinase leakiness. The conventional wisdom in the field is that destabilizing a recombinase would catastrophically reduce its function and so would not be effective. This would seem to make sense given that the stability of recombinases is essential to their function.

[0066] Hence, implementations of the invention disclosure herein are based on a counterintuitive and novel scientific hypothesis that a destabilized recombinase would have sufficient efficiency because relatively little functional recombinase is needed to facilitate the one-time recombination event, and so, lowered recombinase levels were hypothesized to remain effective. This could also be achieved by simply lowering recombinase gene transcription levels; however, this is often not an option since the recombinase is usually placed under the transcriptional control of endogenous promoters of experimental interest. Furthermore, transcription levels can be variable in different tissue types. In many situations the recombinase is co-expressed with other gene products that require relatively high levelsof production. Thus, by destabilizing the recombinase itself alteration of the intrinsic properties of the enzyme (independent of gene expression conditions) to facilitate higher temporal fidelity in broad cellular contexts has been achieved and the methods are disclosed herein.

[0067] In implementations of the present invention, a destabilizing motif targets a recombinase to the proteasome for degradation thus lowering the half-life of this new recombinant enzyme.

[0068] The tamoxifen inducible CreERT2 system is one of, or perhaps the single most widely used version of the ligand inducible recombinase system. In implementations of the present invention, the CreERT2 enzyme has been modified with a destabilizing motif designed to that target proteins to the proteasome.

[0069]

[0056] In certain implementations this destabilizing motif is a PEST domain used to destabilize the recombinase system protein. A PEST domain was added to the C- terminus of the CreERT2 recombinase and analyzed compared to unaltered CreERT2. Data show that the destabilized CrcERT2-pcst system shows significantly reduced leakiness while retaining the ability to efficiently recombine out gene set 1 to facilitate gene set 2 expression in transduced precursor cells (FIG. 1-4).

[0070] Implementations of the present invention improve the utility of the current state of the art in temporal control of genetic recombination. Implementations of the present invention allow for improved data generation through the reduction of “noise” in the system from premature recombination. Implementations of the present invention allow researchers to better test hypotheses regarding gene function. In addition, implementations of the present invention allow for better therapeutic applications by promoting more control of the initiation / termination / alteration of a therapeutic gene.

[0071] The bacteriophage derived Cre recombinase is used to recombine genetic sequences between enzyme recognitions sites (loxP). Previously, this system was modified to allow for ligand induced initiation of the recombination process through fusion with a mutant estrogen receptor (ER) that binds to tamoxifen (TAM). This CreERT2 enzyme binds to heat shock protein 90 (HSP90) which retains the complex in the cytoplasm of the cell where it cannot recombine loxP sites in the nuclear DNA. Upon addition of TAM (denoted by “T” in the figure), HSP90 dissociates from the complex allowing for entry of CreERT2 into thenucleus to facilitate recombination (FIG. 1). Because this complex is relatively stable, it can accumulate to fairly high levels in the cytoplasm. This can lead to free CreERT2 (not bound to IISP90) to enter the nucleus and facilitate premature recombination (FIG 1). This is referred to as leakiness. A ligand-inducible recombinase was modified with a destabilizing ubiquitin-ligase domain (PEST) that has been shown to reduce the half-life of proteins by targeting them to the proteasome for degradation. Counterintuitively, since only small amounts of the recombinase are needed to facilitate a single recombination event, lowered levels reduce the leakiness but retain the ability to recombine genetic elements in the presence of TAM (FIG. 1).

[0072] A retroviral gene transfer vector (Regulator) was used that delivers the CreERT2 enzyme along with polycistronic expression of the tetracycline responsive trans activator (tTA). The tTA allows gene expression from co-transduction of the cell with the Reporter retroviral vector (FIG. 2A). The Regulator plasmid was engineered by fusing amino acids 422-461 of the degradation domain of murine ornithine decarboxylase to the carboxy terminus of CrcERT2. Genetic maps of exemplary comparisons of the regulator plasmid compared to one of the modified plasmids can be seen in FIG. 3.

[0073] In one example, the constructed protein (“CreERT2-pest”) had the sequence:

[0074] Retroviral vectors expressing the Regulator or Regulator-pest as well as a reporter retrovirus expressing a loxP flanked GFP expression cassette (FIG. 2A) were prepared. Upon recombination mediated by Cre activity the green expression cassette is removed allowing for expression of a red fluorescent protein (DSR). Thus green and red cellular fluorescence can be used to assess the recombinogenic state of the reporter vector. Oligodendrocyte precursor cells were transduced with the Reporter vector and with either the Regulator or Regulatorpest vector. After this the cells were either left untreated for seven days or were treated with TAM on day four post transduction (FIG. 2B). The proportion of green and red fluorescent cells were assessed by quantitative fluorescent microscopy, which showed that in the absence of induction with TAM the Regulator resulted in 40% of the transduced cells switching to red fluorescence (FIG. 4 A). Alternatively, the Regulator-pest version showed only 1% of transduced cells turning red. This indicated a dramatic reduction of leakiness of the system.

[0075] When assessing the recombination efficiency after induction in the presence of TAM, it was found that comparable levels of switch were observed between Regulator and Regulator-pest transduced cells (FIG. 4B). Thus the utility of systems, methods, and compositions provided herein has been shown for facilitating significantly reduced leakiness while retaining sufficient recombinase activity.Example 2

[0076] Implementations of the present invention are amenable to various adaptations and modifications of this approach. In certain implementations, other destabilization motifs are used in place of or in addition to the PEST domain described herein.

[0077] Other destabilization motifs include a non-cleavable ubiquitin (Ub) N -terminal moiety

[0078] Furthermore, in certain implementations, the location of the destabilization motif can be changed within the recombinant recombinase sequence, for example placement at the N-terminus of the recombinase as compared to the C-terminus.

[0079] In certain implementations, the destabilizing motif comprises nuclear export sequence (NES) motifs in order to reduce access of the recombinase to the target DNA sequences in the nucleus (i.e., reduced leakiness), for example the sequence found in the HIV-1 rev protein (LQLPPLERLTL).

[0080] In certain implementations, more than one destabilizing motif is used.Example 3

[0070] ReferencesStiffer and Greter 2020, PMID: 32125704) (Stiffer and Greter, 2020)The bacteriophage derived Cre recombinase is used to recombine genetic sequences between enzyme recognitions sites (loxP) (Kim et al. 2018)(PMC38252, PMC41457) (Metzger et al., 1995; Fell et al., 1996)((Li et al. 1998, PMID: 9857028))PEST domain has been used to destabilize proteins and reduce their half-life in cells (Li et al. 1998, PMID: 9857028).Nuclear export sequence (NES) motifs (PMID: 17574289) reduce access to nucleus (Kakar et al., 2007). c-myc or c-fos 3’-UTR (PMID: 15716309) (Voon et al., 2005)SLDE domain (PMID: 8943001) from the G-CSF 3’-UTR(Brown et al., 1996) Brown CY, Lagnado CA, Goodall GJ (1996) A cytokine mRNA-destabilizing element that is structurally and functionally distinct from A+U-rich elements. Proceedings of the National Academy of Sciences of the United States of America 93:13721-13725. PMID: 8943001Feil R, Brocard J, Mascrez B, LeMeur M, Metzger D, Chambon P (1996) Ligand- activated site-specific recombination in mice. Proceedings of the National Academy of Sciences of the United States of America 93:10887-10890. PMID: 8855277Kakar M, Davis JR, Kem SE, Lim CS (2007) Optimizing the protein switch: altering nuclear import and export signals, and ligand binding domain. Journal of controlled release : official journal of the Controlled Release Society 120:220- 232. PMID: 17574289Kim H, Kim M, Im SK, Fang S (2018) Mouse Cre-LoxP system: general principles to determine tissue-specific roles of target genes. Lab Anim Res 34:147-159. PMID: 30671100Li X, Zhao X, Fang Y, Jiang X, Duong T, Fan C, Huang CC, Kain SR (1998) Generation of destabilized green fluorescent protein as a transcription reporter. The Journal of biological chemistry 273:34970-34975. PMID: 9857028Metzger D, Clifford J, Chiba H, Chambon P (1995) Conditional site-specific recombination in mammalian cells using a ligand- dependent chimeric Cre recombinase. Proceedings of the National Academy of Sciences of the United States of America 92:6991-6995. PMID: 7624356Stiffer SA, Greter M (2020) STOP floxing around: Specificity and leakiness of inducible Cre / loxP systems. European journal of immunology 50:338-341. PMID: 32125704Voon DC, Subrata LS, Baltic S, Leu MP, Whiteway JM, Wong A, Knight SA, Christiansen FT, Daly JM (2005) Use of mRNA- and protein-destabilizing elements to develop a highly responsive reporter system. Nucleic acids research 33:e27. PMID: 15716309Example 4

[0082] Implementations of the Present Invention

Claims

WE CLAIM:

1. A composition comprising:(a) a recombinase, and(b) a first destabilization motif, a first nuclear export sequence (NES) motif, or a combination thereof.

2. The composition of Implementation 1 wherein the recombinase is a ligand inducible recombinase.

3. The composition of Implementation 1 wherein the recombinase is a Cre recombinase.

4. The composition of Implementation 1 wherein the recombinase is a ligand inducible Cre recombinase.

5. The composition of Implementation 1 wherein the recombinase comprises SEQ ID NO:1 or 2.

6. The composition of Implementation 1 wherein the first destabilization motif is N- tenninal to the recombinase.

7. The composition of Implementation 1 wherein the first destabilization motif is C- terminal to the recombinase.

8. The composition of Implementation 1 further comprising a second destabilization motif.

9. The composition of Implementation 1 further comprising a second NES motif.

10. The composition of Implementation 1 wherein the first destabilization motif comprises a PEST domain, a non-cleavable ubiquitin (Ub) N-terminal moiety, or an RNA destabilizing element.

11. The composition of Implementation 8 wherein the second destabilization motif comprises a PEST domain, a non-cleavable ubiquitin (Ub) N-terminal moiety, or an RNA destabilizing element.

12. The composition of Implementation 1 wherein the first destabilization motif comprises a PEST domain.

13. The composition of Implementation 1 wherein the first destabilization motif comprises a non-cleavable ubiquitin (Uh) N-terminal moiety.

14. The composition of Implementation 1 wherein the first destabilization motif comprises an RNA destabilizing element.

15. The composition of Implementation 14 wherein the RNA destabilizing element is a c-myc 3’-UTR, a c-fos 3’-UTR, or a SLDE domain from the G-CSF 3’-UTR.

16. The composition of Implementation 1 wherein the first destabilization motif comprises SEQ ID NO:3, 5, or 6.

17. The composition of Implementation 1 wherein the first nuclear export sequence (NES) motif comprises SEQ ID NO:4.

18. A protein comprising an amino acid sequence of SEQ ID NO:7, 8, 9, 10, 11, 12, 13, 14, 15, or 15.

19. A composition comprising a protein with an amino acid sequence that has at least 95% sequence identity with SEQ ID NO:7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

20. A method for genetic recombination comprising destabilization of a ligand inducible recombinase with a one or more destabilization motif, one or more nuclear export sequence (NES) motif, or any combination thereof.

21. A method comprising:(a) genetic recombining with a recombinase, and(b) destabilizing the recombinase by fusing a a first destabilization motif, a first nuclear export sequence (NES) motif, or a combination thereof, thereto.

22. The method of Implementation 21 wherein the recombinase is a ligand inducible recombinase.

23. The method of Implementation 21 wherein the recombinase is a Cre recombinase.

24. The method of Implementation 21 wherein the recombinase is a ligand inducible Cre recombinase.

25. The method of Implementation 21 wherein the recombinase comprises SEQ ID NO:1 or 2.

26. The method of Implementation 21 wherein the first destabilization motif is N-terminal to the recombinase.

27. The method of Implementation 21 wherein the first destabilization motif is C-tcrminal to the recombinase.

28. The method of Implementation 21 further comprising destabilizing the recombinase by fusing a second destabilization motif thereto.

29. The method of Implementation 21 further comprising destabilizing the recombinase by fusing a second NES motif thereto.

30. The method of Implementation 21 wherein the first destabilization motif comprises a PEST domain, a non-cleavable ubiquitin (Ub) N-terminal moiety, or an RNA destabilizing element.

31. The method of Implementation 28 wherein the second destabilization motif comprises a PEST domain, a non-cleavable ubiquitin (Ub) N-terminal moiety, or an RNA destabilizing element.

32. The method of Implementation 30 wherein the RNA destabilizing element is a c- myc 3’-UTR, a c-fos 3’-UTR, or a SLDE domain from the G-CSF 3’-UTR.

33. The method of Implementation 21 wherein the first destabilization motif comprises SEQ ID NO:3, 5, or 6.

34. The method of Implementation 21 wherein the first nuclear export sequence (NES) motif comprises SEQ ID NO:4.

35. A composition for selective destabilization of a ligand inducible recombinase comprising:(a) a ligand inducible CRE recombinase, and(b) a PEST domain.

36. The composition of SEQ ID NO:7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

37. A protein comprising SEQ ID NO:7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

38. A composition comprising the amino acid sequence:

39. An amino acid comprising the sequence encoded in SEQ ID NO:7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

40. A nucleic acid encoding the sequence of SEQ ID NO:7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

41. A composition comprising:(a) at least one of: a recombinase, a ligand inducible recombinase, a CRE recombinase, or a ligand inducible CRE recombinase; and(b) at least one of: a destabilizing motif, a PEST domain, or a modified PEST domain.

42. A composition comprising a recombinase and a PEST domain.

43. A composition comprising a CRE recombinase and a destabilizing motif.

44. A composition comprising a CRE recombinase and a PEST domain.

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