CN112391366B

CN112391366B - Dre recombination system activated by light induction

Info

Publication number: CN112391366B
Application number: CN201910759889.4A
Authority: CN
Inventors: 李大力; 李慧莹; 吴英尹; 刘明耀
Original assignee: East China Normal University; Bioray Laboratories Inc
Current assignee: East China Normal University; Bioray Laboratories Inc
Priority date: 2019-08-16
Filing date: 2019-08-16
Publication date: 2022-10-14
Anticipated expiration: 2039-08-16
Also published as: CN112391366A

Abstract

The invention provides a Dre recombination system activated by light induction. The light-induced activated Dre recombination system has higher space-time specificity, can be applied to multiple fields of lineage tracing, cell clearing and the like, combines the specificity of a promoter, and realizes more accurate gene operation by utilizing local blue light irradiation.

Description

Dre recombination system activated by light induction

Technical Field

The invention relates to the technical field of biology, in particular to a Dre recombination system activated by light induction.

Background

The Dre recombinase is derived from the D6 bacteriophage and belongs to the tyrosine recombinase family like the common Cre recombinase. It can specifically recognize a DNA sequence (rox) with the length of 32bp, so that the gene sequence between rox sites is deleted or recombined.

The inducible recombinase system that is currently used is DreER, which expresses the ligand binding domain of the estrogen receptor fused to Dre recombinase to form a cytoplasmic-localized fusion protein. The Dre recombinase dissociates from the dockerin into the nucleus upon the appearance of the ligand tamoxifen to exert a recombination function. Alternatively, RU 486-induced DrePBD system was used to regulate the expression of the recombinase in a time-specific manner. However, these methods have certain limitations, one of them is that the induction by chemical drugs may disturb the endogenous signal pathway of cells, for example, tamoxifen may bind to endogenous estrogen receptor, thereby affecting the normal function of the body. The induction of two chemical drugs takes a long time, the animals need to be treated by the drugs for at least one week, and the drugs are easy to be metabolized rapidly so as to inhibit the induction effect. The induction range of the three drugs is too large, the space specificity can be realized to a certain extent by virtue of a specific promoter, but the precise operation cannot be realized for cell populations which are distributed in the whole body and lack of tissue characteristics.

Therefore, there is an urgent need in the art to establish a system for inducing expression of Dre recombinase by spatio-temporal specific regulation.

Disclosure of Invention

The invention aims to establish a Dre recombinase expression induction system capable of regulating and controlling the Dre recombinase in a space-time specificity manner.

In a first aspect of the invention there is provided a resolved protein comprising a Z1 element or a Z2 element, wherein the Z1 element is an N-terminal fragment of Dre recombinase; the Z2 element is a C-terminal fragment of Dre recombinase;

and, when Z1 is Dre _1-60 When fragment, Z2 is Dre _61-343 A fragment;

when Z1 is Dre _1-65 When fragment, Z2 is Dre _66-343 A fragment;

when Z1 is Dre _1-66 When fragment, Z2 is Dre _67-343 A fragment;

when Z1 is Dre _1-67 When fragment, Z2 is Dre _68-343 A fragment;

when Z1 is Dre _1-103 When fragment, Z2 is Dre _104-343 A fragment;

when Z1 is Dre _1-104 When fragment, Z2 is Dre _105-343 A fragment;

when Z1 is Dre _1-105 When fragment, Z2 is Dre _106-343 A fragment;

when Z1 is Dre _1-107 When fragment, Z2 is Dre _108-343 A fragment;

when Z1 is Dre _1-108 When fragment, Z2 is Dre _109-343 A fragment;

when Z1 is Dre _1-109 When fragment, Z2 is Dre _110-343 A fragment;

when Z1 is Dre _1-150 When fragment, Z2 is Dre _151-343 A fragment;

when Z1 is Dre _1-151 When fragment, Z2 is Dre _152-343 A fragment;

when Z1 is Dre _1-152 When fragment, Z2 is Dre _153-343 A fragment;

when Z1 is Dre _1-157 When fragment, Z2 is Dre _158-343 A fragment;

when Z1 is Dre _1-190 When fragment, Z2 is Dre _191-343 A fragment;

when Z1 is Dre _1-192 When fragment, Z2 is Dre _193-343 A fragment;

when Z1 is Dre _1-193 When fragment, Z2 is Dre _194-343 A fragment;

when Z1 is Dre _1-194 When fragment, Z2 is Dre _195-343 A fragment;

when Z1 is Dre _1-230 When fragment, Z2 is Dre _231-343 A fragment;

when Z1 is Dre _1-234 When fragment, Z2 is Dre _235-343 A fragment;

when Z1 is Dre _1-237 When fragment, Z2 is Dre _238-343 A fragment;

when Z1 is Dre _1-240 When fragment, Z2 is Dre _241-343 A fragment;

when Z1 is Dre _1-246 When fragment, Z2 is Dre _247-343 And (3) fragment.

In a second aspect, the invention provides a polypeptide which is an active fragment obtained by cutting at the following sites of Dre recombinase shown in SEQ ID NO. 10:

60 th, 65 th, 66 th, 67 th, 103 th, 104 th, 105 th, 107 th, 108 th, 109 th, 150 th, 151 th, 152 th, 157 th, 190 th, 192 th, 193 th, 194 th, 230 th, 234 th, 237 th, 240 th, and/or 246 th bits.

In another preferred embodiment, the polypeptide is an active fragment obtained by cutting the Dre recombinase shown in SEQ ID NO. 10 at the following sites:

bits

60, 150, 151, 152, 157, 190, 192, 193, 194, 234 and/or 246.

In another preferred embodiment, the polypeptide is an active fragment obtained by cleavage of the Dre recombinase shown in SEQ ID No. 10 at the following sites:

60, 150 and/or 246.

In a third aspect, the present invention provides a fusion protein selected from the group consisting of: a first fusion protein, a second fusion protein, or a combination thereof; wherein the structure of the first fusion protein is shown as formula I or formula I':

Z1-L1-A1 (I)；

A1-L1-Z1 (I’)；

the structure of the second fusion protein is shown as formula II or formula II':

Z2-L2-A2 (II)；

A2-L2-Z2 (II’)；

in the formula (I), the compound is shown in the specification,

z1 is an N-terminal fragment of Dre recombinase;

l1 is an optional first linker peptide;

a1 is a first element of a light sensitive protein;

z2 is a C-terminal fragment of Dre recombinase;

a2 is a second element of the light sensitive protein;

l2 is an optional second linking peptide;

each "-" is independently a linking peptide or non-peptide bond.

In another preferred embodiment, the N-terminal fragment of Dre recombinase is selected from the group consisting of: dre _1-60 Fragment, dre _1-65 Fragment, dre _1-66 Fragment, dre _1-67 Fragment, dre _1-103 Fragment, dre _1-104 Fragment, dre _1-105 Fragment, dre _1-107 Fragment, dre _1-108 Fragment, dre _1-109 Fragment, dre _1-150 Fragment, dre _1-151 Fragment, dre _1-152 Fragment, dre _1-157 Fragment, dre _1-190 Fragment, dre _1-192 Fragment, dre _1-193 Fragment, dre _1-194 Fragment, dre _1-230 Fragment, dre _1-234 Fragment, dre _1-237 Fragment, dre _1-240 Fragment, dre _1-246 Fragments, or combinations thereof.

In another preferred embodiment, the N-terminal fragment of the Dre recombinase is based on the N-terminal fragment of SEQ ID No. 10.

In another preferred embodiment, the N-terminal fragment of Dre recombinase is selected from the group consisting of: dre _1-60 Fragment, dre _1-150 Fragment, dre _1-151 Fragment, dre _1-152 Fragment, dre _1-157 Fragment, dre _1-190 Fragment, dre _1-192 Fragment, dre _1-193 Fragment, dre _1-194 Fragment, dre _1-234 Fragment, dre _1-246 Fragments, or combinations thereof.

In another preferred embodiment, the N-terminal fragment of Dre recombinase is selected from the group consisting of: dre _1-60 Fragment, dre _1-150 Fragment, dre _1-246 Fragments, or combinations thereof.

In another preferred embodiment, the lengths of L1 and L2 are each independently 1 to 80aa, preferably 5 to 60aa, and more preferably 10 to 50aa.

In another preferred embodiment, L1 and L2 are each independently selected from the group consisting of:

(1) A polypeptide having an amino acid sequence as set forth in any one of SEQ ID No. 1-5;

(2) 1-5 by one or more, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-8, more preferably 1-3, most preferably 1 amino acid residue, and having (1) the function of the polypeptide, and a polypeptide derived from the polypeptide of any one of the amino acid sequences shown in SEQ ID No. 1-5.

In another preferred embodiment, the light sensitive protein is selected from the group consisting of: a blue light-sensitive protein, a red light-sensitive protein, an ultraviolet light-sensitive protein, or a combination thereof.

In another preferred embodiment, the blue light sensitive protein is selected from the group consisting of: LOV2, VVD, CRY2 cluster, magnet, CRY2/CIB1, or combinations thereof.

In another preferred embodiment, the red light sensitive protein is selected from the group consisting of: bphP1/Q-PAS1, phyB/PIF3& PhyB/PIF6, cph1, or combinations thereof.

In another preferred example, the ultraviolet light sensitive protein comprises UVR8/COP1.

In another preferred embodiment, the first element of the light-sensitive protein comprises nMag.

In another preferred embodiment, the second element of the light sensitive protein comprises pMag.

In another preferred embodiment, the C-terminal fragment of Dre recombinase is selected from the group consisting of: dre _61-343 Fragment, dre _66-343 Fragment, dre _67-343 Fragment, dre _68-343 Fragment, dre _104-343 Fragment, dre _105-343 Fragment, dre _106-343 Fragment, dre _108-343 Fragment, dre _109-343 Fragment, dre _110-343 Fragment, dre _151-343 Fragment, dre _152-343 Fragment, dre _153-343 Fragment, dre _158-343 Fragment, dre _191-343 Fragment, dre _193-343 Fragment, dre _194-343 Fragment, dre _195-343 Fragment, dre _231-343 Fragment, dre _234-343 Fragment, dre _238-343 Fragment, dre _241-343 Fragment, dre _247-343 Fragments, or combinations thereof.

In another preferred embodiment, the C-terminal fragment of the Dre recombinase is based on the C-terminal fragment of SEQ ID NO. 10.

In another preferred embodiment, the C-terminal fragment of Dre recombinase is selected from the group consisting of: dre _61-343 Fragment, dre _151-343 Fragment, dre _152-343 Fragment, dre _153-343 Fragment, dre _158-343 Fragment, dre _191-343 Fragment, dre _193-343 Fragment, dre _194-343 Fragment, dre _195-343 Fragment, dre _235-343 Fragment, dre _247-343 Fragments, or combinations thereof.

In another preferred embodiment, the C-terminal fragment of Dre recombinase is selected from the group consisting of: dre _61-343 Fragment, dre _151-343 Fragment, dre _247-343 Fragments, or combinations thereof.

In another preferred embodiment, the fusion protein further comprises a nuclear localization signal.

In another preferred embodiment, the nuclear localization signal is selected from the group consisting of: NLS ^sv40 、NLS ^EGL-13 、NLS ^NLP 、NLS ^c-Myc 、PY-NLSs、snRNPs、truncated NLS ^sv40 、NLS ^TUS Or a combination thereof.

In another preferred embodiment, the amino acid sequences of the nuclear localization signals are each independently selected from the group consisting of:

(1) A polypeptide having an amino acid sequence as set forth in any one of SEQ ID No. 6-9;

(2) A polypeptide derived from the polypeptide of the amino acid sequence shown in any one of SEQ ID No. 6-9, which has the function of the polypeptide of (1), formed by substituting, deleting or adding one or more, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-8, more preferably 1-3, and most preferably 1 amino acid residue in the amino acid sequence shown in any one of SEQ ID No. 6-9.

In another preferred embodiment, the N-terminal fragment of Dre recombinase and the C-terminal fragment of Dre recombinase together constitute active Dre recombinase.

In another preferred embodiment, the first element of the light-sensitive protein and the second element of the light-sensitive protein together constitute an active light-sensitive protein.

In another preferred embodiment, the first element of the light sensitive protein binds to the second element of the light sensitive protein when the first fusion protein binds to the second fusion protein.

In another preferred embodiment, the fusion protein has the amino acid sequence as shown in any one of SEQ ID No. 11-12.

In another preferred embodiment, the first fusion protein and the second fusion protein combine to form a complex comprising the first fusion protein and the second fusion protein.

In a fourth aspect, the present invention provides a polynucleotide encoding the resolution protein of the first aspect, the polypeptide of the second aspect, or the fusion protein of the third aspect of the present invention.

In another preferred embodiment, said polynucleotide additionally comprises an auxiliary element selected from the group consisting of: a signal peptide, a secretory peptide, a tag sequence (e.g., 6 His), or a combination thereof.

In another preferred embodiment, the polynucleotide is selected from the group consisting of: a DNA sequence, an RNA sequence, or a combination thereof.

In another preferred embodiment, the polynucleotide further comprises a promoter.

In another preferred embodiment, the promoter is selected from the group consisting of: EF1 α, CMV, PGK, CAG, LP1 (liver specific promoter), ctnt (heart specific promoter), syn (interneuron specific promoter), or combinations thereof.

In another preferred embodiment, the nucleic acid construct has a nucleotide sequence as set forth in any one of SEQ ID No. 13-14.

In a fifth aspect, the present invention provides a vector comprising a polynucleotide according to the fourth aspect of the present invention.

In another preferred embodiment, the vector comprises one or more promoters operably linked to the nucleic acid sequence, enhancer, transcription termination signal, polyadenylation sequence, origin of replication, selectable marker, nucleic acid restriction site, and/or homologous recombination site.

In another preferred embodiment, the vector comprises a plasmid, a viral vector.

In another preferred embodiment, the viral vector is selected from the group consisting of: adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpes virus, SV40, poxvirus, or combinations thereof.

In another preferred embodiment, the vector comprises an expression vector, a shuttle vector and an integration vector.

In a sixth aspect, the invention provides a host cell comprising a vector according to the fifth aspect of the invention or having a polynucleotide according to the fourth aspect of the invention integrated into its genome.

In another preferred embodiment, the host cell includes prokaryotic cells and eukaryotic cells.

In another preferred embodiment, the eukaryotic cell comprises a higher eukaryotic cell.

In another preferred embodiment, the cells are from the following species: human, non-human mammal, poultry, plant.

In another preferred embodiment, the non-human mammal includes a rodent (e.g., mouse, rat, rabbit), cow, pig, sheep, horse, dog, cat, non-human primate (e.g., monkey).

In another preferred embodiment, the cell comprises: somatic cells, stem cells, germ cells, non-dividing cells, or combinations thereof.

In another preferred embodiment, the cell comprises: kidney cells, epithelial cells, endothelial cells, or a combination thereof.

In another preferred embodiment, the genetically engineered cell is selected from the group consisting of: human cells, chinese hamster ovary cells, insect cells, wheat germ cells, rabbit reticulocytes, yeast cells, or a combination thereof.

In another preferred embodiment, the genetically engineered cell is selected from the group consisting of: HEK-293, hela, hep2, SH-SY5Y, or a combination thereof.

In a seventh aspect, the present invention provides a method of producing a split protein according to the first aspect of the invention, a polypeptide according to the second aspect of the invention, or a fusion protein according to the third aspect of the invention, comprising the steps of:

culturing the host cell of the sixth aspect of the invention under conditions suitable for expression, thereby expressing the split protein, polypeptide or fusion protein; and/or isolating the split protein, polypeptide or fusion protein.

The eighth aspect of the present invention provides a gene editing reagent comprising the fusion protein according to the third aspect of the present invention.

The ninth aspect of the present invention provides a kit comprising the gene editing reagent according to the eighth aspect of the present invention.

In another preferred embodiment, the kit further comprises a label or instructions.

In a tenth aspect, the invention provides a use of the fusion protein of the third aspect of the invention for preparing a reagent or a kit for improving gene editing efficiency.

The eleventh aspect of the present invention provides a method for gene editing, comprising the steps of:

(a) Providing a first fusion protein and a second fusion protein, wherein the structures of the first fusion protein and the second fusion protein are as set forth in the third aspect of the invention;

(b) Under light, the first fusion protein and the second fusion protein form a complex, thereby performing gene editing in the presence of the complex.

In another preferred example, the illumination comprises blue illumination.

In another preferred embodiment, the illumination intensity is 0.1-20mw/cm ² Preferably, 0.2 to 10mw/cm ² More preferably, 1 to 5mw/cm ² 。

In another preferred embodiment, the illumination time is 2h to 72h, preferably 10 h to 48h, and more preferably 15 h to 40h.

In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

In another preferred embodiment, the gene editing comprises: site-specific cleavage, site-specific insertion, and site-specific recombination.

In another preferred embodiment, said gene editing is performed in a cell in vitro.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows functional verification of different fragment sites of Dre recombinase.

Left panel: schematic of plasmid structure right panel: flow cytometric analysis results

FIG. 2 shows activity screening after segmented Dre recombinase fusion with light sensitive proteins.

Left panel: plasmid structure schematic right panel: results of luciferase Activity detection

FIG. 3 shows the relative positional adjustment of the segmented Dre recombinase to the light-sensitive protein.

FIG. 4 shows a comparison of the activity of the segmented Dre recombinase of different linkers.

FIG. 5 shows a comparison of the activity of the segmented Dre recombinases for different NLSs.

FIG. 6 shows a comparison of recombinase activity for a two plasmid system versus a single plasmid system.

Fig. 7 shows a schematic diagram of the PA-Dre operation.

Fig. 8 shows the illumination time dependence of the PA-Dre system.

Fig. 9 shows the illumination intensity dependence of the PA-Dre system.

FIG. 10 shows that the PA-Dre system is applicable to different promoters.

FIG. 11 shows that the PA-Dre system is suitable for different cell lines.

FIG. 12 shows the detection of luciferase activity in mice at different illumination times when low doses of PA-Dre plasmid were injected.

Left panel: right image of mouse live imaging results: results of luciferase Activity detection

FIG. 13 shows the luciferase activity assay of mice after light irradiation when high doses of PA-Dre plasmid were injected.

Left panel: right image of mouse in vivo imaging results: mouse liver living body imaging result statistics

Detailed Description

The inventor of the invention has conducted extensive and intensive research, and found for the first time, unexpectedly, that the complete Dre recombinase is split into an N-terminal segment and a C-terminal segment, and the N-terminal segment and the C-terminal segment are fused and expressed with photosensitive proteins nMAG and pMag respectively through a linker, so that a light-activated Dre recombinase system, also called PA-Dre, is formed. In the absence of blue light, the segmented Dre recombinase has no catalytic activity and rapidly dimerizes with the light-sensitive protein to form a catalytically active recombinase when the system is stimulated with blue light. Moreover, the applicant also unexpectedly finds that compared with a traditional induction system such as a Dre recombinase induced by tamoxifen, the PA-Dre system of the invention does not affect an endogenous signal path of a cell, has higher space-time specificity, can be applied to multiple fields such as lineage tracing, cell clearing and the like, combines the specificity of a promoter, and realizes more accurate gene operation by local blue light irradiation. On this basis, the present inventors have completed the present invention.

Term(s) for

In order that the disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning given below, unless explicitly specified otherwise herein. Other definitions are set forth throughout the application.

The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition, as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of or" consisting of 823030A ".

Sequence identity (or homology) is determined by comparing two aligned sequences along a predetermined comparison window (which may be 50%, 60%, 70%, 80%, 90%, 95% or 100% of the length of the reference nucleotide sequence or protein) and determining the number of positions at which identical residues occur. Typically, this is expressed as a percentage. The measurement of sequence identity of nucleotide sequences is a method well known to those skilled in the art.

In the present invention, dre is used _1-60 Fragment, dre _61-343 The fragments are exemplified and explained.

Wherein, dre _1-60 Fragment refers to the 1-60 position of the amino acid sequence of Dre recombinase based on SEQ ID NO. 10.

Dre _61-343 The fragment refers to the 61-343 position of the amino acid sequence of Dre recombinase based on SEQ ID NO. 10.

Dre recombinase

The Dre recombinase is derived from the D6 phage, belongs to the family of tyrosine recombinases, and has a molecular weight of about 38kDa. It can specifically recognize rox sites and catalyze the recombination of sequences between two sites, thereby causing the phenomena of DNA insertion, deletion, translocation and the like.

In the present invention, a preferred Dre recombinase is codon-optimized Dre recombinase.

Light-sensitive proteins

The photosensitive protein is mostly from animals, plants, bacteria or fungi, and can rapidly change conformation when receiving light stimulation of specific wavelength, so as to convert light into cell information.

Fusion proteins

As used herein, "fusion protein of the invention", or "polypeptide" both refer to a fusion protein according to the first aspect of the invention. The fusion protein of the invention is selected from the group consisting of: a first fusion protein, a second fusion protein, or a combination thereof;

wherein the structure of the first fusion protein is shown as the following formula I or I':

Z1-L1-A1 (I)；

A1-L1-Z1 (I’)；

Z2-L2-A2 (II)；

A2-L2-Z2 (II’)；

in the formula (I), the compound is shown in the specification,

z1 is an N-terminal fragment of Dre recombinase;

l1 is an optional first linker peptide;

a1 is a first element of a light sensitive protein;

z2 is C-terminal fragment of Dre recombinase;

a2 is a second element of the light-sensitive protein;

l2 is an optional second linker peptide;

each "-" is independently a linking peptide or non-peptide bond.

In the present invention, the length of the connecting peptide has an effect on the activity of the fusion protein, and preferably the length of the first connecting peptide and the second connecting peptide is 1 to 80aa, preferably 5 to 60aa, and more preferably 10 to 50aa.

A preferred linker peptide is shown in SEQ ID NO. 1-5.

The term "fusion protein" as used herein also includes variants as shown in any of SEQ ID No. 11-12 having the above-described activity. These variants include (but are not limited to): deletion, insertion and/or substitution of 1 to 3 (usually 1 to 2, more preferably 1) amino acids, and addition or deletion of one or several (usually up to 3, preferably up to 2, more preferably up to 1) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition or deletion of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the structure and function of the protein. In addition, the term also includes monomeric and multimeric forms of the polypeptides of the invention. The term also includes linear as well as non-linear polypeptides (e.g., cyclic peptides).

The invention also includes active fragments, derivatives and analogs of the above fusion proteins. As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that substantially retains the function or activity of a fusion protein of the invention. The polypeptide fragment, derivative or analogue of the present invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which an antigenic peptide is fused to another compound (such as a compound that increases the half-life of the polypeptide, e.g., polyethylene glycol), or (iv) a polypeptide in which an additional amino acid sequence is fused to the polypeptide sequence (a fusion protein in which a tag sequence such as a leader sequence, a secretory sequence or 6His is fused). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the teachings herein.

A preferred class of reactive derivatives refers to polypeptides formed by the replacement of up to 3, preferably up to 2, more preferably up to 1 amino acid with a qualitatively similar or analogous amino acid compared to the amino acid sequence of formula I or formula II or a combination thereof. These conservative variants are preferably produced by amino acid substitutions according to Table A.

TABLE A

Initial residue(s)	Representative substitutions	Preferred substitutions
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala	Leu

The invention also provides analogs of the fusion proteins of the invention. These analogs may differ from the polypeptide of any of SEQ ID Nos. 11-12 by amino acid sequence differences, by modifications that do not affect the sequence, or by both. Analogs also include analogs having residues other than the natural L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It is to be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

Modified (generally without altering primary structure) forms include: chemically derivatized forms of the polypeptide, such as acetylated or carboxylated, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modification may be accomplished by exposing the polypeptide to an enzyme that effects glycosylation, such as a mammalian glycosylase or deglycosylase. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides modified to increase their resistance to proteolysis or to optimize solubility.

In a preferred embodiment, the amino acid sequence of the fusion protein of the invention is as shown in any one of SEQ ID No. 11-12.

Expression vectors and host cells

The invention also relates to vectors comprising the polynucleotides of the invention, as well as genetically engineered host cells transformed with the vectors of the invention or the coding sequences of the fusion proteins of the invention, and methods for producing the polypeptides of the invention by recombinant techniques.

The polynucleotide sequences of the present invention may be used to express or produce recombinant fusion proteins by conventional recombinant DNA techniques. Generally, the following steps are provided:

(1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) of the invention encoding a fusion protein of the invention, or with a recombinant expression vector comprising the polynucleotide;

(2) A host cell cultured in a suitable medium;

(3) Isolating and purifying the protein from the culture medium or the cells.

In the present invention, the polynucleotide sequence encoding the fusion protein may be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors well known in the art. Any plasmid or vector may be used as long as it can replicate and is stable in the host. An important feature of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translation control elements.

Methods well known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding a fusion protein of the invention and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. Representative examples of such promoters are: lac or trp promoter of E.coli; a lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, LTRs of retrovirus, and other known promoters which can control the expression of genes in prokaryotic or eukaryotic cells or viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli.

Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.

The host cell may be a prokaryotic cell (e.g., E.coli), or a lower eukaryotic cell, or a higher eukaryotic cell, such as a yeast cell, a plant cell, or a mammalian cell (including human and non-human mammals). Representative examples are: escherichia coli, wheat germ cells, insect cells, SF9, hela, HEK293, CHO, yeast cells, etc. In a preferred embodiment of the present invention, a yeast cell (e.g., pichia pastoris, kluyveromyces lactis, or a combination thereof; preferably, the yeast cell comprises Kluyveromyces lactis, more preferably Kluyveromyces marxianus, and/or Kluyveromyces lactis) is selected as the host cell.

When the polynucleotide of the present invention is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 bp in length, that act on a promoter to increase gene transcription. Examples include the SV40 enhancer on the late side of the replication origin at 100 to 270 bp, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

It will be clear to one of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl ₂ Methods of treatment, the steps used are well known in the art. Another method is to use MgCl ₂ . If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

The main advantages of the invention include:

(1) The invention discovers for the first time that the complete Dre recombinase is split into an N-terminal segment and a C-terminal segment, and the N-terminal segment and the C-terminal segment are respectively fused and expressed with photosensitive proteins nMAG and pMag through a linker to form a light-activated Dre recombinase system, also called PA-Dre. In the absence of blue light, the segmented Dre recombinase has no catalytic activity, and when the system is stimulated by blue light, the segmented Dre recombinase is quickly dimerized under the drive of photosensitive protein to form the recombinase with catalytic activity, so that the activity is high, the background is few, and the induction multiple is up to 102 times.

(2) Compared with the traditional induction system such as a Dre recombinase induced by tamoxifen, the PA-Dre system not only does not influence the endogenous signal path of cells, but also has higher space-time specificity, can be applied to multiple fields such as lineage tracing, cell clearing and the like, combines the specificity of a promoter, and realizes more accurate gene operation by using local blue light irradiation.

(3) The PA-Dre system established by the invention can realize space-time specific gene operation by controlling the time and space of illumination, makes up the defects of the traditional induction system and provides a more appropriate tool for subsequent research.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Unless otherwise specified, the materials and reagents used in the examples of the present invention are commercially available products.

Example 1 determination of the segmental sites of the Dre recombinase

First, 23 potential splitting sites are selected (table 1), and the N-terminal and the C-terminal of Dre recombinase are respectively connected with two interacting proteins Cho2 and Docs through a linker to form a fusion protein. Three plasmids were co-transfected into HEK293 cells using CAG-rox-stop-rox-ZsGreen as the reporter plasmid, and 11 of these cleavage sites were found to be working (fig. 1). Then N end and C end of Dre recombinase formed by the 11 splitting sites are respectively fused and expressed with blue light-sensitive proteins pMag and nMAG, CMV-rox-stop-rox-LUC is taken as a report plasmid, three plasmids are co-transferred into HEK293 cells, and the activity of the segmented Dre recombinase formed by 3 splitting sites (R60/Q61, L150/L151 and S246/N247) is found to be dependent on blue light regulation, and the three sites are taken as candidate sites of a PA-Dre system. (FIG. 2).

Example 2 construction and optimization of PA-Dre System

(1) Adjusting the relative direction

In order to adjust the relative positions of the light sensitive protein and the segmented Dre recombinase, 4 different plasmids were constructed for the three segmented sites, and the plasmids were co-transformed into HEK293 cells by combining a + B, a + D, C + B, and C + D, and a + D was found to be the optimal configuration, when the activity of the segmented Dre recombinase was the highest (fig. 3). Considering that the enzyme activity of the R60/Q61 segmented site is low, the subsequent experiments are optimized on two segmented sites of L150/L151 and S246/N247.

(2) Change different linker

In the process of constructing fusion proteins, linker is important because it is involved in the activity of the protein. We selected 5 different linkers (table 2) and verified that L4 was most effective on HEK293 cells (fig. 4). When the segmented Dre recombinase and the photosensitive protein are connected by the L4, the enzyme activity is higher and the background is greatly reduced. The two segmented Dre recombinase induction folds of L150/L151 and S246/N247 are similar, but the S246/N247 enzyme activity is higher, so this site is taken as the final selection.

(3) Changing different NLS

To increase the activity of the segmented Dre recombinase, we selected 4 different nuclear localization signal sequences (table 3) that were linked to the C-terminus of the segmented Dre recombinase, respectively. The best effect of NLP was confirmed on HEK293 cells, and the induction factor of the segmented Dre recombinase was increased from the initial 26-fold to 70-fold (FIG. 5).

(4) Construction of Single plasmid System

In order to make the segmented Dre recombinase system more stable and efficient, we used the self-hydrolyzed peptide T2A to fuse and express the C-terminal and the N-terminal of the segmented Dre to form a single plasmid system, and the induction fold of Dre recombinase was increased by about 30-fold (fig. 6). We define this final single particle as PA-Dre (FIG. 7).

Example 3 illustrates the features of the PA-Dre System

(1) The PA-Dre system has illumination time dependency

The PA-Dre and CMV-rox-stop-rox-LUC reporter plasmids were co-transferred into HEK293 cells. After 24 hours, one group was dark and protected from light, and the other group was used at an intensity of 2mw/cm ² Blue light (30 s on, 3min off), illuminate 4h, 8h, 16h, 24h, 32h, 36h and 48h, respectively. The enzyme activity of the LUC is detected after 48h, and the PA-Dre system is found to have illumination time dependency, and the enzyme activity of the LUC is higher along with the increase of illumination time (figure 8).

(2) The PA-Dre system has the dependence of illumination intensity

The PA-Dre and CMV-rox-stop-rox-LUC reporter plasmids were co-transfected into HEK293 cells. After 24 hours, one group was dark and protected from light, and the other group was irradiated at an intensity of 4mw/cm, respectively ² 、2mw/cm ² 、1mw/cm ² 、0.5mw/cm ² And 0.05mw/cm ² Blue light (30 s on, 3min off). The enzyme activity of the LUC is detected after 48h, and the PA-Dre system is found to have illumination intensity dependency, and the enzyme activity of the LUC is higher along with the increase of illumination intensity (figure 9).

(3) The PA-Dre system is adapted to different promoters

To verify whether the PA-Dre system is compatible with different promoters, we constructed plasmids driving expression by EF 1. Alpha., CMV, and PGK. These were separately co-transfected in equimolar amounts with the CMV-rox-stop-rox-LUC reporter plasmid into HEK293 cells. After 24 hours, one group was dark and protected from light, and the other group was used at an intensity of 2mw/cm ² Blue light (30 s on, 3min off) illumination. After 48h, the enzyme activity of LUC was tested, and the PA-Dre system was found to be adapted to different promoters, among which CAG promoter is expressed most strongly (FIG. 10).

(3) The PA-Dre System is adapted to different cell lines

To verify whether the PA-Dre system is compatible with different cell lines, we performed the validation on HEK-293A, hela, hep2 and SH-SY5Y cells, respectively. The results indicate that the PA-Dre system is adapted to different cell lines, probably affected by transfection efficiency, and works best on HEK-293 cells (FIG. 11).

Example 4 validation of PA-Dre System in WT mice

(1) Injection of Low dose PA-Dre plasmid

We used the hydrodynamic tail vein injection of plasmid to co-inject low doses of PA-Dre and CMV-rox-stop-rox-LUC reporter plasmid into mice. After 8h, one group was dark and protected from light, and the other group was used at an intensity of 20mw/cm ² Blue light (1 min on, 3min off), 0.5h, 1h, 2h and 4h, respectively. After 24h, live images were taken and the results showed that the PA-Dre system was also light time dependent in mice when low doses of PA-Dre plasmid were injected and that the catalytic efficiency of the recombinant enzyme was highest at 4h (FIG. 12).

(2) Injection of high dose of PA-Dre plasmid

High doses of PA-Dre and CMV-rox-stop-rox-LUC reporter plasmid were co-injected into mice. After 8h, one group was dark and protected from light, and the other group was used at an intensity of 20mw/cm ² Blue light (1 min on, 3min off) for 1min. After 24h, in vivo imaging was taken and the results showed that PA-Dre had higher catalytic efficiency when injected with high doses of PA-Dre plasmid, even with short exposure times (FIG. 11). The liver of the mouse was taken and imaged in vivo and the LUC signal was found to be indeed very strong, demonstrating that the PA-Dre system works well not only at the cellular level but also in animals (FIG. 13).

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes or modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the appended claims of the present application.

Sequence listing

<110> university of east China

Shanghai Bangyao Biological Technology Co.,Ltd.

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aacaccctga gagacctgag gctggtgttc cactcctggg ctagatggtg ccacgctaga 180

cagctggcct ggttccctat ctctcctgag atggctaggg agtacttcct tcagctgcac 240

gatgctgacc tggcctctac caccatcgac aagcactacg ccatgctgaa catgctgctg 300

tcccactgtg gcctgcctcc tctgtctgat gacaagtctg tgagcctggc catgaggaga 360

atccggagag aggctgccac cgagaaggga gagagaaccg gccaggccat ccctctgaga 420

tgggatgacc tgaagctgct ggatgtgctg ctgtctagat ctgagagact ggtggacctg 480

aggaataggg ccttcctgtt tgtggcctac aacaccctga tgaggatgtc tgagatctct 540

aggatcagag tgggagacct ggaccagacc ggagacaccg tgaccctgca catctcccac 600

accaagacca tcaccaccgc tgctggcctg gacaaagtgc tgtctaggag gaccaccgct 660

gtgctgaatg actggctgga tgtgtctggc ctgagagagc accctgacgc tgtgctgttc 720

cctcctatcc accggagcgc accgactctg gcggatctag cggtggatct agcggctctg 780

agacccctgg aacatccgaa tccgccactc cagagagcag cggaggctct tctgcatact 840

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tgtgacctga aacagaaaga caccccgata gtttacgcga gtgaagcctt cctctacatg 180

acaggttaca gcaacgcaga ggtgctgggc cggaattgcc ggtttctgca aagccctgac 240

ggcatggtga agcccaagag cacccggaag tacgtggata gtaacacaat caatactatg 300

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gctcttctga acaaggctag gatcaccacc acccctctga ccgcccctgc catggagaag 600

atttttagcg atgcctgggt gctgctgaac aagagggatg ccacccctaa caagggccgc 660

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caagtgtcca tggtggagat catgcaggag ggcacctgga aaaagcctga gacactgatg 780

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Claims

1. A fusion protein, wherein the fusion protein is a combination of a first fusion protein and a second fusion protein; wherein the first fusion protein has a structure shown in formula I or formula I':

Z1-L1-A1 (I)；

A1-L1-Z1 （I’）；

Z2-L2-A2 （II）；

A2-L2-Z2（II’）；

in the formula (I), the compound is shown in the specification,

z1 is an N-terminal fragment of Dre recombinase;

l1 is an optional first linker peptide;

a1 is a first element of a light sensitive protein;

z2 is a C-terminal fragment of Dre recombinase;

a2 is a second element of the light sensitive protein;

l2 is an optional second linking peptide;

each "-" is independently a linking peptide or a peptide or non-peptide bond;

the N-terminal fragment of the Dre recombinase is based on SEQ ID No.:10, the C-terminal fragment of the Dre recombinase is based on SEQ ID No.:10, and the N-terminal fragment of the Dre recombinase is Dre _1-60 A fragment, wherein the C-terminal fragment of the Dre recombinase is Dre _61-342 A fragment; or the N-terminal fragment of the Dre recombinase is Dre _1-150 A fragment, wherein the C-terminal fragment of the Dre recombinase is Dre _151-342 A fragment; or the N-terminal fragment of the Dre recombinase is Dre _1-246 A fragment, wherein the C-terminal fragment of the Dre recombinase is Dre _247-342 And (3) fragment.

2. The fusion protein of claim 1, wherein the fusion protein further comprises a nuclear localization signal.

3. The fusion protein of claim 2, wherein the nuclear localization signal is selected from the group consisting of: NLS ^sv40 、NLS ^EGL-13 、NLS ^NLP 、NLS ^c-Myc 、PY-NLSs、snRNPs、truncated NLS ^sv40 、NLS ^TUS Or a combination thereof.

4. The fusion protein of claim 1, wherein the fusion protein has an amino acid sequence as set forth in any one of SEQ ID No. 11-12.

5. A polynucleotide encoding the fusion protein of claim 1.

6. A vector comprising the polynucleotide of claim 5.

7. A host cell comprising the vector of claim 6, or having the polynucleotide of claim 5 integrated into its genome.

8. A method of producing the fusion protein of claim 1, comprising the steps of:

culturing the host cell of claim 7 under conditions suitable for expression, thereby expressing the fusion protein; and/or isolating the fusion protein.

9. A gene editing reagent comprising the fusion protein of claim 1.

10. A kit comprising the gene editing reagent according to claim 9.

11. Use of the fusion protein of claim 1 for the preparation of a reagent or kit for increasing gene editing efficiency.

12. A method of performing gene editing comprising the steps of:

(a) Providing a first fusion protein and a second fusion protein, wherein the structures of the first fusion protein and the second fusion protein are as set forth in claim 1;

(b) Under illumination, the first fusion protein and the second fusion protein form a complex, thereby performing gene editing in the presence of the complex.

13. The method of claim 12, wherein the illumination intensity is from 0.1 to 20mw/cm ² 。

14. The method of claim 12, wherein the gene editing is performed in a cell in vitro.