CN116574729A - Engineered homodromous repeat sequence, gRNA thereof and application thereof - Google Patents

Engineered homodromous repeat sequence, gRNA thereof and application thereof Download PDF

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CN116574729A
CN116574729A CN202310509459.3A CN202310509459A CN116574729A CN 116574729 A CN116574729 A CN 116574729A CN 202310509459 A CN202310509459 A CN 202310509459A CN 116574729 A CN116574729 A CN 116574729A
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grna
sequence
gene
gene editing
homodromous
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韩红兵
汪林丽
艾越
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China Agricultural University
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China Agricultural University
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Abstract

The invention discloses an engineered homodromous repeat sequence, gRNA thereof and application thereof. Specifically disclosed is the RNA having the nucleotide sequence of SEQ ID No.5 at positions 14-36 or SEQ ID No. 5. The invention also discloses a gRNA comprising the RNA and a targeting sequence of a target gene, a gene editing system and application thereof, and a method for improving the gene editing efficiency. The invention carries out engineering transformation on the wild type homodromous repeated sequence to obtain the homodromous repeated sequence DRf-4. The result shows that the homodromous repeated sequence DRf-4 of the invention improves the stability of the stem-loop structure of the gRNA, obviously improves the editing activity of the Cas protein, and obviously improves the gene editing efficiency of the Cas protein under the action of the Cas protein by the gRNA formed by the homodromous repeated sequence DRf-4 and the targeting sequence. The homodromous repeated sequence and the gRNA thereof have wide application prospect in the field of gene editing.

Description

Engineered homodromous repeat sequence, gRNA thereof and application thereof
Technical Field
The invention relates to the field of gene editing, relates to an engineered homodromous repeated sequence, gRNA and application thereof, and in particular relates to an engineered homodromous repeated sequence, gRNA and a gene editing system capable of improving Cas enzyme activity.
Background
CRISPR/Cas is an adaptive immune system generated by prokaryotes against viral infection or phage invasion that protects bacteria from viral repeat attacks mainly by adapting, expressing, interfering with 3 basic phases. Since being discovered, researchers continuously optimize and upgrade the CRISPR/Cas system, so that the CRISPR/Cas system becomes one of important gene editing tools in the field of molecular biology, and the gene editing is realized mainly through 3 steps of specific site recognition, target gene cutting and repair. At present, the CRISPR/Cas9 technology is widely applied to a plurality of species such as microorganisms, animals and plants and the like, and breaks through the limitation of traditional breeding, and animal varieties which cannot be bred or are difficult to breed by the traditional breeding method can be cultivated in a short time, so that the progress of animal genetic improvement is accelerated. CRISPR/Cas system plays an important role in the fields of genetic engineering and screening, mammal gene therapy, animal and plant breeding and the like.
The CRISPR/Cas system consists of a CRISPR/Cas protein and a guide RNA (gRNA) comprising a repeat sequence (repeat) and a targeting sequence (spacer), which is complementary to a target sequence of a gene of interest, and which can target the gene of interest, wherein the repeat sequence forms a stem-loop structure that can interact with the Cas protein. In the participation of the gRNA and the Cas protein, the target gene to be edited is precisely sheared.
Disclosure of Invention
The technical problem to be solved by the invention is how to improve the efficiency of gene editing. The technical problems to be solved are not limited to the described technical subject matter, and other technical subject matter not mentioned herein will be clearly understood by those skilled in the art from the following description.
In order to solve the technical problems, the invention firstly provides RNA, and the nucleotide sequence of the RNA can be shown as 14 th to 36 th positions of SEQ ID No.5 or SEQ ID No. 5.
The RNA can be a cognate repeat (e.g., SEQ ID No. 5) used to construct the gRNA, which can form a stem-loop structure that can bind to the Cas protein.
The RNA can also be a truncated orthostatic sequence (such as positions 14-36 of SEQ ID No. 5), which is a residue that is modified by Cas12i3 after transcription of the full-length orthostatic sequence (i.e., a functionally functional mature body) that directs gene editing of the Cas12i3 protein.
The invention also provides a gRNA that can include the RNA and a targeting sequence that targets a gene of interest.
The targeting sequence may be an RNA that is complementary to a target sequence of the gene of interest.
The gRNA may comprise, in sequence from the 5 'to the 3' end, the RNA and a targeting sequence that targets the gene of interest.
The invention also provides DNA molecules encoding said RNAs or said grnas.
The present invention also provides a biomaterial which may be any one of the following:
b1 An expression cassette containing said DNA molecule;
b2 A recombinant vector comprising said DNA molecule, or a recombinant vector comprising B1) said expression cassette;
b3 A recombinant microorganism comprising said RNA, said gRNA or said DNA molecule, or a recombinant microorganism comprising B1) said expression cassette, or a recombinant microorganism comprising B2) said recombinant vector;
b4 A recombinant host cell comprising said RNA, said gRNA or said DNA molecule, or a recombinant host cell comprising B1) said expression cassette, or a recombinant host cell comprising B2) said recombinant vector.
Further, the DNA molecule may be expressed by B1) the expression cassette, B2) the recombinant vector, B3) the recombinant microorganism, and B4) the recombinant host cell.
The microorganism described herein may be a bacterium, fungus, actinomycete, protozoan, algae or virus. Wherein the bacteria may be derived from Escherichia sp, erwinia sp, agrobacterium sp, flavobacterium sp, etc., but are not limited thereto. The fungus may be a yeast, which may be from the genus Saccharomyces, kluyveromyces, pichia, etc., but is not limited thereto. The actinomycetes may be derived from Streptomyces sp, nocardia sp, micromonospora sp, etc., but are not limited thereto. The algae may be derived from Fucus sp, aspergillus sp, etc., but is not limited thereto. The virus may be rotavirus, herpes virus, influenza virus, adenovirus, etc., but is not limited thereto.
The host cell (also referred to as a recipient cell) described herein may be a plant cell or an animal cell. The host cell is understood to mean not only the particular recipient cell, but also the progeny of such a cell, and such progeny may not necessarily correspond, in their entirety, to the original parent cell, but are included in the scope of the host cell, due to natural, accidental, or deliberate mutation and/or alteration. Suitable host cells are known in the art, wherein: the plant cell may be, but is not limited to, plant cells such as arabidopsis thaliana (Arabidopsis thaliana), tobacco (Nicotiana tabacum), maize (Zea mays), rice (Oryza sativa), wheat (Triticum aestivum), etc.; the animal cells may be mammalian cells (e.g., chinese hamster ovary cells (CHO cells), vero cells, baby hamster kidney cells (BHK cells), mouse breast cancer cells (C127 cells), human embryonic kidney cells (HEK 293 cells), human HeLa cells, fibroblasts, bone marrow cell lines, T cells, NK cells, etc.), avian cells (e.g., chicken or duck cells), amphibian cells (e.g., xenopus laevis cells or giant salamander cells (Andrias davidianus) cells), fish cells (e.g., grass carp, rainbow trout or catfish cells), insect cells (e.g., sf21 cells or Sf-9 cells), etc., but are not limited thereto. In one or more embodiments of the invention, the host cell is a sheep fibroblast.
The invention also provides any one of the following uses of the RNA, the gRNA, the DNA molecule or the biological material:
a1 Use in gene editing;
a2 Use in the preparation of a gene editing system;
a3 Use in the preparation of a gene editing product;
a4 Use in improving gene editing efficiency;
a5 Use in increasing Cas protein activity;
a6 For the detection of a gene of interest, or for the preparation of a product for the detection of a gene of interest.
The gene edits described herein may be gene edits for eukaryotes or prokaryotes, or gene edits for eukaryotic cells or prokaryotes. The eukaryote may include an animal or a plant.
The gene editing products described herein may include, but are not limited to, cell models, animal or plant models, new varieties of animals or plants, and the like.
The present invention also provides a gene editing system, which may include at least any one of:
e1 The RNA;
e2 -said gRNA;
e3 A) the DNA molecule;
e4 A recombinant vector as described in B2).
Further, the gene editing system may further comprise a Cas protein.
The gene editing system may be a CRISPR/Cas system.
Further, the gene editing system described herein can be a Cas12i3 protein-mediated gene editing system (CRISPR/Cas 12i3 gene editing system).
The invention also provides application of the gene editing system in gene editing, preparing a gene editing product, improving gene editing efficiency, improving activity of Cas protein or preparing a product for target gene detection.
The methods of gene detection of interest described herein can include contacting a sample with a Cas protein and the gRNA, or contacting a sample with the gene editing system, detecting a detectable signal produced by cleavage of a gene of interest by the Cas protein, thereby detecting the gene of interest.
The detectable signal may be achieved by: visual-based detection, fluorescent signal-based detection, sensor-based detection, color detection, gold nanoparticle-based detection, fluorescence polarization or fluorescent signal, colloidal phase change/dispersion, electrochemical detection, and semiconductor-based detection.
The present invention also provides a method of gene editing, which may include the step of gene editing using the recombinant vector and/or the gene editing system in the RNA, the gRNA, the DNA molecule, B2).
Further, the method of gene editing may include: contacting the sample to be edited with the RNA, the gRNA, the DNA molecule, the recombinant vector in B2) and/or the gene editing system for gene editing.
The sample to be edited may include cells (e.g., animal cells or plant cells), tissues, organs, or a combination thereof.
The sample may be from an animal, plant or microorganism (including bacteria, viruses, etc.).
The gene edits described herein include in vitro gene edits, in vivo gene edits, or a combination thereof.
The gene editing described herein may include gene knockout, gene knock-in, gene mutation, gene fragment substitution, or gene modification.
The invention also provides a method for improving the efficiency of gene editing, which can comprise the step of mutating U at the 24 th position in the homodromous repeated sequence shown in SEQ ID No.1 into C and mutating A at the 33 th position into G.
The orthostatic sequence shown in SEQ ID No.1 is based on the full-length Direct-Repeat (DRf) of Cas12i3 protein, which can be divided into 5 segments, the first segment being CUCUGACCACCUG; the second section is AGAGAAU; the third segment is GUGUG; the fourth segment is CAUAGU; the fifth section is CACAC. The first segment is a sequence modified and sheared by a special RNase functional domain carried by Cas12i3 after transcription of the full-length homodromous repeat sequence. The third and fifth segments are the locations of the stem regions that make up the stem-loop structure.
Further, the method may be a method of increasing efficiency of Cas protein gene editing.
Further, the method for improving gene editing efficiency may further include a step of constructing the DNA encoding the gRNA into a vector containing a Cas protein gene, preparing a gene editing vector.
The methods described herein can be based on a CRISPR/Cas system.
Cas proteins described herein may include, but are not limited to, cas12i3, cas9, cas12a, cas12b, cas13a, cas14 proteins.
Further, the Cas protein described herein may be a Cas12i3 protein. The amino acid sequence of the Cas12i3 protein may be SEQ ID No.21 and the nucleotide sequence of the Cas12i3 protein coding sequence may be SEQ ID No.22.
The grnas described herein include a homeotropic sequence (DRf) under the name DRf-4 (SEQ ID No. 5) and a targeting sequence (spacer sequence). The target sequence (spacer sequence) is complementary to the target sequence of the target gene, and targets the target of the target gene.
In one embodiment of the invention, the gene of interest is sheep endogenous gene ZFX (GenBank accession No. NC-056080.1, position 22500545-22537460 (Update Date 4-Nov-2022)). The target sequence of the target gene ZFX is SEQ ID No.10. The nucleotide sequence of the ZFX-gRNA-4 can be SEQ ID No.11 and the encoding DNA sequence can be SEQ ID No.12, for the gRNA name of the ZFX gene, ZFX-gRNA-4.
In another embodiment of the present invention, the target gene is tdTomato gene (GenBank accession No. KT878736.1, positions 2529-3959 (Update Date 06-OCT-2015)). The target sequence of the target gene tdTomato is SEQ ID No.18. The tdTomato gene has a gRNA name tdTomato-gRNA-4, the nucleotide sequence of tdTomato-gRNA-4 can be SEQ ID No.19, and the encoding DNA sequence can be SEQ ID No.20.
In order to improve the gene editing efficiency of the Cas protein, the invention is widely and deeply researched to engineer a wild type homodromous repeated sequence (SEQ ID No. 1) to determine the base pairing in the modified stem ring, and the base pairing is changed from U-A pairing to G-C or C-G pairing. The mutation modification mode finally screened is as follows: the 24 th U in the same direction repeated sequence shown in SEQ ID No.1 is mutated into C, the 33 rd A is mutated into G, and the engineered same direction repeated sequence DRf-4 (SEQ ID No. 5) is obtained. It is divided into 5 sections, the first being CUCUGACCACCUG; the second section is AGAGAAU; the third segment is GUGCG; the fourth segment is CAUAGU; the fifth section is CGCAC. Wherein the first segment is modified and cut off by a special RNase functional domain carried by Cas12i3 after the transcription of the full-length homodromous Repeat sequence, and the rest is a truncated homodromous Repeat sequence (DRt), named DRt-4, and the nucleotide sequence of the truncated homodromous Repeat sequence DRt-4 is 14 th-36 th position of SEQ ID No. 5. DRt-4 is a component of mature gRNA, is a part of DRf-4 which plays a functional role after transcription in organisms, and guides Cas12i3 protein to carry out gene editing.
Experimental results show that the engineered homodromous repeated sequence disclosed by the invention is used for modifying the U-A pairing into the C-G pairing, so that the editing activity of Cas12i3 is obviously improved. The homodromous repeated sequence DRf-4 (SEQ ID No. 5) and the truncated homodromous repeated sequence DRt-4 (14 th-36 th positions of SEQ ID No. 5) of the invention improve the stability of the stem loop structure of the gRNA, thereby improving the activity of the Cas enzyme, and the gRNA formed by the same and a targeting sequence (complementary to a target sequence of a target gene) finally and obviously improves the gene editing efficiency of the Cas protein under the action of the Cas protein. The homodromous repeated sequence and the gRNA thereof have wide application prospects in the aspects of accelerating gene editing breeding, gene editing and the like.
Drawings
FIG. 1 shows the T7E1 assay for the efficiency of editing of different engineered orthostatic repeats in sheep fibroblasts (48 h post transfection).
FIG. 2 is a graph showing the proportion of tdTomato average fluorescence intensity quenching (48 h post transfection) of tdTomato labeled sheep fibroblasts by flow analysis of different engineered orthostatic repeats.
FIG. 3 shows the weak tdTomato fluorescence intensity of Weak tdTomato labeled sheep fibroblasts by flow analysis of different engineered homodromous repeats<10 3 ) Is a change in the cell number ratio (48 h after transfection).
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Definition of the definition
In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Further, the procedures of molecular genetics, nucleic acid chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics and recombinant DNA, etc., as used herein, are all conventional procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.
gRNA
In general, the guide RNA can comprise or consist essentially of a Direct Repeat (DR) and a guide sequence (spacer), also referred to in the context of endogenous CRISPR systems. The homeotropic repeats are also known as "framework regions", "protein binding segments", "protein binding sequences"; the guide sequence may also be referred to as a "targeting sequence of a targeting nucleic acid" or a "targeting segment of a targeting nucleic acid". In certain instances, a targeting sequence is any polynucleotide sequence that has sufficient complementarity to a target sequence to hybridize to the target sequence and direct specific binding of a CRISPR/Cas complex to the target sequence.
Cas proteins
The expression "Cas protein" refers to a CRISPR protein, the terms "CRISPR/Cas protein", "Cas effect protein", "Cas enzyme", "single effect nuclease" are used interchangeably. A ribonucleoprotein complex of Cas protein and gRNA or mature crRNA, once bound to a feature sequence to be detected (target sequence), comprises a guide sequence hybridized to the target sequence and bound to the Cas protein. The ribonucleoprotein complex is capable of recognizing and cleaving a polynucleotide that hybridizes to a gRNA or mature crRNA.
Orthotropic repeat sequences
Regularly clustered, spaced short palindromic repeats (clustered regularly interspaced short palindromic repeats sequences, CRISPR) are DNA repeats that provide adaptive immunity to viruses and plasmids for bacteria and archaea. CRISPR gene sequences consist mainly of leader (leader), repeat (repeat) and spacer (spacer). Repeat and spacer transcribed to form a pre-crRNA array, which is digested by RNase to form mature crrnas. If the repeat sequence directions are identical, the repeat sequence is called Direct Repeat (DR).
Spacer sequence
The CRISPR array consists of a repeating sequence and a spacer sequence. Spacer sequences, also known as "Spacer", "target sequence", "target", space determines the targeting position of CRISPR/Cas.
Stem ring structure
Stem-loop (Stem-loop) refers to an intramolecular base pairing process that can occur with single-stranded DNA, but is more common in RNA molecules. When the loop formed is small, it is also called a hairpin or hairpin loop. The stem-loop structure of the invention is directed to intramolecular base pairing structures formed by partial co-repeat sequences in the gRNA.
Carrier body
Refers to a DNA molecule capable of self-replication in which a DNA fragment (gene of interest) is transferred to a recipient cell in a genetically engineered recombinant DNA technique. Three of the most commonly used vectors are bacterial plasmids, phages and animal and plant viruses. Vectors can be functionally divided into cloning vectors and expression vectors. Cloning vectors are the simplest vectors, mainly used for cloning and amplifying DNA fragments, and mainly include plasmid vectors, phage vectors, and viral vectors. The expression vector has DNA elements such as a promoter and a terminator necessary for transcription and translation in addition to the basic elements of the cloning vector. The promoters referred to herein are the U6 and CBh promoters, the U6 promoter belongs to the pol III type promoter, the sequence length of the drive is very small, and the currently common promoters are gRNA and siRNA for expression by the U6 promoter. The sequence driven by the U6 promoter will terminate when pol (U) is encountered. The CBh promoter is an artificially constructed combined promoter consisting of a cytomegalovirus (the cytomegalovirus, CMV), an early enhancer (early enhancer element), a chicken beta-actin (chicken beta-actin) promoter, and a mixed sequence of chicken beta-actin (CBA) and murine parvovirus (minute virus of mice, MMV) introns for driving high level expression of genes in mammalian vectors.
T7E1 enzymatic cleavage
T7E1, which is known as T7 Endonuclease I, is a relatively specific DNA Endonuclease capable of recognizing and cleaving incompletely paired DNA, cross-structured DNA, holliday structure, etc. T7E1 is commonly used for CRISPR/Cas, TALEN, and other editing tool-formed mutant detection.
The sheep fibroblasts in the following examples were prepared as follows: a small amount of ear tissue of sheep within 2 weeks of birth was taken and placed in PBS. In an ultra clean bench, the ear tissue was sterilized in 75% alcohol for 1min, washed 3 times with PBS, and minced to 1mm with sterile scissors 3 Size, 200. Mu.L of foetal calf serum was added, transferred to a cell culture dish, and incubated at 37℃with 5% CO 2 The incubator was placed upside down for 1 hour. The complete medium was carefully added, taking care not to rinse up the tissue mass. After about 1 week of culture, fibroblasts were climbed out of the tissue mass. And after the growth is completed, pancreatin is digested, and the culture is expanded, and then frozen for later use.
The PX458 vector in the examples below is derived from the Addgene plasmid shared information library (numbered 48138).
The amino acid sequence of the Cas12i3 protein in the following examples is SEQ ID No.21, and the nucleotide sequence of the Cas12i3 protein coding sequence is SEQ ID No.22.
Example 1 optimization of the homodromous repeat sequence
The invention is based on the wild type full-length Direct-Repeat (DRf) engineering of CRISPR/Cas12i3 system, the full-length Direct-Repeat (DRf) of the Wild Type (WT) is 5'-CUCUGACCACCUGAGAGAAUGUGUGCAUAGUCACAC-3' (SEQ ID No. 1), which can be divided into 5 segments, the first segment is CUCUGACCACCUG; the second section is AGAGAAU; the third segment is GUGUG; the fourth segment is CAUAGU; the fifth section is CACAC. Wherein the first segment is modified and cut off by a special RNase functional domain carried by Cas12i3 after transcription of the full-length homodromous Repeat sequence, and the rest becomes a truncated homodromous Repeat sequence (DRt), and the sequence of DRt-WT is 5'-AGAGAAUGUGUGCAUAGUCACAC-3'. Wherein the third and fifth segments constitute a stem region of a stem-loop structure. The base pairing of the stem region in the stem-loop structure is altered by simultaneously altering the bases at corresponding positions of the third and fifth segments. The stem-loop structural sequences are GUGUG and CACAAC, and 8 engineered full-length homodromous repeated sequences are generated by changing the pairing of U-A into G-C or C-G pairing. Specific mutant engineering patterns and mutated DRf are shown in table 1.
TABLE 1, 8 engineered full length homodromous repeats
Example 2, T7E1 enzymatic cleavage assay for the efficiency of editing engineered full Length Co-repeat
1. Construction of design target sequence, gRNA sequence and gene editing vector
The gRNA includes a cognate repeat and a targeting sequence (spacer), wherein the cognate repeat is DRf-WT, DRf-1, DRf-2, DRf-3, DRf-4, DRf-13, DRf-14, DRf-23, and DRf-24, respectively, in example 1. The target sequence (spacer sequence) is complementary to the target sequence of the target gene, and targets the target of the target gene.
In this example, sheep endogenous gene ZFX (GenBank Accession No. NC_056080.1, position 22500545-22537460 (Update Date 4-Nov-2022)) was selected, and a target sequence targeting the ZFX gene gRNA (gRNA 1) was designed, the target sequence being 5'-CAGTACAGCAAGAGTGGATGAAT-3' (SEQ ID No. 10).
The gRNA designed for the ZFX gene and its coding DNA sequence are as follows:
ZFX-gRNA-WT: CUCUGACCACCUGAGAGAAUGUGUGCAUAGUCACACCAGUACA GCAAGAGUGGAUGAAU, which encodes the DNA sequence: CTCTGACCACCTGAGAGAATGTGTGCAT AGTCACACCAGTACAGCAAGAGTGGATGAAT;
ZFX-gRNA-1: CUCUGACCACCUGAGAGAAUGGGUGCAUAGUCACCCCAGUACAGC AAGAGUGGAUGAAU, which encodes the DNA sequence: CTCTGACCACCTGAGAGAATGGGTGCATAG TCACCCCAGTACAGCAAGAGTGGATGAAT;
ZFX-gRNA-2: CUCUGACCACCUGAGAGAAUGCGUGCAUAGUCACGCCAGUACAGC AAGAGUGGAUGAAU, which encodes the DNA sequence: CTCTGACCACCTGAGAGAATGCGTGCATAG TCACGCCAGTACAGCAAGAGTGGATGAAT;
ZFX-gRNA-3: CUCUGACCACCUGAGAGAAUGUGGGCAUAGUCCCACCAGUACAGC AAGAGUGGAUGAAU, which encodes the DNA sequence: CTCTGACCACCTGAGAGAATGTGGGCATAG TCCCACCAGTACAGCAAGAGTGGATGAAT;
ZFX-gRNA-4: CUCUGACCACCUGAGAGAAUGUGCGCAUAGUCGCACCAGUACAGC AAGAGUGGAUGAAU (SEQ ID No. 11), which encodes the DNA sequence: CTCTGACCACCTGAGAGA ATGTGCGCATAGTCGCACCAGTACAGCAAGAGTGGATGAAT (SEQ ID No. 12);
ZFX-gRNA-13: CUCUGACCACCUGAGAGAAUGGGGGCAUAGUCCCCCCAGUACAG CAAGAGUGGAUGAAU, which encodes the DNA sequence: CTCTGACCACCTGAGAGAATGGGGGCATA GTCCCCCCAGTACAGCAAGAGTGGATGAAT;
ZFX-gRNA-14: CUCUGACCACCUGAGAGAAUGGGCGCAUAGUCGCCCCAGUACAG CAAGAGUGGAUGAAU, which encodes the DNA sequence: CTCTGACCACCTGAGAGAATGGGCGCATA GTCGCCCCAGTACAGCAAGAGTGGATGAAT;
ZFX-gRNA-23: CUCUGACCACCUGAGAGAAUGCGGGCAUAGUCCCGCCAGUACAG CAAGAGUGGAUGAAU, which encodes the DNA sequence: CTCTGACCACCTGAGAGAATGCGGGCATA GTCCCGCCAGTACAGCAAGAGTGGATGAAT;
ZFX-gRNA-24: CUCUGACCACCUGAGAGAAUGCGCGCAUAGUCGCGCCAGUACAG CAAGAGUGGAUGAAU, which encodes the DNA sequence: CTCTGACCACCTGAGAGAATGCGCGCATA GTCGCGCCAGTACAGCAAGAGTGGATGAAT.
The vector was constructed as follows:
PX458 (U6-sgRNA-CBh-Cas 9-T2A-EGFP-bGH polyA) was double digested with restriction enzymes BbsI (NEB (Beijing) limited) and XbaI (NEB (Beijing) limited) to remove the sgRNA scaffold sequence. And (3) enzyme cutting system: PX458 5 μg, bbsI 25units,XbaI 25units,cutsmart 10 μl, ddH 2 O was added to 100. Mu.L. Reaction conditions: incubate at 37℃for 6h. The concentration was recovered and measured by a recovery kit (Guangzhou Mei-based Biotechnology Co., ltd., product No. D2111-02). The synthetic primers 5'-CACCACTAGTT-3' and 5'-CTAGAACTAGT-3' anneal to form a DNA duplex complementary to the linear PX458 vector after cleavage as described above. Annealing system: 5'-CACCACTAGTT-3' (100. Mu.M) 2.5. Mu.L, 5'-CTAGAACTAGT-3' (100. Mu.M) 2.5. Mu.L, T4 ligase buffer 1. Mu.L, ddH 2 O was added to 10. Mu.L. Annealing procedure: the metal bath was set at 95℃for 5min, then the metal bath lid was opened, the metal bath was closed, and the metal bath was cooled to room temperature. The recovered linear PX458 vector (after BbsI and XbaI double cleavage) was ligated with the annealed product by T4 ligase kit (Takara Shuzo Co., ltd.) to U6-CBh-Cas9-T2A-EGFP-bGH polyA.
The above plasmid U6-CBh-Cas9-T2A-EGFP-bGH polyA was double digested with restriction enzymes AgeI (NEB (Beijing) Co.) and FseI (NEB (Beijing) Co.) to remove the Cas9 coding sequence. And (3) enzyme cutting system: 5. Mu.g of the above plasmid (U6-CBh-Cas 9-T2A-EGFP-bGH polyA), ageI 20units,FseI 20units,cutsmart10. Mu.L and ddH2O were added to 100. Mu.L. Reaction conditions: incubate at 37℃for 6h. The concentration was recovered and measured by a recovery kit (Guangzhou Mei-based Biotechnology Co., ltd., product No. D2111-02). The recombinant vectors obtained by assembling the digestion products (AgeI and FseI double digestion plasmids U6-CBh-Cas9-T2A-EGFP-bGH polyA) and mammal codon optimized Cas12i3 protein coding DNA (SEQ ID No. 22) through a seamless cloning kit are U6-CBh-Cas12i3-T2A-EGFP-bGH polyA respectively.
The vector expressing Cas12i3 constructed above (U6-CBh-Cas 12i3-T2A-EGFP-bGH polyA) was double digested with KpnI (NEB (beijing) limited) and SpeI (NEB (beijing) limited) and recovered (U6 promoter followed by SpeI and KpnI cleavage recognition sites). And (3) enzyme cutting system: 5 μg of the above plasmid, speI 50units,KpnI 50units,cutsmart10μL，ddH 2 O was added to 100. Mu.L. Reaction conditions: incubate at 37℃for 6h. Then 5 μLBeyoAP alkaline phosphatase (Biyun biotechnology Co., ltd., cat. D7027) was added and incubation continued for 10min at 37 ℃. The concentration was recovered and measured by a recovery kit (Guangzhou Mei-based Biotechnology Co., ltd., product No. D2111-02).
The coding DNA sequence of the ZFX gene gRNA was amplified for expression by the following primers (table 2) (where uppercase letters indicate direct repeat + target sequence + transcription termination signal and lowercase letters indicate vector homology sequence). PCR amplification system: f1. Mu.L, R1. Mu.L, primeSTAR 15. Mu.L, ddH 2 O13. Mu.L. PCR amplification procedure: pre-denaturation at 98℃for 3min; denaturation at 98℃for 10s, annealing at 60℃for 15s, extension at 72℃for 5s (33 cycles); extending at 72℃for 5min. After completion of PCR, the PCR product was recovered by a product recovery kit (Guangzhou Mei-based Biotechnology Co., ltd., product No. D2111-02) and the concentration was measured.
TABLE 2 primers for amplification of DNA fragments expressing gRNA (targeting the ZFX gene)
The DNA fragment expressing the gRNA is homologous and recombined with SpeI and KpnI double-enzyme-digested vectors (U6-CBh-Cas 12i3-T2A-EGFP-bGH polyA) respectively through a seamless cloning kit to form 9 recombinant vectors containing gRNA (targeted ZFX) with different directional repeated sequences, namely the gene editing vector.
The construction of the gene editing vector for ZFX gene editing comprises a ZFX gene editing target point, gRNA coding DNA designed and modified by the invention and Cas12i3 protein genes, after the gene editing vector is introduced into receptor cells, transcribed gRNA (guide RNA) can target the ZFX gene through base complementation pairing, and expressed Cas12i3 protein breaks DNA double chains at the upstream and downstream of the ZFX gene target point, so that the gene editing function is realized.
2. Electroporation transfection of sheep fibroblasts
Sheep fibroblasts in good condition were transferred to a 10cm dish and cultured until the cell confluence was about 80%. Cells were harvested by pancreatin digestion into EP tubes. mu.L of electrotransfer solution (Beijing Yinggan Biotechnology Co., ltd., cat. No. 98668-20) was suspended and 7. Mu.g of plasmid (9 kinds of gene-editing vectors for ZFX gene editing constructed as described above) was added thereto, and mixed well. Put into Lonza Amaxa Nucleofector B cell nuclear transfection instrument, adjust to procedure A-033, electrotransfection. After completion of the electric transfer, 500. Mu.L of DMEM high-sugar medium was added to the standing horse, and the cell culture incubator was allowed to stand at 37℃for 10 minutes. Cells were plated into 6-well plates with complete medium containing 20% FBS. After 6h, the medium was changed to complete medium containing 15% FBS.
3. T7E1 detection editing efficiency
Cells after 48h of electrotransformation were subjected to genomic DNA extraction using a genomic extraction kit (Guangzhou Meiyi Biotechnology Co., ltd., product No. D3018-02). 100ng of the extracted sheep genomic DNA was used as a template for PCR amplification. Amplification reaction system and amplification procedure: the total volume of the amplification reaction was 50. Mu.L, and the primers in Table 3 were used for amplification, each of which had the following components: 100ng of DNA template, 1. Mu.L of 10. Mu. Mol/L upstream and downstream primers, 25. Mu.L PrimeSTAR (Takara Shuzo Co., ltd.) and 50. Mu.L of the primer were supplemented with sterilized deionized water; the PCR reaction procedure was: pre-denaturation at 98℃for 3min; denaturation at 98℃for 10s, annealing at 60℃for 15s, extension at 72℃for 30s (33 cycles); finally, the extension is carried out for 5min at 72 ℃. After the PCR is completed, the PCR product is recovered by a product recovery kit and the concentration is determined.
TABLE 3 amplification primers
Taking the product recovered by the previous step of PCR, and preparing an enzyme digestion system as follows: 500ng,cutsmart 1.1. Mu.L of amplified product, ddH 2 O was added to 11.5. Mu.L. After mixing well, following hybridization procedure: 95 ℃ for 10min; -2 ℃/s down to 85 ℃; -0.1 ℃/s down to 25 ℃. Adding 0.5 μl of T7E1 (NEB), enzyme cutting at 37deg.C for 15min, immediately adding2. Mu.L of Loading Buffer was added, 2% agarose was prepared for electrophoresis analysis, and the result after digestion was observed and analyzed in a gel imaging system.
As observed by agarose gel electrophoresis (FIG. 1), the editing efficiency of DRf-4 (SEQ ID No. 5) was 10.3% as compared to 9.9% of the WT control group, improving editing efficiency.
Example 3 efficiency of editing of engineered full Length Co-repeat
1. Construction of tdTomato red fluorescence labeled sheep fibroblasts
1-1, construction of CRISPR/Cas9 Gene targeting vector
1-1-1 and enzyme-cut PX458 carrier
And (3) enzyme cutting system: PX458 vector 5. Mu.g, bbsI 50units,cutsmart 10. Mu.L, ddH 2 O was made up to 100. Mu.L. And enzyme cutting at 37 ℃ for 5 hours. After the cleavage, the cleavage product was purified by a product purification kit (guangzhou mei biotechnology limited) to obtain a purified PX458 BbsI cleavage product. Target (oligo) was designed for goat ZFY gene sequences, and target sequences were synthesized according to table 4.
TABLE 4 sgRNA target sequences
1-1-2, oligo annealing
The designed oligo was annealed according to the following annealing system and annealing procedure, and annealed to form an annealed product (double-stranded DNA).
Annealing system: ZFY-sgRNA-F (100. Mu.M) 2.5. Mu.L, ZFY-sgRNA-R (100. Mu.M) 2.5. Mu.L, T4 library buffer 1. Mu.L, ddH 2 O was made up to 10. Mu.L. Annealing procedure: the metal bath was kept at 95℃for 5min, the metal bath was closed, the lid was opened, and the metal bath was taken out after the temperature was lowered to room temperature.
1-1-3, connection
The annealed product was diluted 50-fold and ligated with the PX458BbsI cleavage product of step 1-1-1 according to the ligation system and ligation procedure as follows.
The connection system is as follows: PX458BbsI cleavage product 90ng, annealed product (after dilution) 1. Mu.L, T4 ligase 0.5. Mu.L, T4 ligase buffer 1. Mu.L, ddH 2 O was made up to 10. Mu.L. And (3) connection procedure: the reaction was carried out at 25℃for 1h.
10 mu L of the connecting product is used for transformation, and is subjected to bacterial picking sequencing and plasmid large extraction, so that a CRISPR/Cas9 gene targeting vector (i.e. sgRNA expression vector) is constructed and obtained, and the CRISPR/Cas9 gene targeting vector is named as PX458-ZFY-sgRNA.
1-2 construction of donor plasmids
The laboratory stores pCBh-tdTomato-SV40polyA plasmid, the construction process of which: pROSA 26-precursor (Addgene 21710) was digested with SpeI and XbaI to obtain digested pROSA 26-precursor, and a DNA molecule (SEQ ID No. 13) (tdTomato-SV 40polyA sequence) was joined to digested pROSA 26-precursor by a seamless cloning assembly technique to obtain pROSA26-tdTomato-SV40 polyA. And (3) carrying out double-enzyme digestion on px458 by KpnI and AgeI to obtain a CBh promoter, amplifying sequences except the ROSA26 promoter by using pROSA26-tdTomato-SV40 polyA as a template and using a primer F (5'-tttttttcaggttggaccggTGCCACCATGGACTAGTATGGTGAGCAAGGGCGA-3') and a primer R (5'-taccgtaagttatgtaacggggtacCCAGCTTTTGTTCCCTTTAGT-3'), and constructing the pCBh-tdTomato-SV40polyA by using the sequences and the CBh promoter sequence through a seamless cloning assembly technology.
The plasmid can normally express red fluorescence in primary goat fibroblasts. The sequences on both sides of the cleavage site (3-4 bp upstream of PAM) of ZFY target nuclease are used as homology arms (left homology arm (HA-L) is 925bp, nucleotide sequence is SEQ ID No.14, right homology arm (HA-R) is 958bp, and nucleotide sequence is SEQ ID No. 15). The primers in Table 5 were amplified by PCR and then recovered by a PCR product recovery kit (Meiy Biotechnology Co., ltd.).
The plasmid pCBh-tdTomato-SV40 polyA is digested, and left and right homologous arms of a ZFY target are correspondingly cloned to two ends of the pCBh-tdTomato-SV40 polyA through a seamless cloning assembly technology, so that a plasmid HA-L-CBh-tdTomato-SV40polyA-HA-R is constructed. Next, recognition sequences for ZFY targets are added on the outer sides of the left and right Homology arms to construct the type of donor plasmid required for HMEJ (Homology-arm mediated end ligation, homolog-mediated end joining). In addition, the homology of the left and right homology arms on the constructed donor plasmid was 96.11% and the homology of the right homology arm was 97.66% by NCBI BLAST, though it was derived from goat, but it had extremely high homology to the corresponding site of sheep. Wherein the nucleotide sequence of the sheep left homology arm is SEQ ID No.16, and the nucleotide sequence of the sheep right homology arm is SEQ ID No.17.
Table 5, ZFY left and right homology arm primers
1-3 construction of tdTomato red fluorescent labeled sheep fibroblasts
The CRISPR/Cas9 gene targeting vector PX458-ZFY-sgRNA constructed in this example and donor plasmid HA-L-CBh-tdTomato-SV40polyA-HA-R (carrying the exogenous gene tdTomato gene, although the homology arm is derived from goats, the homology arm corresponding to sheep HAs high homology, and thus is expected to be used in sheep) were used to integrate exogenous gene (tdTomato gene) into targeting sites of ZFY gene by HMEJ-method-mediated recombination site-directed, and sheep fibroblast line with site-directed exogenous gene integration in ZFY gene was constructed. The method comprises the following specific steps:
1-3-1, gene editing plasmid and donor plasmid cotransfection of sheep fibroblasts
The constructed donor plasmid HA-L-CBh-tdTomato-SV40 polyA-HA-R5000 ng and the gene targeting vector PX458 (PX 458-ZFY-sgRNA) 9536ng (molar ratio 1:1.5) were taken and electrotransformed (electrotransformation step is the same as the electrotransformation step of step 2 in example 2, only the added plasmid was different) into primary sheep fibroblasts, and after 24h tdTomato and EGFP positive primary sheep fibroblasts were flow-sorted and plated into cell culture dishes at about 500 cells per dish. After 2 weeks of culture, the cells in the cell culture dish were monoclonal digested by cloning loop into 96-well plates for culture.
1-3-2 sheep fibroblast screen of site-directed integration tdTomato
After the cell clone of the 96-well plate is full, the cells are digested, half of the original wells are left for culture, and the other half of the cells are taken into a 1.5mL centrifuge tube. 12000rpm, centrifuging for 3min, discarding the supernatant, adding 50. Mu.L of cell identification lysate (cell identification lysate preparation: tris-HCl (1M, pH=8.0) 2mL, triton X-100.45 mL, NP-40.45 mL, proteinase K0.02 g, adding deionized water to dissolve and volume to 50mL,0.22 μm filter), and fully suspending the cells, lysing according to the following procedure: 65 ℃ for 30min;95 ℃ for 15min;16 ℃ and infinity. The obtained lysate was used as a DNA template. Primers were designed and PCR identified as per Table 6.
TABLE 6 site-directed integration identification primers
Amplification reaction system and amplification procedure: the total volume of the amplification reaction was 50. Mu.L, and the respective components were: 1. Mu.L of DNA template, 1. Mu.L of 10. Mu. Mol/L upstream and downstream primer, 10. Mu.L PrimeSTAR (Takara Shuzo Co., ltd.) were filled to 20. Mu.L with sterilized deionized water. The PCR reaction procedure was: pre-denaturation at 98℃for 3min; denaturation at 98℃for 10s, annealing at 62℃for 15s, extension at 72℃for 50s (33 cycles); finally, the extension is carried out for 5min at 72 ℃. And after the PCR is finished, detecting a result by agarose gel electrophoresis.
The results showed that 16 cell monoclonals were lysed and 3 clones were identified by PCR as ZFY site-directed integrated cell monoclonals. At the same time, three clones all fluoresced red when viewed by fluorescence microscopy. It shows that the exogenous gene (tdTomato gene) is subjected to site-directed integration at the targeting site, and the tdTomato red fluorescent labeled sheep fibroblasts are successfully constructed.
2. Construction of design target sequence, gRNA sequence and gene editing vector
In this example, the cell clone of ZFY site-directed integration tdmamato constructed in step 1 above was selected as a cell line for evaluating the editing efficiency of the engineered full-length co-repeat sequence by flow analysis. In this example, tdTomato-targeting gRNA (gRNA 2) was designed for the tdTomato coding sequence (Ge nBank Accession No. KT878736.1, positions 2529-3959 (Update Date 06-OCT-2015)) with target sequence 5'-AAGACCATCTACATGGCCAAGAA-3' (SEQ ID No. 18).
The gRNA and its coding DNA sequence designed for tdTomato gene are as follows:
tdTomato-gRNA-WT: CUCUGACCACCUGAGAGAAUGUGUGCAUAGUCACACAAGA CCAUCUACAUGGCCAAGAA, which encodes the DNA sequence: CTCTGACCACCTGAGAGAATGTGTG CATAGTCACACAAGACCATCTACATGGCCAAGAA;
tdTomato-gRNA-1: CUCUGACCACCUGAGAGAAUGGGUGCAUAGUCACCCAAGACC AUCUACAUGGCCAAGAA, which encodes the DNA sequence: CTCTGACCACCTGAGAGAATGGGTGCA TAGTCACCCAAGACCATCTACATGGCCAAGAA;
tdTomato-gRNA-2: CUCUGACCACCUGAGAGAAUGCGUGCAUAGUCACGCAAGACC AUCUACAUGGCCAAGAA, which encodes the DNA sequence: CTCTGACCACCTGAGAGAATGCGTGCA TAGTCACGCAAGACCATCTACATGGCCAAGAA;
tdTomato-gRNA-3: CUCUGACCACCUGAGAGAAUGUGGGCAUAGUCCCACAAGACC AUCUACAUGGCCAAGAA, which encodes the DNA sequence: CTCTGACCACCTGAGAGAATGTGGGCA TAGTCCCACAAGACCATCTACATGGCCAAGAA;
tdTomato-gRNA-4: CUCUGACCACCUGAGAGAAUGUGCGCAUAGUCGCACAAGACC AUCUACAUGGCCAAGAA (SEQ ID No. 19), which codes for the DNA sequence: CTCTGACCACCTGAG AGAATGTGCGCATAGTCGCACAAGACCATCTACATGGCCAAGAA (SEQ ID No. 20);
tdTomato-gRNA-13: CUCUGACCACCUGAGAGAAUGGGGGCAUAGUCCCCCAAGAC CAUCUACAUGGCCAAGAA, which encodes the DNA sequence: CTCTGACCACCTGAGAGAATGGGGGC ATAGTCCCCCAAGACCATCTACATGGCCAAGAA;
tdTomato-gRNA-14: CUCUGACCACCUGAGAGAAUGGGCGCAUAGUCGCCCAAGAC CAUCUACAUGGCCAAGAA, which encodes the DNA sequence: CTCTGACCACCTGAGAGAATGGGCGC ATAGTCGCCCAAGACCATCTACATGGCCAAGAA;
tdTomato-gRNA-23: CUCUGACCACCUGAGAGAAUGCGGGCAUAGUCCCGCAAGAC CAUCUACAUGGCCAAGAA, which encodes the DNA sequence: CTCTGACCACCTGAGAGAATGCGGGC ATAGTCCCGCAAGACCATCTACATGGCCAAGAA;
tdTomato-gRNA-24: CUCUGACCACCUGAGAGAAUGCGCGCAUAGUCGCGCAAGAC CAUCUACAUGGCCAAGAA, which encodes the DNA sequence: CTCTGACCACCTGAGAGAATGCGCGC ATAGTCGCGCAAGACCATCTACATGGCCAAGAA.
By passing throughThe following primers (Table 7) amplify the coding DNA sequence (wherein capital letters identify direct repeat sequence + target sequence + transcription termination signal and capital letters identify vector homologous sequences) expressing the tdTomato gene gRNA. PCR amplification system: f1. Mu.L, R1. Mu.L, primeSTAR 15. Mu.L, ddH 2 O13. Mu.L. PCR amplification procedure: pre-denaturation at 98℃for 3min; denaturation at 98℃for 10s, annealing at 60℃for 15s, extension at 72℃for 5s (33 cycles); extension at 72℃is 5 min. After completion of PCR, the PCR product was recovered by a product recovery kit (Guangzhou Mei-based Biotechnology Co., ltd., product No. D2111-02) and the concentration was measured.
TABLE 7 primers for amplification of DNA fragments expressing gRNA (targeting tdTomato Gene)
The DNA fragment expressing the gRNA is respectively homologous and recombined with SpeI and KpnI double-digested vectors (U6-CBh-Cas 12i3-T2A-EGFP-bGH polyA) in the embodiment 2 through a seamless cloning kit to form 9 recombinant vectors containing gRNA (targeting tdTomato) with different directional repeated sequences, namely the gene editing vector.
The construction of the gene editing carrier for tdTomato gene editing comprises a tdTomato gene editing target, gRNA coding DNA designed and modified by the invention and Cas12i3 protein genes, after the gene editing carrier is introduced into a receptor cell, the transcribed gRNA (guide RNA) can target the tdTomato gene through base complementary pairing, and the expressed Cas12i3 protein breaks the DNA double strand at the upstream and downstream of the tdTomato gene target, so that the gene editing function is realized.
3. Flow cytometry analysis of tdmamato fluorescence changes
The above-described different plasmids (gene editing vector for tdmamto gene editing) were separately transfected into tdmamto red fluorescence-labeled sheep fibroblasts constructed in step 1 of this example. The electrotransport step is the same as in example 2. After 48h, EGFP-positive cells of WT type (control group) and different engineered homodromous repeats were analyzed by flow cytometry (see EGFP-positive cells only because of the top)The control vector and 8 gene editing vectors are respectively provided with EGFP expression sequences, the sequences are PX458 vectors, EGFP positive cells represent cells of successfully transfected plasmids, so that errors caused by cell transfection can be reduced, specifically, the proportion of the disappearance of the tdTomato average fluorescence intensity is calculated, and the fluorescence intensity is weaker (the tdTomato fluorescence intensity in EGFP positive cells is less than 10) 3 ) Is a ratio of the number of cells.
By calculating the ratio of tdTomato average fluorescence intensity quenching of EGFP positive cells (FIG. 2), compared with a DRf-WT control group, the ratio of tdTomato average fluorescence intensity quenching of the engineered DRf-4 (SEQ ID No. 5) is remarkably improved, which shows that the DRf-4 has higher editing efficiency on the tdTomato gene, causes functional inactivation of tdTomato proteins in more cells, finally causes weakening and even quenching of red fluorescence of more cells, and reduces the overall cell tdTomato fluorescence intensity, namely the ratio of tdTomato average fluorescence intensity quenching is larger. Similarly, by calculating the weak tdTomato fluorescence intensity in EGFP-positive cells<10 3 ) Compared with the DRf-WT control group, the DRf-4 obtained by engineering obviously improves the weak tdTomato fluorescence intensity<10 3 ) The cell number ratio (FIG. 3).
The nucleotide sequence of the engineered full-length homodromous repeated sequence DRf-4 is as follows:
5’-CUCUGACCACCUGAGAGAAUGUGCGCAUAGUCGCAC-3' (SEQ ID No. 5). It is divided into 5 sections, the first being CUCUGACCACCUG; the second section is AGAGAAU; the third segment is GUGCG; the fourth segment is CAUAGU; the fifth section is CGCAC. Wherein the first segment is modified and cut off by a special RNase functional domain carried by Cas12i3 after the transcription of the full-length homodromous Repeat sequence, and the rest is a truncated homodromous Repeat sequence (DRt), named DRt-4, and the nucleotide sequence of the truncated homodromous Repeat sequence DRt-4 is 14 th-36 th position of SEQ ID No. 5. DRt-4 is a component of mature gRNA, is a part of DRf-4 which plays a functional role after transcription in organisms, and guides Cas12i3 protein to carry out gene editing.
The 24 th nucleotide 'cytosine (C)' of SEQ ID No.5 is mutated from 'uracil (U)' and the 33 th nucleotide 'guanine (G)' is mutated from 'adenine (A)' i.e. U-A pairing is transformed into C-G pairing, and after the mutation transformation, the stability of the stem-loop structure of gRNA is improved, so that the activity of Cas enzyme is improved, and finally the gene editing efficiency is improved.
The application changes the base pairing of the stem-loop structure through base mutation, and the detection by a T7E1 enzyme digestion method and a flow analysis method shows that DRf-4 significantly improves the editing activity of Cas12i3 relative to DRf-WT. The gRNA formed by the homologous repeated sequence engineering modification body DRf-4 and the targeting sequence (the spacer sequence is complementary with the target sequence of the target gene) can obviously improve the gene editing efficiency under the action of the Cas protein, and has wide application prospects in the aspects of accelerating gene editing breeding, gene editing and the like.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Claims (10)

  1. RNA, characterized in that the nucleotide sequence of the RNA is shown in positions 14-36 of SEQ ID No.5 or SEQ ID No. 5.
  2. A gRNA, comprising the RNA of claim 1 and a targeting sequence that targets a gene of interest.
  3. 3. A DNA molecule encoding the RNA of claim 1 or the gRNA of claim 2.
  4. 4. A biomaterial characterized in that the biomaterial is any one of the following:
    b1 A cassette comprising the DNA molecule of claim 3;
    b2 A recombinant vector comprising the DNA molecule of claim 3, or a recombinant vector comprising the expression cassette of B1);
    b3 A recombinant microorganism comprising the RNA of claim 1, the gRNA of claim 2 or the DNA molecule of claim 3, or a recombinant microorganism comprising the expression cassette of B1), or a recombinant microorganism comprising the recombinant vector of B2);
    b4 A recombinant host cell comprising the RNA of claim 1, the gRNA of claim 2 or the DNA molecule of claim 3, or a recombinant host cell comprising the expression cassette of B1), or a recombinant host cell comprising the recombinant vector of B2).
  5. 5. Use of any one of the RNA of claim 1, the gRNA of claim 2, the DNA molecule of claim 3 or the biological material of claim 4:
    A1 Use in gene editing;
    a2 Use in the preparation of a gene editing system;
    a3 Use in the preparation of a gene editing product;
    a4 Use in improving gene editing efficiency;
    a5 Use in increasing Cas protein activity;
    a6 For the detection or diagnosis of a gene of interest, or for the preparation of a product for the detection or diagnosis of a gene of interest.
  6. 6. A gene editing system, comprising at least any one of:
    e1 The RNA of claim 1;
    e2 The gRNA of claim 2;
    e3 A DNA molecule according to claim 3;
    e4 A recombinant vector as claimed in claim 4.
  7. 7. The gene editing system of claim 6, further comprising a Cas protein.
  8. 8. Use of the gene editing system of claim 6 or 7 in gene editing, preparing a gene editing product, increasing gene editing efficiency, increasing Cas protein activity, or preparing a product for gene detection or diagnosis of interest.
  9. 9. A method of gene editing comprising the step of gene editing using the RNA of claim 1, the gRNA of claim 2, the DNA molecule of claim 3, the recombinant vector of claim 4, and/or the gene editing system of claim 6 or 7.
  10. 10. A method for improving gene editing efficiency, comprising mutating U at position 24 in the homodromous repeat sequence shown in SEQ ID No.1 to C and mutating a at position 33 to G.
CN202310509459.3A 2023-05-08 2023-05-08 Engineered homodromous repeat sequence, gRNA thereof and application thereof Pending CN116574729A (en)

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