CN115074390A

CN115074390A - Method for efficiently constructing genome large fragment deletion mutant and application

Info

Publication number: CN115074390A
Application number: CN202210758278.XA
Authority: CN
Inventors: 祖尧; 王宏杰
Original assignee: Shanghai Ocean University
Current assignee: Shanghai Ocean University
Priority date: 2022-06-30
Filing date: 2022-06-30
Publication date: 2022-09-20

Abstract

The invention discloses a method for efficiently constructing a genome large-fragment deletion mutant and application thereof, wherein a target sequence of gene deletion is determined in a gene cluster, a specific CRISPR gRNA target sequence and an amplification primer are designed, gRNA framework plasmid is used as a template for PCR amplification, purification and in vitro transcription, gRNA and Cas9 protein are subjected to microinjection and genome extraction for PCR amplification, a glue pattern obtained by T7E1 enzyme digestion and agarose gel electrophoresis is sequentially used for detecting strip gray values, the brightness volume calculation knockout efficiency of each strip is obtained, an optimal target combination is formed for microinjection and genome extraction, and the genome is stably cultured through double outer side primer PCR amplification and agarose gel electrophoresis. According to the invention, CRISPR single-site knockout efficiency evaluation is adopted for the first time, and the CRISPR target combination is screened to generate efficient genome large fragment deletion, so that the method can be used for efficient deletion of gene large fragments in genomes of vertebrates.

Description

Method for efficiently constructing genome large fragment deletion mutant and application

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a method for efficiently constructing a genome large fragment deletion mutant and application thereof, in particular to a method for efficiently constructing a disease animal model for researching the disease caused by genome large fragment deletion by utilizing a CRISPR system to delete genome large fragments in zebra fish ttn1-ttn2, nppa-nppb and hox gene clusters.

Background

The CRISPR/Cas system is an RNA-mediated adaptive defense system evolved from bacteria and archaea, and its main function is to protect organisms from invading viruses and plasmids, and to cut exogenous genes with guide RNA nucleases. Compared to Zinc Finger Nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR systems have been widely used to perform mutations in various organisms by causing small insertions and deletions (indels) to disrupt target genes by non-homologous end joining that results in repair errors. The CRISPR/Cas9 system has the advantages of simple operation, easy synthesis, high targeting efficiency, accurate targeting, low cytotoxicity and the like, and can cause the mutation of germ cells while ensuring the mutation of genes in somatic cells, thereby transmitting the mutated genes to the next generation.

However, insertion deletion marker (indel) mutagenesis is not suitable for all targets: multiple transcriptional variants or alternative initiation codons may be present in the same gene (whether annotated or not), making it difficult to determine whether a functional protein product is disrupted by a limited local mutation; for non-coding RNA (ncRNA) genes, untranslated regions or regulatory regions, local mutations are incapable of destroying the function of these genes or cis-elements, it is also difficult to remove multiple genes or a group of adjacent genes on a chromosome simultaneously by only targeting a single locus, and deletion of a long fragment of a genome by CRISPR combination provides an effective tool for realizing the function of coding genes and non-coding genes.

Currently, large fragment deletion techniques have been applied to mammals, fish, plants, bacteria, fungi, etc. (fig. 12). In CRISPR/Cas9 mediated fragment deletion, the adopted general method is to set a target point at each end of a pre-deleted fragment, and a few methods are to set two pairs at each end, David et al designs deletion of 1.2kbp aiming at an upstream key regulatory sequence of a TYR pigment gene in a mammal mouse, and carries out microinjection on mouse fertilized eggs through a pair of gRNAs and Cas9 with different concentrations, successfully constructs a mouse with reduced pigment and verifies the function of the regulatory sequence, and meanwhile, the efficiency of fragment deletion is improved from 23% to 36% when the concentrations of Cas9 mRNA and gRNA are improved. Xiao et al, injected one pair of CRISPRs or two pairs of TALENs into zebrafish embryos to obtain mutants and their progeny for zebrafish genomes, but many were not tested for deletion efficiency. In addition, the 105kbp TYR gene was deleted in rabbits using a mixture of Cas9 mRNA and 2 pairs of gRNA targets with an efficiency of up to 25%. A 245kbp fragment was deleted at 16.7% efficiency by transfer expression of a plasmid comprising Cas9 protein and a pair of grnas in rice. Because the repair capacity of bacteria to double-strand breaks is poor, the strain-Beier et al uses Cas9 Nickase for cutting single strands to delete escherichia coli genes, the cost of the nuclease is high, targeting of one site needs to be matched with two complementary gRNAs, fragments of 36 kb and 97kb are deleted in escherichia coli by mixing 2 pairs of gRNAs and Cas9 Nickase aiming at the two sites, and the deletion success rates are 20% and 1% respectively. In addition, there are reports of large fragment deletions in soybeans. In conclusion, the deletion distance of the genome large fragment is from hundreds of base pairs to nearly one million in each species, and the efficiency of the fragment deletion is between 1% and 36%. The average efficiency of fragment deletion in model organisms such as mice, rabbits, zebrafish, escherichia coli, soybeans, rice and the like is below 25%, and the efficiency is generally low. And the work described above employs Cas9 and 2 injections to perform large fragment deletions on the following grnas, lacking the operational steps to evaluate the optimized gRNA combinations.

The ratio of the number of the genome fragment-deleted diseases to the total number of the diseases is about 17.7%, and the fragment deletion can be used for researching functions of gene families, regulatory elements and the like, so that a disease mechanism is disclosed. The mutant constructed by the model organisms can meet basic research requirements, and meanwhile, drug screening can be performed to help search possible drugs. The ttn1, ttn2 gene mutation coding for the titin is related to human skeletal and heart myopathy, the hox gene cluster codes for a transcription regulation family, the head-tail axis of animals is shaped during the development process, and the hox gene mutation or deletion can cause a series of developmental disorder diseases. The CRISPR is utilized to carry out single-site knockout, so that local gene mutation can be introduced, and thus, the gene expression is abnormal, and normal protein cannot be synthesized. However, the presence of a large number of genes or regulatory elements in the DNA of an organism does not directly express proteins but influences the synthesis of proteins in an indirect manner. Single-site knockouts sometimes do not function to knock out these regulatory elements, and editing these regulatory elements or gene clusters requires the use of fragment deletion means. In plants and animals, fragment deletion can be used to study crop trait genes, delete reverse regulatory elements, and the like to achieve accurate molecular breeding. In the field of microbial industry, antibiotics, pesticides and the like can be manufactured by utilizing bacterial and fungal secondary metabolites, and specific genes can be more durable to store or improved in yield by deleting a plurality of specific genes. Medically, fragment deletions can be used to study the function of long fragments or clustered gene families. Therefore, in the fields of medical treatment, agriculture, industrial production, and the like, it is important to improve the efficiency of gene editing, and at present, the efficiency of fragment deletion is generally low, and further development of efficient genome fragment deletion means is required.

Disclosure of Invention

In order to solve the problem of low genome large fragment deletion efficiency in the prior art, the invention mainly aims to provide a method for efficiently constructing a genome large fragment deletion mutant, and particularly, the genome large fragment deletion is carried out in zebrafish ttn1-ttn2, nppa-nppb and hox gene clusters through a CRISPR system, so that the genome large fragment deletion efficiency is greatly improved.

The invention also aims to provide application of the method for efficiently constructing the genome large fragment deletion mutant in efficiently constructing an animal model for researching diseases caused by genome large fragment deletion.

The purpose of the invention is realized by the following technical scheme:

the method for efficiently constructing the genome large fragment deletion mutant provided by the invention comprises the following steps:

s1, determining a target sequence of gene deletion in a gene cluster of a processing object, and respectively designing not less than three specific CRISPR gRNA target sequences at two ends of a gene fragment to be deleted under the condition of meeting a CRISPR PAM region;

s2, respectively designing amplification primers according to the specific CRISPR gRNA target sequences designed in the step S1;

s3, performing PCR amplification by using a gRNA framework plasmid as a template and using a primer T7-gRNA target as a forward primer and a gRNA reverse primer to obtain a PCR product;

s4, purifying the PCR product obtained in the step S3, and carrying out in-vitro transcription to obtain a plurality of gRNAs;

s5, step S4, the gRNA and Cas9 proteins were microinjected into fertilized eggs of the subject at one-cell stage;

s6, selecting the ovum of the embryo stage of the processing object in the step S5 to extract genome, and respectively carrying out PCR amplification by using a knock-out target detection primer to obtain PCR products;

s7, carrying out enzyme digestion and agarose gel electrophoresis on the PCR product obtained in the step S6 in sequence, detecting the gray value of the bands of the obtained gel Image according to an Image Lab software quantitative tool to obtain the brightness volume of each band of a, b and c, and calculating according to a formula indel% (a + b)/(a + b + c) × 100% to obtain the knockout efficiency;

s8, forming a plurality of groups of optimal target point combinations from the specific CRISPR target points at two ends of the gene fragment to be deleted;

s9, selecting at least three groups of optimal target point combinations in the step S8, and respectively carrying out microinjection and leading the optimal target point combinations into the fertilized eggs in the cell stage of the processing object;

s10, selecting the ovum of the embryo stage of the processing object in the step S9 to extract genome, and performing double outer side primer PCR amplification and agarose gel electrophoresis to perform stable culture to obtain the genome large fragment deletion mutant.

Preferably, in step S8, a specific CRISPR target with high T7E1 enzymatic activity and similarity is selected from the specific CRISPR targets at both ends of the gene fragment to be deleted as an optimal target combination.

Preferably, the method for efficiently constructing the genome large-fragment deletion mutant simultaneously satisfies the following conditions:

(1) the deletion efficiency of the large genome fragment of the treated object is positively correlated with the mutagenesis efficiency of a single specific CRISPR target of the treated object, and is negatively correlated with the difference of the efficiencies of gRNAs at two ends of the gene fragment to be deleted;

(2) the gene length between any two specific CRISPR targets has no obvious influence on the large fragment deletion efficiency;

(3) the genome large fragment deletion efficiency of microinjection of the optimal target combination with high mutagenesis efficiency in step S9 is high compared to the simultaneous microinjection of not less than two specific CRISPR targets.

Preferably, in step S6, the knockout target detection primer is a specific primer that can amplify the target containing primer.

Preferably, in step S5, the final concentration of gRNA is 100-200 ng/. mu.L, the final concentration of Cas9 protein is 800 ng/. mu.L, the final concentration of Cas9 mRNA is 400 ng/. mu.L, and the injection amount is 1 nL.

Preferably, the method for efficiently constructing the genome large fragment deletion mutant is used for genome large fragment deletion in zebrafish ttn1-ttn2, nppa-nppb and hox gene clusters, and comprises the following steps:

(1) determining target sequences of gene knockout in ttn1-ttn2, nppa-nppb and hox gene clusters of zebra fish, and respectively designing not less than three specific CRISPR gRNA target sequences at two ends of a gene fragment to be deleted under the condition of meeting with a CRISPR PAM region;

(2) respectively designing amplification primers according to the specific CRISPR gRNA target sequences designed in the step (1);

(3) performing PCR amplification by taking a gRNA framework plasmid as a template and taking a primer T7-gRNA target as a forward primer and a gRNA reverse primer to obtain a PCR product;

(4) purifying the PCR product obtained in the step (3), and carrying out in-vitro transcription to obtain gRNA;

(5) microinjecting the gRNA obtained in the step (4) and the Cas9 protein into a fertilized egg at a cell stage of the zebra fish;

(6) selecting roe of the zebra fish in the 48h embryonic period in the step (5) to extract genome, and performing PCR amplification by using a knock-out target detection primer to obtain a PCR product;

(7) carrying out T7E1 enzyme digestion detection and agarose gel electrophoresis on the PCR product obtained in the step (6) in sequence, detecting the gray value of the bands of the obtained gel Image according to an Image Lab software quantitative tool to obtain the brightness volume of each band of a, b and c, and calculating according to a formula of (a + b)/(a + b + c) multiplied by 100% indel% to obtain the knockout efficiency;

(8) selecting at least three groups of specific CRISPR targets from the specific CRISPR targets at two ends of the gene fragment to be deleted to form an optimal target combination;

(9) respectively carrying out microinjection on the optimal target point combination obtained in the step (8) and guiding the optimal target point combination into fertilized eggs of the zebra fish in a cell stage;

(10) and (4) selecting the roe of the zebra fish in the 48h embryonic period in the step (9) to extract a genome, and performing double-outer-side primer PCR amplification and agarose gel electrophoresis in sequence to obtain the zebra fish genome large-fragment deletion mutant through stable culture.

Preferably, in step (1), the target sequences are as shown in the following table:

preferably, in step (2), the sequences of the amplification primers are shown in the following table:

the invention also provides application of the method for efficiently constructing the genome large-fragment deletion mutant in gene large-fragment deletion in the genome of vertebrates.

The invention also provides application of the method for efficiently constructing the genome large fragment deletion mutant in constructing an animal model for disease treatment and drug screening caused by genome large fragment deletion.

Compared with the prior art, the invention has the beneficial effects that:

firstly, screening gRNAs with high mutation efficiency, then selecting gRNAs with smaller efficiency difference values to carry out combined microinjection into fertilized eggs of zebra fish in a cell stage, and screening CRISPR combinations to generate efficient genome large fragment deletion by adopting CRISPR single-site efficiency evaluation for the first time, wherein the requirements are met: 1) the deletion efficiency of the large genome fragment is positively correlated with the mutagenesis efficiency of a single specific CRISPR target spot, and is negatively correlated with the difference of the efficiencies of gRNAs at two ends of a gene fragment to be deleted; 2) the gene length between any two specific CRISPR targets has no obvious influence on the large fragment deletion efficiency; 3) the optimal target point combination with high mutagenesis efficiency by microinjection has high genome large fragment deletion efficiency compared with the simultaneous microinjection of not less than two specific CRISPR target points.

Secondly, by designing gRNAs aiming at different genome sites of zebra fish, co-injecting a pair of gRNAs and Cas9 proteins aiming at a specific gene cluster into an embryo in a cell phase, deleting large genome fragments in the zebra fish ttn1-ttn2, nppa-nppb and hox gene clusters by using a CRISPR/Cas system, wherein the deleted genome fragment has the length range of 5-340kb, the average efficiency reaches 64.5 percent, the highest efficiency reaches 100 percent (20/20), and meanwhile, the 340kb ttn1-ttn2 cluster ultra-long fragment is successfully deleted at 95 percent, so that the genome large fragment deletion efficiency is greatly improved. The deletion distance of the existing report fragment is about hundreds of base pairs to about one million, the efficiency of fragment deletion is between 1% and 36%, the average efficiency of fragment deletion in model organisms such as mice, rabbits, zebra fish, escherichia coli, soybeans, rice and the like is below 25%, the efficiency is generally low, large fragment deletion is performed on the following gRNAs by injecting Cas9 and 2, and the operation steps for evaluating and optimizing the gRNA combination are lacked.

Thirdly, the method realizes efficient fragment deletion in the gene clusters of hoxab (33.3kb), hoxca (138.1kb), hoxcb (48.3kb), hoxda (53.3kb) and nppb-nppa of the zebra fish, and the obtained zebra fish genome large fragment deletion mutant can be used as an animal model for researching genome large fragment deletion diseases. In addition, the hoxca gene cluster deletion mutant is obtained from the zebra fish for the first time, and the corresponding dorsal fin deletion phenotype is generated, so that a model is provided for the development research of the dorsal fin of the fish.

Drawings

FIG. 1 is a schematic representation of the deletion of large fragments of the genome in the zebrafish ttn1-ttn2, nppa-nppb and hox gene clusters in the examples.

Fig. 2 is a schematic diagram of agarose gel electrophoresis and ImageLab gray scale value (brightness volume) quantification in the CRISPR-mediated deletion method of the zebra fish genome in the example, and the single-site digestion efficiency is calculated as indel% (a + b)/(a + b + c) × 100%.

FIG. 3 is a gel diagram of efficient T7E1 cleavage of the target genes ttn1, ttn2, hoxa13b, hoxa2b, hoxc1a, hoxc4a, hoxc6a, hoxc9a, hoxc11a, hoxc13a, hoxc6b, hoxc13b, nppb, and nppa in examples.

Fig. 4 is a schematic representation of hoxcb deletion and statistical histograms of hoxcb cluster single site efficiency and fragment deletion efficiency, single site efficiency: T7E1 efficiency, fragment deletion efficiency: knockout efficiency; wherein: (A) schematic diagram of hoxc6b-hoxc13b gene cluster and target; (B) single site efficiencies and mean and fragment deletion efficiencies of hoxc6b T1, hoxc13b T2; (C) single site efficiencies and mean and fragment deletion efficiencies of hoxc6b T1, hoxc13b T1; (D) single site efficiencies and mean and fragment deletion efficiencies of hoxc6b T2, hoxc13b T2; (E) single site efficiencies and mean and fragment deletion efficiencies of hoxc6b T2, hoxc13b T1. The deletion efficiency of the segment from (B) to (E) increases with the increase of the mean of the single-site efficiency, and is positively correlated.

FIG. 5 shows the deletion results of the tn1-ttn2 gene cluster in example; wherein: (a) ttn1-ttn2 cluster fragment deletion efficiency (fra-del efficiency) and target efficiency (indel efficiency difference) histogram, wherein in the deletion of two fragments in ttn1-ttn2 cluster, the difference between the fragment deletion efficiency and the target efficiency is in negative correlation; (b) test gel chart of tt1-ttn2 target spot knockout efficiency; (c) ttn1-ttn2 clusters and the detection of the efficiency of knock-out of each fragment.

FIG. 6 shows the results of deletion of the hoxab gene cluster in the examples; wherein: (a) histogram of difference between hoxb cluster fragment deletion efficiency and target efficiency, where the difference between fragment deletion efficiency and target efficiency is negatively correlated in the deletion of two types of fragments in a hoxb cluster; (b) detecting a gel chart of hoxab target knockout efficiency; (c) gel plots for detection of the efficiency of knockdown of individual fragments of the hoxab cluster.

FIG. 7 shows the results of different hoxca fragment length deletions: (A) schematic target point diagrams of hoxca gene cluster and four deletion modes of 55.7Kb, 71.4Kb, 91.6Kb and 133.9 Kb; (B) the fragment generated by the combination of the four targets of hoxca deleted the pectin-forming pattern. (C) A histogram of single-site deletion efficiency statistics of five hoxca targets; (D) a statistical graph of the deletion efficiency of four fragments of the hoxca cluster; (E) and (3) a hoxca cluster fragment deletion efficiency and deletion length line graph, wherein the fragment deletion efficiency and the fragment deletion length are irrelevant.

FIG. 8 is a graph comparing the efficiency of deletion of a plurality of target spots and a pair of target fragments in the examples; wherein: (A) segment deletion pattern diagram for different target point combinations, first row: the target point A1 is located at the 5 'end of gene1, and the target point A2 is located at the 3' end of gene n; a second row: the target point A1 is located at the 5 'end of gene1, and the target points A3 and A2 are located at the 3' end of gene n; third row: targets A1 and A4 are located at the 5 'end of gene1, and target A2 is located at the 3' end of gene n; fourth row: targets A1 and A4 are located at the 5 'end of gene1, and targets A3 and A2 are located at the 3' end of gene n; (B) deleting efficiency histograms of four target point combination fragments of the hoxca cluster, wherein the four combinations from top to bottom respectively correspond to a first row to a fourth row in the pattern diagram (A); (C) ttn cluster four target point combination fragment deletion efficiency histogram, wherein the top four combinations correspond to the first row to the fourth row in the pattern diagram (A).

FIG. 9 is a photograph of the no dorsal fin phenotype of mutant zebrafish F1 with deletion of the hoxca cluster in the example; wherein: (A) a hoxca cluster deletion pattern map, with the front target located on the CDS of the first exon of hoxc13a and the back target located on the CDS of the second exon of hoxc1 a; (B) the integral photograph of the offspring generated by the wild type outcrossing of the hoxca fragment deletion mutant is three months old, the picture shows that the offspring is male fish with dorsal fin (left), and the genotype is subsequently identified as WT sibling; no dorsal fin phenotype, genotype subsequently identified as hoxca ^+/ -; (C) after the tail of the fish without dorsal fin phenotype is cut, a hoxca cluster double-outside primer PCR is used for amplifying an electrophoretogram, wherein the weight of the fish without dorsal fin phenotype is 1-5: in the form of a strip (star) with a backNo banding on fin sitting C1, C2, C3; (D) sequencing result, Ref is NCBI reference wild type sequence, and #3 is sequencing result of fish No. 3 in (C).

FIG. 10 shows the result of deletion of hoxcb cluster fragments in examples; wherein: (A) the hoxc13b T1 and hoxc6b T1 fragments delete the electrophoretogram with an efficiency of 14/20; (B) randomly picking the amplification product in the step (A) for sequencing result; (C) the hoxc13b T2 and hoxc6b T2 fragments delete the electrophoretogram with an efficiency of 13/20; (D) the hoxc13b T2 and hoxc6b T1 fragments delete the electrophoretogram with an efficiency of 13/20; the bottom is the result of randomly picking the amplified product in (D) for sequencing.

FIG. 11 is the result of deleting the 340kb ttn1-ttn2 ultralong fragment with high efficiency in the examples; (A) ttn 1T 5 and ttn 2T 3, T7E1 enzyme digestion detection knockout efficiency agarose gel electrophoresis picture; (B) ttn 1T 5 and ttn 2T 3 target co-injection generated fragment deletion result efficiency detection glue picture; (C) statistics of long fragment deletion efficiency for each species.

FIG. 12 is a statistical graph of the efficiency of fragment deletion for each species, where is the efficiency of fragment deletion for zebrafish by the methods of the invention, and the rest are the statistical efficiency of fragment deletion in Bacillus thuringiensis, E.coli, brewer's yeast, rice, soy, mouse, rabbit.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the drawings of the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the described embodiments of the invention without inventive step, are within the scope of protection of the invention.

In the following examples, genome large fragment deletion is performed in zebrafish ttn1-ttn2, nppa-nppb and hox gene clusters by the method for efficiently constructing genome large fragment deletion mutants according to the invention as shown in fig. 1, anti-fragment knockout is performed by taking ttn gene cluster as an example, scissors indicate the position of Cas9 cleavage, and the length of the gene cluster and the distance between two genes are marked on the figure (fig. 1, a); taking the Hox gene cluster as an example, the length of each gene cluster of Hox and the distance between the genes at two ends of each gene cluster are completely marked in the knock-out mode (FIG. 1, B); taking nppa-npb as an example, the knock-out pattern is complete with length and distance between genes at both ends of the gene cluster (FIG. 1, C)

Example 1

In this example, a zebra fish genome large fragment deletion mutant was constructed according to the method described in fig. 1, specifically as follows:

(I) a subject. The wild zebra fish is AB type, each mutant fish line is constructed by the inventor and is carried out according to the ethical relevant provisions of animals of Shanghai oceanic university (IACUC SHOU-DW-2021-042), and the experimental conditions are as follows: the zebra fish culture system has perfect zebra fish culture equipment, the living environment of the zebra fish is a circulating water system which is treated by UV and aeration, the water temperature is 28.5 ℃, the pH value and the conductivity are within normal indexes, and the illumination is dark 10h + illumination 14h every day according to the strict requirements. The zebra fish mating and spawning in a special spawning tank, the collected zebra fish spawns are placed in a constant-temperature incubator at 28.5 ℃ for culture, generally, the membrane rupture is started about 48h, water is changed once in the morning and at night before the membrane rupture and dead spawns are sucked off, water is changed once every day after the membrane rupture, paramecium is fed after 5 days, fairy shrimp is fed after 20 days, the zebra fish enters a circulating system for feeding about one month, the zebra fish is fed in tanks for one half and a half, and sex differentiation is noticed.

(II) plasmids. gRNA backbone plasmids used were from literature: chang N, Sun C, Gao L, Zhu D, Xu X, Zhu X, Xiong JW, Xi JJ. genome editing with RNA-guided Cas9 nucleic in zebrafish embryo, Cell Res,2013,23(4): 465-.

(III) main reagents. The experimental reagents are shown in table 1:

TABLE 1

Reagent	Source and goods number
		Taq enzyme	Vazyme，P213-03
Endonuclease Xba I enzyme	NEB，R0145S
		T7E1 endonuclease	Vazyme，EN303-01
MAXIscript T7	invitrogen，00688273
		mMESSAGEmMACHINE T7 ULTRA kit	invitrogen，AM1345
GenCrispr NLS-Cas9-NLS Nuclease	GenScript，Z03389-100

And (IV) main instruments. Ultraviolet spectrophotometer (Thermo Scientific company-Nanodrop 2000C), pure water instrument (Mercury company-Milli-Q Direct 8), -40 ℃ low temperature refrigerator (Haier-DW-40L 508), large Centrifuge (eppendorf company-Centrifuge 5810R), constant temperature incubator (SANYO company-MIR-262), high pressure steam sterilizer (SANYO company-MLS-3780), electrophoresis apparatus (BIO-RAD-PowerPac Basic), 4 ℃ refrigerator (Haier-HYC-610), Gel mixer (BIO-RAD company-Gel Doc EZ Imager), shaker (VORTEX-GENIE company-G560E), PCR amplification apparatus (O-company-C1000 Touch), 80 ℃ ultra low temperature refrigerator (WPE-14053: TW 32), macro capillary tube 19, model No. 5: TW-19), vertical pin drawing instrument (brand: NARISHIGE, model: PC-10).

(V) constructing large fragment deletion mutant of zebra fish genome

1. gRNA synthesis

(1) Target design

Searching a zebra fish gene sequence on an Ensembl website, searching a proper gRNA on a ZIFIT website, and after the gRNA design is completed, firstly carrying out BLAST detection specificity on an NCBI website. When the primer is sent to a company to synthesize the target point primer, whether the target point is a sense strand or an antisense strand is confirmed, and design errors are avoided. The target sequence information is shown in table 2, the T7 promoter sequence TAATACGACTCACTATA is added at the 5 'end, the gRNA backbone reverse primer sequence GTTTTAGAGCTAGAAAT is added at the 3' end, and the primers are synthesized by the company.

TABLE 2

(2) Design of detection primers

The detection primers comprise two end targets, the upstream detection primer is positioned outside the front end 200bp of the upstream target, the downstream detection primer is positioned outside the rear end 200bp of the downstream target, and the PCR amplification product is ensured to have the length of more than 400bp so as to clearly distinguish different strips. Double outer primers are selected when detecting the efficiency of the large fragment, for example, F of an upstream target and R of a downstream target form a pair of primers. The primers were synthesized by Shanghai Bioengineering Co., Ltd, and the information of the primers is shown in Table 3.

TABLE 3

(3) Synthetic gRNA in vitro transcription template

a. And (3) carrying out PCR reaction with forward primers and gRNA reverse primers corresponding to the target spots in the table 2 by taking gRNA framework plasmids as templates.

b. The PCR reaction conditions are as follows: pre-denaturation at 94 deg.C for 3 min; denaturation at 94 ℃ for 30 s; annealing at 65 ℃ for 30 s; extension at 72 ℃ for 30 s; 35 cycles were performed; then 72 ℃ for 10 min; and finally, preserving the heat in a refrigerator of 4 ℃.

c. The PCR product was detected by 120V, 20min, 2% agarose Gel electrophoresis (Gel EP) with bands slightly higher than the marker band of 100bp length.

d. Purification was carried out using a DNA purification kit, and then elution was carried out by dissolving with RNase-Free water.

e. Taking 0.5-1uL of the solution, and detecting the concentration by using a Nanodrop spectrophotometer.

(4) In vitro transcription

Under the RNase-Free condition, the PCR purified products are respectively subjected to in vitro transcription to obtain gRNAs, and a transcription system refers to the specification of an in vitro transcription kit, wherein T7 enzyme mix is added finally as shown in Table 4; the reaction conditions are as follows: water bath at 37 deg.C for 90 min; then adding 1 mu L of TURBO DNase, and carrying out water bath at 37 ℃ for 15 min.

TABLE 4

RNase free water	to 20μL
		gRNA plasmid Template	1μg
10×Transcription Buffer	2μL
		10mM ATP	1μL
10mM CTP	1μL
		10mM GTP	1μL
10mM UTP	1μL
		T7 Enzyme Mix	2μL

Mixing, centrifuging for a short time, and incubating at 37 deg.C for 80 min; then, 1. mu.L of TURBO DNase was added to the system and mixed well, centrifuged briefly and incubated at 37 ℃ for 15 min.

(5) Precipitation and purification of gRNA

After in vitro transcription, the gRNA was purified by LiCl precipitation, the procedure was:

a. adding 2 μ L of 4M LiCl into the reaction product, and then adding 200 μ L of 100% ethanol; the mixture was stored in a refrigerator at-80 ℃ for 2-12 hours (overnight treatment is possible).

b. Centrifuge at 12000rpm for 30min at 4 deg.C, pour or remove supernatant with pipette.

c. The sediment is washed by sucking precooled 70% ethanol by a pipette gun and is suspended by slightly blowing the sediment. The supernatant was then removed by centrifugation at 12000rpm for 10min at 4 ℃ and the procedure was repeated once in order to remove impurities and air dried for 5min at room temperature in a clean room.

d. Adding 10 μ L RNase-free water to dissolve.

e. Detecting the concentration of the Nanodrop; and (4) detecting the synthesis quality by electrophoresis of 1% agarose gel at 150V for 15 min.

2. Microinjection

Cas9 protein or Cas9 mRNA and gRNA are prepared into a mixed solution according to a certain proportion, and the final concentration is as follows: the gRNA is 100-200 ng/. mu.L, the Cas9 protein is 800 ng/. mu.L, the Cas9 mRNA is 400 ng/. mu.L, the gRNA is injected into an animal pole at the 1 cell stage of the zebra fish embryo, the detection can be carried out after 1nL is injected for 24h, and a part of fish eggs which are not injected need to be reserved as a control during injection.

3. T7E1 enzyme digestion detection knockout efficiency

a. And (3) taking 3 groups after 24 hours, putting a group of 5 embryos into a PCR (polymerase chain reaction) tubule, and extracting a genome by an alkaline cracking method: adding 50uL of 50mM NaOH solution into each tube, taking out after 10min at 95 ℃, and fully shaking and crushing tissues on a shaking instrument; continuing to perform heating at 95 ℃ for 10 min; after 5uL of 1M Tris-HCl (pH 8) was added, the mixture was shaken and mixed, and centrifuged at 10000rpm for 5 min.

b. PCR amplification of DNA fragments comprising a target

The target fragment was amplified using the double outer primers corresponding to the injection set in Table 3, and the PCR reaction system is shown in Table 5 below:

TABLE 5

ddH ₂ O	9.5μL
		PCR mix
2×	12.5μL
		F	1μL
R	1μL
		Template	1μL

And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30sec, annealing at 60 ℃ for 30sec, and extension at 72 ℃ for 40sec for 35 cycles; further extension for 10min at 72 ℃; storing at 12 deg.C. 2% agarose gel 120V electrophoresis for 25 min.

c. Knockout efficiency detection of T7E1 enzyme

The reaction system for the enzyme digestion detection of T7E1 is shown in Table 6:

TABLE 6

PCR product	5μL
		Reaction Buffer	1.1μL
ddH ₂ O	up to 10μL

The reaction conditions are as follows: at 95 ℃ for 30 s; 5min at 85 ℃; 4 ℃, 5 min; after the reaction, 0.25. mu. L T7E1 enzyme was added at 37 ℃ for 45 min. 120V, 25min, 2% agarose gel electrophoresis detection.

d. Agarose gel electrophoresis

Imaging the electrophoresis result by using a Bio-Rad gel camera, observing the number of the strips, wherein the WT is one strip, and the experimental group (enzyme digestion group) is 2 to 3 strips; the electropherograms were subjected to grey scale analysis using ImageLab software to calculate the knockout efficiency (fig. 2).

4. Results of the experiment

(1) "hoxa 13", "hoxa 2", "hoxc 1", "hoxc 4", "hoxc 6", "hoxc 9", "hoxc 11", "hoxc 13", "hoxc 6", "hoxc 13", "nppb", "nppa gene target efficiency T7E enzyme digestion is shown in FIG. 3, and the target efficiency enzyme digestion patterns of (T, T, T, T), hoxa13 (T, T), hoxa 2(T, T), hoxc1 (T), hoxc4 (T), hoxc6 (T), hoxc9 (T), hoxc11 (T), hoxc13 (T, T), hoxc6 (T, T), hoxc13 np (T, T), nppa (T, T, T, T) are shown from top to bottom.

(2) Statistical results of hoxc6b-hoxc13b fragment deletion efficiency and single-site efficiency

Two targets of T1 and T2 are arranged on hoxc6b, and two targets of T1 and T2 are arranged on hoxc13b (FIG. 4, A). And deleting the four combinations of the four target points to obtain the deletion efficiency. The mean single-site efficiencies of the hoxc6B T1 and hoxc13B T2 combinations were 41% and the fragment deletion efficiency was 55% (fig. 4, B), the mean single-site efficiencies of the hoxc6B T1 and hoxc13B T1 combinations were 47% and the fragment deletion efficiency was 60% (fig. 4, C), the mean single-site efficiencies of the hoxc6B T2 and the hoxc13B T2 combinations were 57.5% and the fragment deletion efficiency was 65% (fig. 4, D), the mean single-site efficiencies of the hoxc6B T2 and the hoxc13B T1 combinations were 63.5% and the fragment deletion efficiency was 70% (fig. 4, E). Statistical results show that the efficiency of segment deletion is positively correlated with the average of the unit-site efficiency.

(3) ttn1-ttn2 fragment deletion results

First, randomly selecting 6 targets from ttn clusters designed by us to verify the knockout efficiency of a single site. The selected target points are ttn 1T 1, ttn 1T 2, ttn 1T 3, ttn 2T 1, ttn 2T 2 and ttn 2T 5 respectively. Wherein ttn 1T 1, ttn 2T 5 are inefficient targets; ttn 1T 3 control group had a miscellaneous band. Therefore, ttn 1T 2, ttn 2T 1 and ttn 2T 2 are selected for fragment knockout. the column comparison of the difference between the deletion efficiency and the target efficiency of the ttn1-ttn2 cluster fragment is shown in FIG. 5, a, and the digestion efficiencies of ttn 1T 2, ttn 2T 1 and ttn 2T 2 are 46.2%, 33.3% and 44.2%, respectively (FIG. 5, b). ttn1-ttn2 cluster fragment knockout efficiency was statistically determined, and it was found that the knockout efficiency of ttn 1T 2-ttn 2T 1 fragment was 11/22 (50%), and ttn 1T 2-ttn 2T 2 fragment was 14/22 (63.6%) (FIG. 5, c).

(4) Deletion result of hoxa13b-hoxa2b fragment

Because the first exon and the last exon of the hoxa gene cluster are shorter, more targets cannot be found to accurately delete the whole gene cluster, two targets are respectively arranged at two ends for screening, and the targets are hoxa2b T1, hoxa2b T2, hoxa13b T1 and hoxa13b T2. The bar comparison of the difference between the deletion efficiency and the target efficiency of the hoxa13b-hoxa2b cluster fragment is shown in fig. 6, a, the cleavage efficiencies of the hoxa2b T1, the hoxa2 1T 1, the hoxa13 1T 1 and the hoxa13 1T 1 are respectively 84.8%, 54.5%, 59.5% and 65.8% (fig. 6, b), the knock-out efficiencies of the hoxa13 1-hoxa 21 clusters are counted, and the results show that the knock-out efficiencies of the hoxa13 1T 1 and the hoxa2 1T 1 are 1 (45%), the knock-out efficiencies of the hoxa13 1T 1, the hoxa2 1T 1 are 1 (75%), the hoxa13 1T 1 and the hoxa2 1T 1 are 1 (85%), the hoxa 13T 1 and the hoxa 2T 1 are 1 (100%).

(5) Statistical result of deletion efficiency of different-length fragments of hoxca gene cluster

Four deletion patterns of 55.7Kb, 71.4Kb, 91.6Kb and 133.9Kb were set for the hoxca gene cluster (FIG. 7, A), and the efficiency of fragment deletion was examined after double outer primer PCR (FIG. 7, B), and the efficiency of fragment deletion was hoxc1a-hoxc4a: 65%, hoxc1a-hoxc6a: 70%, hoxc1a-hoxc9a: 35%, and hoxc1a-hoxc13a: 60%, respectively. The single site efficiencies of five targets used on the hoxc1a, hoxc4a, hoxc6a, hoxc9a, and hoxc13a genes were also counted (FIG. 7, C), and the single site efficiencies of hoxc1a: 57%, hoxc4a: 88%, hoxc6a: 56%, hoxc9a: 69%, and hoxc13a: 72%. Histograms of different fragment deletions were counted (fig. 7, D). It was found by means of line graph statistics that the segment deletion length does not affect the segment deletion efficiency, i.e. the segment deletion efficiency is not related to the segment deletion length (fig. 7, E).

(6) Comparison of multiple target sites with the efficiency of deletion of a pair of target site fragments for fragment deletion patterns with different target site combinations (fig. 8, a), first row: the target point A1 is located at the 5 'end of gene1, and the target point A2 is located at the 3' end of gene n; a second row: the target point A1 is located at the 5 'end of gene1, and the target points A3 and A2 are located at the 3' end of gene n; third row: targets A1 and A4 are located at the 5 'end of gene1, and target A2 is located at the 3' end of gene n; fourth row: targets A1, A4 are located at the 5 'end of gene1, and targets A3, A2 are located at the 3' end of gene n.

The four target point combination fragment deletion efficiencies of the hoxca cluster (fig. 8, B) correspond to the first row to the fourth row in the pattern diagram from top to bottom respectively. ttn histogram of deletion efficiency of four target combinations in cluster (FIG. 8, C). The four combinations from top to bottom correspond to the first to fourth rows in the pattern diagram (a), respectively.

(7) Mutant zebrafish deleted of hoxca cluster produce a dorsal fin-free phenotype

The hoxca cluster deletion pattern front target is located on the CDS of the first exon of hoxc13a and the back target is located on the CDS of the second exon of hoxc1a (fig. 9, a).

The overall picture of the offspring generated by the wild type outcrossing of the hoxca fragment deletion mutant, three months old, is shown as male fish (figure 9, B), has dorsal fin (left), and the genotype is subsequently identified as WT sibling; no dorsal fin phenotype, genotype subsequently identified as hoxca ^+/ -。

After tail snipping of fish without dorsal fin phenotype, electropherograms were amplified using a hoxca cluster of double outside primers (fig. 9, C). 1-5 of fish without back fin, which is in the shape of a strip (marked with a star), and has no strips of back fin filing C1, C2 and C3.

Ref is the NCBI reference wild type sequence (FIG. 9, D) and #3 is the sequencing result of fish number 3 in (C) (FIG. 9).

(8) hoxcb cluster fragment deletion results

The efficiency of deletion electrophoresis of the hoxc13B T1 and the hoxc6B T1 fragments is 14/20 (FIG. 10, A), and amplification products are randomly picked for sequencing results (FIG. 10, B); the efficiency of the hoxc13b T2 and hoxc6b T2 fragment deletion electrophoresis was 13/20 (fig. 10, C); the efficiency of deletion electrophoresis of the hoxc13b T2 and the hoxc6b T1 fragments is 13/20 (FIG. 10, D), and the lower part of the D picture shows the results of randomly picking amplification products for sequencing.

(9) High-efficiency fragment deletion result of 340kb ttn1-ttn2 cluster

the cleavage efficiency of ttn 1T 5 was 89.2%, and that of ttn 2T 3 was 75.1% (FIG. 11, A). Ttn 1T 5 and ttn 2T 3 and Cas9 protein were co-injected, 48h embryos were randomly picked for double outer primer PCR amplification, and the knockout efficiency was detected to be 95% (20/20) (FIG. 11, B). This fragment deletion is the longest fragment deletion in the present invention, and the fragment deletion length (i.e., the distance between two targets) reaches 340kb (FIG. 11, C, labeled column), is longer than that performed in other species, and is more efficient than that (FIG. 11, C).

(10) Statistical fragment deletion efficiency results

The length of the longest fragment ttn1-ttn2 for fragment deletion reaches 340Kb, and the deletion efficiency is 100 percent (FIG. 11, C); the average efficiency of fragment deletion reached 64.5% (figure 12,) which was higher than the average efficiency of fragment deletion in other species.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.

Sequence listing

<110> Shanghai ocean university

<120> method for efficiently constructing genome large fragment deletion mutant and application

<141> 2022-06-30

<160> 153

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ggaggccctg gcaacagaag 20

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ggaagcagca gctccggggg 20

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ggggaaggct ggatcgcata 20

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cattgatctc cattacaacc 20

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ggcggaaaga gaagtaaagg 20

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ggggtgtttc gggagcaaac 20

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ggaagacagt aaaaaaaggg 20

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<400> 8

gggtagtgct gcaacgttcg 20

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<400> 9

ccaatggtgc tggacaagcc 20

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ggatggtgat ttggctcata 20

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<212> DNA

<213> Artificial Sequence

<400> 11

atcagaccag tgaagtctcc 20

<210> 12

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 12

gggcccaacc gttctcacct 20

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gggccatgat gtctttttaa 20

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ggatagatta aacctgaggt 20

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<212> DNA

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<400> 15

caacaggtag agaggctccc 20

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<211> 20

<212> DNA

<213> Artificial Sequence

<400> 16

tattgagaca accgaggacc 20

<210> 17

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 17

ggtgtgaaag agaccgatgc 20

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<400> 18

gagcgctgtt tgcatttacc 20

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<212> DNA

<213> Artificial Sequence

<400> 19

aaattcctcc ataacacc 18

<210> 20

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 20

ggttcattgt atacagtgcg 20

<210> 21

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 21

ggtgtttcct agcgtggtgc 20

<210> 22

<211> 20

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<400> 22

aacccgtcac ccactgttcc 20

<210> 23

<211> 20

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<213> Artificial Sequence

<400> 23

gcttacacct ctcagatgcc 20

<210> 24

<211> 20

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<400> 24

ggttctgctt gggccggctg 20

<210> 25

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 25

ccattcccgc tggattgacc 20

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<211> 20

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<213> Artificial Sequence

<400> 26

agacatgaat ctctactgcc 20

<210> 27

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 27

acacgcacag catcagttcc 20

<210> 28

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 28

ggcaagacac ttagaaaaat 20

<210> 29

<211> 20

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<400> 29

atgatttgac ccgccatccc 20

<210> 30

<211> 20

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<400> 30

ggcccacggc tctcaatatt 20

<210> 31

<211> 19

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<400> 31

ggctgttaga gctctttac 19

<210> 32

<211> 20

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<400> 32

ggaactcttc gttcaggtga 20

<210> 33

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 33

ggggattttc ccgcctgacc 20

<210> 34

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 34

tgcaagggac agtggctacc 20

<210> 35

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 35

ccccttcggc tgcatcttcc 20

<210> 36

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 36

acatgccact gaactcgacc 20

<210> 37

<211> 20

<212> DNA

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<400> 37

aaaaccggat ttacgcgtcc 20

<210> 38

<211> 20

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<400> 38

atatcctcca gctcacgccc 20

<210> 39

<211> 20

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<400> 39

ggagactgtt tgggctcgaa 20

<210> 40

<211> 20

<212> DNA

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<400> 40

ggtcctgcat ccacgctggg 20

<210> 41

<211> 20

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<213> Artificial Sequence

<400> 41

ggcaccatag cgactcacca 20

<210> 42

<211> 20

<212> DNA

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<400> 42

cgtcacgatg cattgatccc 20

<210> 43

<211> 20

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<400> 43

agacgagcag cgctttgccc 20

<210> 44

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 44

ctttaagaat ggctgtctcc 20

<210> 45

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 45

cttagcttga atccttcccc 20

<210> 46

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 46

atgcctgggc aacgaaaacc 20

<210> 47

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 47

ggcaagacta tggaaccgaa 20

<210> 48

<211> 20

<212> DNA

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<400> 48

ggtaggagct ggccaagaca 20

<210> 49

<211> 20

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<400> 49

gggagcctcg cgagtcacca 20

<210> 50

<211> 20

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<400> 50

ggcctcgtcg gggacttcac 20

<210> 51

<211> 20

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<400> 51

tctttgtcta cgtttccacc 20

<210> 52

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 52

accagccgcg atcaatcata 20

<210> 53

<211> 20

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<400> 53

ggttagctgc aatacgcgga 20

<210> 54

<211> 19

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<400> 54

agaaaccagc cgcgatcaa 19

<210> 55

<211> 20

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<400> 55

gctgcaatac gcggatacag 20

<210> 56

<211> 20

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<400> 56

accgcttagt catgttgggg 20

<210> 57

<211> 22

<212> DNA

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<400> 57

cgcatcaaga taagtgcaca aa 22

<210> 58

<211> 20

<212> DNA

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<400> 58

cattcccgct tcaaagcaca 20

<210> 59

<211> 22

<212> DNA

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<400> 59

caggtcccaa gaagaactga ag 22

<210> 60

<211> 20

<212> DNA

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<400> 60

ttcacattcc cgtagtcggc 20

<210> 61

<211> 22

<212> DNA

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<400> 61

aggtcccaag aagaactgaa gt 22

<210> 62

<211> 20

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<400> 62

agccgacaaa tctgaaccga 20

<210> 63

<211> 20

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<400> 63

acaaacgacg acatgggtgt 20

<210> 64

<211> 19

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<400> 64

gctgggggtg caagtgatt 19

<210> 65

<211> 20

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<400> 65

ccagttccag aggtgagctg 20

<210> 66

<211> 21

<212> DNA

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<400> 66

aggggcatat aagagcaagc a 21

<210> 67

<211> 20

<212> DNA

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<400> 67

acacaaggga gccaagtgag 20

<210> 68

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 68

cgccaggcta gggttgaaaa 20

<210> 69

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 69

tacaaacaag cagcccaggg 20

<210> 70

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 70

gctaccaaga cttgcacgag 20

<210> 71

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 71

ccccagaatt attagcgccc 20

<210> 72

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 72

ccaattggga gtggcttgga 20

<210> 73

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 73

taagggaatg ggcagcagag 20

<210> 74

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 74

attgttgctg aggcgctaga 20

<210> 75

<211> 20

<212> DNA

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<400> 75

aagtcatgct gtgggcaatg 20

<210> 76

<211> 21

<212> DNA

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<400> 76

gctgtgggca atgtgtcaat g 21

<210> 77

<211> 22

<212> DNA

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<400> 77

tagacaaacg cagtgacttg aa 22

<210> 78

<211> 21

<212> DNA

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<400> 78

agccgaagtt aacggttatg g 21

<210> 79

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 79

aatcctgtgg gcatgtgcac 20

<210> 80

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 80

cccttcacag tgctcaaggt a 21

<210> 81

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 81

caccccttcc tacgttaagc a 21

<210> 82

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 82

gttcttaccg tggcttgtgc 20

<210> 83

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 83

gccgaagtta acggttatgg a 21

<210> 84

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 84

agccgaagtt aacggttatg g 21

<210> 85

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 85

aatcctgtgg gcatgtgcac 20

<210> 86

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 86

cttgcaaaag gtgcaaggtg a 21

<210> 87

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 87

attaaagccg gaggcctcac 20

<210> 88

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 88

aggattggcc tctcctggaa 20

<210> 89

<211> 20

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<400> 89

acggtttgag tttgagggct 20

<210> 90

<211> 20

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<213> Artificial Sequence

<400> 90

aggattggcc tctcctggaa 20

<210> 91

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 91

acggtttgag tttgagggct 20

<210> 92

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 92

gactcagaac ggcgtacact 20

<210> 93

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 93

tttgtatcgc tcgacggcaa 20

<210> 94

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 94

acaatgtatc aggggcgctt 20

<210> 95

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 95

tttgtatcgc tcgacggcaa 20

<210> 96

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 96

tacgaattcg agcgagagac g 21

<210> 97

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 97

gtatagtgga ccagggtcgg 20

<210> 98

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 98

gtgcctgctc tcgtccaata 20

<210> 99

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 99

cggcagctat tacccgtgta 20

<210> 100

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 100

tgttcggccg tgcaaaatac 20

<210> 101

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 101

cggagacaca ccattgtgtt 20

<210> 102

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 102

tgcatgtctc gcttgtgcat 20

<210> 103

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 103

agcactcagc acttctagcc 20

<210> 104

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 104

agcactcagc acttctagcc 20

<210> 105

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 105

catagaacca cccaccacca 20

<210> 106

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 106

cacttcaaag tggtccttct gtg 23

<210> 107

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 107

tgtcacccaa caatgcagag a 21

<210> 108

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 108

actgtcaccc aacaatgcag a 21

<210> 109

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 109

ttgtctcttt ccacaaatgg ct 22

<210> 110

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 110

atagcatacc tgccaacaca ct 22

<210> 111

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 111

agcaagacaa ccagagcatg 20

<210> 112

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 112

aaacagctgt cagtggagca 20

<210> 113

<211> 23

<212> DNA

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<400> 113

gttagcactt ttgcaatccc tga 23

<210> 114

<211> 20

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<400> 114

acaccgaaag cagcaagtca 20

<210> 115

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 115

tttacgacag gccatgacgg 20

<210> 116

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 116

ctttacaggg ttgctcgcta gt 22

<210> 117

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 117

taactctctc tctccgtctg cc 22

<210> 118

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 118

gcgtcagtat aactgtgcaa gc 22

<210> 119

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 119

aggcattggt aattacaccc ac 22

<210> 120

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 120

acaagctcga tattcctccg ta 22

<210> 121

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 121

gaggaccgta cgagtatgga tc 22

<210> 122

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 122

gagagggcga taggaaaggt at 22

<210> 123

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 123

ttttatggcc tcgtaaacca ct 22

<210> 124

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 124

ggatgcatgt aaaagagaag gg 22

<210> 125

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 125

agccgtcgac attagagaaa ag 22

<210> 126

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 126

tccatacact caccaacctc ac 22

<210> 127

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 127

tctcctaaga agccaagtct gg 22

<210> 128

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 128

aacccactct cttcgggcta 20

<210> 129

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 129

tctgagttac ggctacccct 20

<210> 130

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 130

aacccactct cttcgggcta 20

<210> 131

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 131

gctcttacca ggctgttcca 20

<210> 132

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 132

gttacggcta cccctttgga 20

<210> 133

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 133

acaggttatg actgcgcatc c 21

<210> 134

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 134

tcaccaggga tccgtgtact 20

<210> 135

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 135

cgcatccact agctctgcat 20

<210> 136

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 136

tcgagctgtt attgtatcca ctc 23

<210> 137

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 137

tatgacgact tcgctggtcc 20

<210> 138

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 138

tcgagctgtt attgtatcca ctc 23

<210> 139

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 139

tatgacgact tcgctggtcc 20

<210> 140

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 140

tcgagctgtt attgtatcca ctc 23

<210> 141

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 141

tatgacgact tcgctggtcc 20

<210> 142

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 142

ctatgacccg gtgcgacatt 20

<210> 143

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 143

aacgtcttga tgccttgcag 20

<210> 144

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 144

ctcacaggag ttggctatgg g 21

<210> 145

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 145

gtgtgttgct tcgccactt 19

<210> 146

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 146

agcatcgaca acccatgacc 20

<210> 147

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 147

tggctatttg gacgtcccag 20

<210> 148

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 148

tgggcggaca cactgattta 20

<210> 149

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 149

atccaggcat gaagcgttgt 20

<210> 150

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 150

caaagagcgt tcgtgttggt ag 22

<210> 151

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 151

gcttcttttg gtaacgtcga aact 24

<210> 152

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 152

accagccgcg atcaatcata 20

<210> 153

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 153

acaatccagc ctgatctcac g 21

Claims

1. The method for efficiently constructing the genome large fragment deletion mutant is characterized by comprising the following steps of:

s5, step S4 gRNA and Cas9 protein or Cas9 mRNA are respectively injected into a fertilized egg at one cell stage of the treated object by microinjection;

s6, selecting the ovum of the processed object in the embryonic stage in the step S5 to extract genome, and respectively carrying out PCR amplification by using a knock-out target detection primer to obtain PCR products;

2. The method for efficiently constructing the deletion mutant of the large genome fragment according to claim 1, wherein in step S8, specific CRISPR targets with high T7E1 enzyme activity and similarity are selected from specific CRISPR targets at two ends of a gene fragment to be deleted as an optimal target combination.

3. The method for efficiently constructing the genome large-fragment deletion mutant according to claim 1, wherein the method for efficiently constructing the genome large-fragment deletion mutant simultaneously satisfies the following conditions:

4. The method for efficiently constructing genome large-fragment deletion mutants according to claim 1, wherein in step S6, the knockout target detection primer is a specific primer capable of amplifying a gene containing the target.

5. The method for efficiently constructing the genome large-fragment deletion mutant according to claim 1, wherein in the step S5, the final concentration of gRNA is 100-200 ng/μ L, the final concentration of Cas9 protein is 800ng/μ L, the final concentration of Cas9 mRNA is 400ng/μ L, and the injection amount is 1 nL.

6. The method for efficiently constructing large genomic fragment deletion mutants according to any one of claims 1 to 5, wherein large genomic fragment deletion is performed in the zebrafish ttn1-ttn2, nppa-nppb and hox gene clusters by the method for efficiently constructing large genomic fragment deletion mutants according to any one of claims 1 to 5, comprising the steps of:

(5) microinjecting the gRNA obtained in the step (4) and Cas9 protein or Cas9 mRNA into a fertilized egg at a cell stage of the zebra fish;

7. The method for efficiently constructing large genomic fragment deletion mutants according to claim 6, wherein in the step (1), the target sequences are shown in the following table:

8. the method for efficiently constructing large genomic fragment deletion mutants according to claim 6, wherein in the step (2), the sequences of the amplification primers are shown in the following table:

9. use of the method of any one of claims 1-8 for efficient construction of large genomic deletion mutants for gene deletion in the genome of a vertebrate.

10. Use of the method for efficiently constructing large genomic fragment deletion mutants according to any one of claims 1 to 8 in constructing animal models for disease treatment and drug screening caused by large genomic fragment deletion.