阅读说明：本技术 一种基因编辑选育ppm1g基因突变型斑马鱼的方法 (Method for breeding ppm1g gene mutant zebra fish by gene editing ) 是由 邓云 欧阳诗 徐婧怡 曹秋香 陈坤 于 2019-12-18 设计创作，主要内容包括：本发明涉及基因敲除技术领域,特别是公开了一种基因编辑选育ppm1g基因突变型斑马鱼的方法,通过CRISPR/Cas9基因编辑技术,在斑马鱼的ppm1g基因上设计合适的打靶位点,在体外合成的特异性sgRNA和Cas9-mRNA,显微共注射进入斑马鱼一细胞内,胚胎培养50h后,通过选取胚胎进行基因型分析,从而证实了所选位点的有效性。本发明能更高效且更精确地在生物体基因组中突变特定基因,且制作简单、成本低,且可同时对靶基因上多个位点进行剪切,突变任意数目的单个基因。且对ppm1g基因表达进行干扰,并且通过遗传学手段研究其功能,有助于进一步揭示心脏发育的整个过程以及调控这些过程的分子机制,在医学上心脏疾病病理的理解和新的治疗方案的研发中具有十分重要的意义。(The invention relates to the technical field of gene knockout, and particularly discloses a method for breeding ppm1g gene mutant zebra fish by gene editing, wherein a CRISPR/Cas9 gene editing technology is adopted, a proper targeting site is designed on a ppm1g gene of the zebra fish, specific sgRNA and Cas9-mRNA synthesized in vitro are injected into a zebra fish cell in a micro-co-injection manner, and after 50h of embryo culture, an embryo is selected for genotype analysis, so that the effectiveness of the selected site is verified. The invention can more efficiently and accurately mutate specific genes in organism genomes, has simple manufacture and low cost, and can simultaneously cut a plurality of sites on a target gene and mutate any number of single genes. The interference on the expression of the ppm1g gene is realized, and the function of the gene is researched by a genetic means, so that the gene is helpful for further disclosing the whole process of cardiac development and the molecular mechanism for regulating and controlling the processes, and has very important significance in understanding the pathology of the cardiac disease and developing a new treatment scheme in medicine.)

1. A method for breeding a ppm1g gene mutant zebra fish by gene editing is characterized by comprising the following steps:

1) respectively designing CRISPR/Cas9 gene knockout target sites and detection primers

Inquiring The genome DNA sequence of zebrafish ppm1g gene on a National Center for Biotechnology Information (NCBI), analyzing The functional structural domain on a website SMART (http:// smart.embl-heidelberg. de /), and designing The target site of ppm1g gene on The website The ZiFiT target (http:// ZiFiT. partners. org/ZiFiT /) according to The CRISPR/Cas knockout principle;

two pairs of specific target site PCR primers were as follows:

f1 (target site a forward primer):

TGTAATACGACTCACTATAGgaagccactatgacaatcgGTTTTAGAGCTAGAAATAGC

f2 (target site b forward primer):

TGTAATACGACTCACTATAGGtttggttctgtccgtagcGTTTTAGAGCTAGAAATAGC

r (common reverse primer): AAGCACCGACTCGGTGCCACT

PCR detection primer

F (5’-CAGGACACTGTTCAAATTCCTTC-3’)

R (5’-GACTTTTCAGCTGATCCTTCAGA-3)；

2) Construction of gRNA expression vector and in vitro synthesis of specific gRNA

Firstly, cloning a gRNA framework onto a p42250 vector;

b, in vitro synthesis of specific gRNAs

This plasmid was linearized with BsaI restriction endonuclease

In general, the total volume of the digestion reaction is 20. mu.L, and the system is as follows:

p42250 vector 2. mu.L

10× Buffer 2μL

BsaI restriction enzyme 1.5. mu.L

ddH2O 14.5μL

Total volume 20. mu.L

Mixing, performing enzyme digestion in 37 deg.C water bath for more than 4 hr

c, taking the linearized p42250 vector as a template, and carrying out PCR through the following specific primers to amplify the double-stranded DNA for synthesizing the specific gRNA

Forward specific target site primer F1 or F2: the T7 promoter +20pb target sequence 20bp + sgRNA upstream backbone; reverse primer R: 20bp sgRNA downstream backbone

The PCR reaction (50. mu.L) was as follows:

ddH2O 18 μL

10 Xbuffer (20 mM Mg2 +) 25. mu.L

dNTPs（10 mM） 1 μL

Primer F1 (or F2, 10 uM) 2 uL

Primer R（10 uM） 2 μL

Template (p 42250 vector) 1. mu.L

Ex-Taq DNA polymerase 1. mu.L

Total volume 50. mu.L

After shaking and mixing, centrifuging at 4 ℃, and carrying out amplification reaction on a PCR instrument

After the reaction is finished, centrifuging the PCR product, taking 1 microliter of sample to sample on 1.8% agarose gel for electrophoresis, and taking a result by a gel imaging system

d, detecting and determining that the band is correct, then carrying out agarose gel DNA recovery, and purifying and recovering a PCR product;

e, determining the concentration of the purified DNA, taking the DNA as a template, and carrying out in-vitro transcription by using a 20 mu L system to synthesize the specific gRNA

The Tip head and the EP tube used in the transcription experiment are both products of DEPC-treated RNase-Free, and the specific operation is as follows:

in vitro transcription reaction system (20 μ L):

template DNA 12. mu.L

10×Buffer 2 μL

rATP（10 mM） 1 μL

rUTP（10 mM） 1 μL

rCTP（10 mM） 1 μL

rGTP（10 mM） 1 μL

T7 RNA polymerase 2. mu.L

Total volume 20. mu.L

Adding the reactants into an EP (advanced technology attachment) tube with 1.5mL of RNase-Free, uniformly mixing, placing the mixture in a water bath kettle with the temperature of 37 ℃ for reaction for 2.5h, adding 1 mu L of DNase into a transcription system, placing the mixture in the water bath kettle with the temperature of 37 ℃ for reaction for 30min to digest a DNA template, taking 1 mu L of gRNA sample, and carrying out agarose gel electrophoresis to detect a transcription result, wherein if the size of a transcription product is consistent with that of an expected product, the successful transcription is indicated;

f, purification of specific gRNA

Purifying successfully transcribed gRNA by using RNeasy Mini kit, and storing at-20 ℃; taking 1 mu L of the purified gRNA solution to determine the concentration;

3) microinjection of zebrafish embryos

Within 30min after fertilization, the embryos were pipetted and transferred to a microinjection petri dish made of agarose

Before microinjection, Cas9 mRNA and gRNA are prepared into a mixed solution and are fully and uniformly mixed, so that the final concentration of Cas9 protein is 5 mug/muL, and the final concentration of gRNA is 30-40 ng/muL

Injecting a mixture of Cas9 protein and gRNA at about 1.8nL into fertilized egg at one cell stage

The injected fertilized eggs are placed in E3 water and incubated at 28 DEG C

Observing embryo phenotype under body type microscope, screening normally developed embryo for target site mutation analysis

Microinjection systems were as follows:

cas9 protein 5 μ g/. mu.L

gRNA 30-40 ng/μL

Nuclease free H2O

Total 3μL

4) Sanger sequencing to test the effectiveness of target sites

After microinjection is carried out on zebra fish embryos, partial early embryos which are normally developed are selected, and whether the ppm1g gene of the early embryos has mutation or not is detected;

a, extracting zebra fish genome

After the zebra fish embryo is fertilized for 50 hours (50 hpf), respectively collecting the wild type and the injected embryo in a 1.5mL Ep tube, extracting genome DNA according to the following method by 5 embryos per tube, and the specific steps are as follows:

adding 100 mu L of cell lysate and 1 mu L of proteinase K into an Ep tube filled with embryos, and placing the Ep tube in a water bath kettle at 55 ℃ for lysis overnight;

after cracking, placing the mixture on an oscillator for full oscillation, adding isopropanol which is cooled in advance in the same volume into an Ep tube, fully and evenly mixing the mixture in a reversed way, centrifuging the mixture for 10min at 12000rpm under the condition of 4 ℃, and pouring out supernate;

adding 500 μ L of 75% ethanol, centrifuging at 4 deg.C and 12000rpm for 5 min, removing supernatant, and air drying at room temperature for 8-10 min;

adding 10 mu L of deionized water, fully and uniformly blowing, and detecting the extraction efficiency by agarose gel electrophoresis;

b, PCR amplification of the target sequence

After extracting genome DNA, designing a primer sequence by using PrimerPremier 5.0 software according to a genome region of about 100-250bp upstream and downstream of a CRISPR target site to amplify a target DNA fragment;

the PCR reaction system is as follows:

2×Es Taq MasterMix 10 μL

Primer F (10 μM) 0.6 μL

Primer R (10 μM) 0.6 μL

template (genomic DNA) 1. mu.L

ddH2O 7.8 μL

Total volume 20. mu.L

After shaking and mixing evenly, centrifuging at 4 ℃, and carrying out amplification reaction on a PCR instrument;

c, detecting the PCR product by using 1.8% agarose gel electrophoresis;

d, sending the target DNA fragment after partial purification to perform Sanger sequencing, and preliminarily obtaining the information of insertion or deletion according to a sequencing peak map;

e, after two months of injection, carrying out tail shearing identification, which is the same as the identification step;

5) TA cloning of sequences of interest

Performing Sanger sequencing on the target sequence which is preliminarily identified to have mutation possibility by PCR, if a sequencing peak diagram has double peaks and a sequencing result shows that the target sequence has an insertion or deletion phenomenon, and then performing TA cloning and then selecting a monoclonal for further detection;

6) sanger sequencing of bacterial solutions

The bacteria liquid with the strip size which is displayed by the PCR result of the bacteria liquid and accords with the expected result is sent to sequencing, the bacteria liquid is compared with a standard target sequence on NCBI according to a peak diagram and a sequence which are given after the sequencing, and the mutation type of each monoclonal is analyzed according to the comparison result;

7) generation F1 to obtain heritable zebrafish mutants

Determining zebra fish mutant F0 generation through a series of screening, respectively hybridizing the mutant F0 generation with wild zebra fish to obtain F1 generation embryos, culturing at 28 ℃, carrying out hereditary identification on the zebra fish F1 generation, and respectively taking 5 embryos from each mutant F1 generation for mutation hereditary identification two days after fertilization; extracting genome of each embryo independently, amplifying a region near a target site with 612bp by PCR, performing Sanger sequencing, and if the mutation can be inherited to F1 generation, wherein a sequencing peak diagram has double peaks; culturing F1 generation of the zebra fish mutant for 2-3 months, respectively carrying out tail shearing, PCR, TA cloning and other steps on each F1 generation of zebra fish adult fish, sequencing the positive bacterial liquid, and knowing through a sequence comparison result that a sequence of 11bp is inserted into the position of a target site a.

2. The method for breeding the ppm1g gene mutant zebra fish by gene editing according to claim 1, wherein the selection of the target site in the step 1) follows the following criteria: 5 '-GG- (N) 18-NGG-3'; wherein the 5 'GG dinucleotide is part of the T7 promoter, ensuring that the 3' end of the target site is NGG; the selected location of the target is within the domain of the gene.

3. The method for breeding zebrafish with ppm1g mutant gene by gene editing according to claim 1, wherein the Tip head and EP tube used in the transcription experiment in step 2) are DEPC-treated RNase-Free.

4. The method for selectively breeding the zebra fish with the ppm1g gene mutation by gene editing according to claim 1, wherein the step c of step 2) is an amplification reaction carried out on a PCR instrument under the following reaction conditions: pre-denaturation at 95 ℃ for 5 min, and repeat the following steps for 30 cycles: denaturation at 95 ℃ for 15 s, annealing at 60 ℃ for 15 s, extension at 72 ℃ for 45 s, and then at 72 ℃ for 8 min; after the reaction is finished, centrifuging the PCR product, and performing electrophoresis.

5. The method for breeding zebrafish with ppm1g gene mutation by gene editing according to claim 1, wherein the E3 water in step 3) is a mixture of 5 mmol/L NaCl, 0.33mmol/L CaCl2, 0.33mmol/L MgSO4 and 0.17mmol/L KCl.

6. The method for selectively breeding the zebra fish with the ppm1g gene mutation by gene editing according to claim 1, wherein the amplification reaction is performed on a PCR instrument in step b) of step 4) under the following reaction conditions: pre-denaturation at 95 ℃ for 5 min, and repeat the following steps for 30 cycles: denaturation at 95 ℃ for 30 s, annealing at 60 ℃ for 30 s, extension at 72 ℃ for 45 s, and then at 72 ℃ for 8 min; after the reaction is finished, centrifuging the PCR product, and performing electrophoresis.

Technical Field

The invention relates to the field of gene knockout, and particularly discloses a method for breeding ppm1g gene mutant zebra fish by gene editing.

Background

The ppm1G (protein phosphatase, Mg2+/Mn2+ dependent, 1G) gene is located on No. 13 chromosome of zebra fish and comprises 10 exons, the full length of cDNA is 1488bp, and meanwhile, through gene differential expression profiling analysis, genome correlation analysis and the like, the ppm1G gene is found to be expressed in a plurality of tissues in the early stage of human embryo, particularly, the gene is closely related to the neural development of the zebra fish.

The zebra fish has high homology with genes and signal paths in the human heart development process, the ppm1g gene is more conservative in evolution, and researches show that the ppm1g has higher expression level in early embryo of the zebra fish. Moreover, compared with other animal models, the zebra fish has the advantages of small size, easy feeding, fast development, strong reproductive capacity, in-vitro fertilization, embryo in-vitro development, transparency and the like.

Through a CRISPR/Cas9 gene editing technology, a proper targeting site is designed on a ppm1g gene of zebra fish, specific sgRNA and Cas9 protein synthesized in vitro are injected into a zebra fish cell in a micro-co-injection mode, and after an embryo is cultured for 50 hours, the effectiveness of the targeted site is identified by selecting the embryo to perform genotype analysis.

Gene targeting technology originated in the end of the 20 th century 80 s, is an important means for studying gene function by site-directed modification of genome, and can also be used for treating various genetic diseases of human. The technology mainly utilizes modes such as deletion mutation, gene inactivation, chromosome large fragment deletion, exogenous gene introduction and the like to change the genetic information of organisms, and stably expresses mutation characters after inheritance in a germ line, so that the function of specific genes in the organisms in the growth and development process is researched, and the technical means become a research hotspot of modern molecular biology. The traditional gene targeting technology is based on the Embryonic Stem Cell (ESC) and homologous recombination technology, so the targeting technology has extremely low efficiency. In the beginning of 2013, a brand new artificial endonuclease clustered regulated short palindromic repeats (CRISPR)/CRISPR-associated (Cas)9 can mutate a specific gene in an organism genome more efficiently and more accurately, is simple to manufacture and low in cost, can simultaneously shear a plurality of sites on a target gene, mutates any number of single genes, and has certain defects and relatively high off-target rate.

Disclosure of Invention

In order to overcome the defects of the technical problems, the invention provides a method for breeding ppm1g gene mutant zebra fish by gene editing, finds out a proper targeting site, and breeds ppm1g gene mutant zebra fish by a CRISPR/Cas9 gene editing technology.

The technical scheme for solving the technical problems is as follows:

1) design CRISPR/Cas9 gene knockout target site and detection primer

The genomic DNA sequence of zebrafish ppm1g gene was queried on The National Center for Biotechnology Information (NCBI), its functional domain was analyzed on The website SMART (http:// smart.embl-heidelberg. de /), and The target site of ppm1g gene was designed on The website The ZiFiT target (http:// ZiFiT. partners. org/ZiFiT /) according to The CRISPR/Cas knockout principle. The selection of the target must follow this criterion: 5 '-GG- (N) 18-NGG-3'. The GG dinucleotide at the 5 'end is part of the T7 promoter, and the target site can be designed without limitation, but the NGG at the 3' end of the target site must be ensured. The target site must be selected within the domain of the gene to ensure that the insertion or deletion of the target site base can affect the entire domain of the ppm1g gene to alter gene expression

Two pairs of specific target site PCR primers were as follows:

f1 (target site a forward primer):

TGTAATACGACTCACTATAGgaagccactatgacaatcgGTTTTAGAGCTAGAAATAGC

f2 (target site b forward primer):

TGTAATACGACTCACTATAGGtttggttctgtccgtagcGTTTTAGAGCTAGAAATAGC

r (common reverse primer): AAGCACCGACTCGGTGCCACT

PCR detection primer

F (5’-CAGGACACTGTTCAAATTCCTTC-3’)

R (5’-GACTTTTCAGCTGATCCTTCAGA-3)；

2) Construction of gRNA expression vector and in vitro synthesis of gRNA

a, first cloning the gRNA backbone onto a p42250 vector

b, in vitro synthesis of specific gRNAs

This plasmid was linearized with BsaI restriction endonuclease. In general, the total volume of the digestion reaction is 20. mu.L, and the system is as follows:

p42250 vector 2. mu.L

10× Buffer 2μL

BsaI restriction enzyme 1.5. mu.L

ddH2O 14.5 μL

Total volume 20. mu.L

Mixing, performing enzyme digestion in 37 deg.C water bath for more than 4 hr

c, taking the linearized p42250 vector as a template, and carrying out PCR through the following specific primers to amplify the double-stranded DNA for synthesizing the specific gRNA

Forward specific target site primer F1 or F2: the T7 promoter +20pb target sequence 20bp + sgRNA upstream backbone; reverse primer R: 20bp sgRNA downstream backbone

The PCR reaction (50. mu.L) was as follows:

ddH2O 18 μL

10 Xbuffer (20 mM Mg2 +) 25. mu.L

dNTPs（10 mM） 1 μL

Primer F1 (or F2, 10 uM) 2 uL

Primer R（10 uM） 2 μL

Template (p 42250 vector) 1. mu.L

Ex-Taq DNA polymerase 1. mu.L

Total volume 50. mu.L

After shaking and mixing, the mixture was centrifuged at 4 ℃ and subjected to amplification reaction on a PCR instrument. After the reaction is finished, centrifuging the PCR product, taking 1 microliter of sample to sample on 1.8% agarose gel for electrophoresis, and taking a result by a gel imaging system

d, detecting and determining that the band is correct, then carrying out agarose gel DNA recovery, and purifying and recovering a PCR product;

e, measuring the concentration of the purified DNA, and taking the DNA as a template to perform in vitro transcription by using a 20 mu L system to synthesize the specific gRNA. The Tip head and the EP tube used in the transcription experiment are both products of DEPC-treated RNase-Free, and the specific operation is as follows:

in vitro transcription reaction system (20 μ L):

template DNA 12. mu.L

10×Buffer 2 μL

rATP（10 mM） 1 μL

rUTP（10 mM） 1 μL

rCTP（10 mM） 1 μL

rGTP（10 mM） 1 μL

T7 RNA polymerase 2. mu.L

Total volume 20. mu.L

Adding the reactants into an EP (advanced technology attachment) tube with 1.5mL of RNase-Free, uniformly mixing, placing the mixture in a water bath kettle with the temperature of 37 ℃ for reaction for 2.5h, adding 1 mu L of DNase into a transcription system, placing the mixture in the water bath kettle with the temperature of 37 ℃ for reaction for 30min to digest a DNA template, taking 1 mu L of gRNA sample, and carrying out agarose gel electrophoresis to detect a transcription result, wherein if the size of a transcription product is consistent with that of an expected product, the successful transcription is indicated;

f, purification of specific gRNA

Purifying successfully transcribed gRNA by using RNeasy Mini kit, and storing at-20 ℃; taking 1 mu L of the purified gRNA solution to determine the concentration;

3) microinjection of zebrafish embryos

Within 30min after fertilization, the embryos were pipetted and transferred to a microinjection petri dish made of agarose

Before microinjection, Cas9 mRNA and gRNA are prepared into a mixed solution, and the mixed solution is fully and uniformly mixed, so that the final concentration of Cas9 protein is 5 mug/muL, and the final concentration of gRNA is 30-40 ng/muL. About 1.8nL of Cas9 protein and gRNA cocktail were injected into fertilized eggs at one cell stage. The injected fertilized eggs were placed in E3 water and incubated at 28 ℃. Observing embryo phenotype under body type microscope, screening normally developed embryo for target site mutation analysis

Microinjection systems were as follows:

cas9 protein 5 μ g/. mu.L

gRNA 30-40 ng/μL

Nuclease free H2O

Total 3μL

4) Sanger sequencing to test the effectiveness of target sites

After microinjection is carried out on zebra fish embryos, partial early embryos which are normally developed are selected, and whether the ppm1g gene of the early embryos has mutation or not is detected;

a, extracting zebra fish genome

After the zebra fish embryo is fertilized for 50 hours (50 hpf), respectively collecting the wild type and the injected embryo in a 1.5mL Ep tube, extracting genome DNA according to the following method by 5 embryos per tube, and the specific steps are as follows:

adding 100 mu L of cell lysate and 1 mu L of proteinase K into an Ep tube filled with embryos, and placing the Ep tube in a water bath kettle at 55 ℃ for lysis overnight;

after cracking, placing the mixture on an oscillator for full oscillation, adding isopropanol which is cooled in advance in the same volume into an Ep tube, fully and evenly mixing the mixture in a reversed way, centrifuging the mixture for 10min at 12000rpm under the condition of 4 ℃, and pouring out supernate;

adding 500 μ L of 75% ethanol, centrifuging at 4 deg.C and 12000rpm for 5 min, removing supernatant, and air drying at room temperature for 8-10 min;

adding 10 mu L of deionized water, fully and uniformly blowing, and detecting the extraction efficiency by agarose gel electrophoresis;

b, PCR amplification of the target sequence

After extracting genome DNA, designing a primer sequence by using PrimerPremier 5.0 software according to a genome region of about 100-250bp upstream and downstream of a CRISPR target site to amplify a target DNA fragment;

the PCR reaction system is as follows:

2×Es Taq MasterMix 10 μL

Primer F (10 μM) 0.6 μL

Primer R (10 μM) 0.6 μL

template (genomic DNA) 1. mu.L

ddH2O 7.8 μL

Total volume 20. mu.L

After shaking and mixing evenly, centrifuging at 4 ℃, and carrying out amplification reaction on a PCR instrument;

c, detecting the PCR product by using 1.8% agarose gel electrophoresis;

d, sending the target DNA fragment after partial purification to perform Sanger sequencing, and preliminarily obtaining the information of insertion or deletion according to a sequencing peak map;

e, after two months of injection, carrying out tail shearing identification, which is the same as the identification step;

5) TA cloning of sequences of interest

Performing Sanger sequencing on the target sequence which is preliminarily identified to have mutation possibility by PCR, if a sequencing peak diagram has double peaks and a sequencing result shows that the target sequence has an insertion or deletion phenomenon, and then performing TA cloning and then selecting a monoclonal for further detection;

6) sanger sequencing of bacterial solutions

The bacteria liquid with the strip size which is displayed by the PCR result of the bacteria liquid and accords with the expected result is sent to sequencing, the bacteria liquid is compared with a standard target sequence on NCBI according to a peak diagram and a sequence which are given after the sequencing, and the mutation type of each monoclonal is analyzed according to the comparison result;

7) generation F1 to obtain heritable zebrafish mutants

Determining zebra fish mutant F0 generation through a series of screening, respectively hybridizing the mutant F0 generation with wild zebra fish to obtain F1 generation embryos, culturing at 28 ℃, carrying out hereditary identification on the zebra fish F1 generation, and respectively taking 5 embryos from each mutant F1 generation for mutation hereditary identification two days after fertilization; extracting genome of each embryo independently, amplifying a region near a target site with 612bp by PCR, performing Sanger sequencing, and if the mutation can be inherited to F1 generation, wherein a sequencing peak diagram has double peaks; culturing F1 generation of the zebra fish mutant for 2-3 months, respectively carrying out tail shearing, PCR, TA cloning and other steps on each F1 generation of zebra fish adult fish, sequencing the positive bacterial liquid, and knowing through a sequence comparison result that a sequence of 11bp is inserted into the position of a target site a.

The invention has the beneficial effects that:

through a CRISPR/Cas9 gene editing technology, a proper targeting site is designed on a ppm1g gene of zebra fish, specific sgRNA and Cas9 protein synthesized in vitro are injected into a zebra fish cell in a micro-co-injection mode, and after an embryo is cultured for 50 hours, the effectiveness of the targeted site is identified by selecting the embryo to perform genotype analysis. The invention can more efficiently and accurately mutate specific genes in organism genomes, has simple manufacture and low cost, and can simultaneously cut a plurality of sites on a target gene and mutate any number of single genes. The ppm1g gene is interfered, and the function of the gene is researched by genetics, which is helpful for further disclosing the whole process of cardiac development and the molecular mechanism for regulating the process, and has very important significance in understanding the pathology of the cardiac disease and developing a new treatment scheme in medicine.

The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

Drawings

Fig. 1 is a schematic diagram of a CRISPR/Cas9 targeting system;

FIG. 2 is a structural diagram of a target site on ppm1g gene;

FIG. 3 is a diagram showing the result of electrophoresis of zebra fish F1 generation;

FIG. 4 is a peak plot demonstrating that zebrafish F0 generation targeting was effective;

FIG. 5 shows a forward alignment of mutant and WT type gene sequences;

FIG. 6 shows a deletion contrast near the target site.

Detailed Description