阅读说明：本技术 一种基因敲除选育Lgr6基因缺失型斑马鱼的方法 (Method for breeding Lgr6 gene-deleted zebra fish through gene knockout ) 是由 邓云 禹青青 曹秋香 廖美 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敲除选育Lgr6基因缺失型斑马鱼的方法,通过CRISPR/Cas9基因编辑技术,在斑马鱼的Lgr6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60h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沉默特定基因,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,沉默任意数目的单个基因。且干扰掉Lgr6基因,并且通过遗传学手段研究其功能,有助于进一步揭示骨骼形态发生的整个过程以及调控这些过程的分子机制,在医学上骨骼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 an Lgr6 gene-deleted zebra fish through gene knockout, wherein a proper targeting site is designed on an Lgr6 gene of the zebra fish through a CRISPR/Cas9 gene editing technology, specific sgRNA and Cas9-mRNA synthesized in vitro are injected into a zebra fish cell together in a microscopic mode, and an embryo is selected for genotype analysis after being cultured for 60 hours, so that the effectiveness of the selected site is verified. The invention can silence specific gene in organism genome more efficiently and accurately, and has simple manufacture and low cost, and can simultaneously cut a plurality of sites on the target gene to silence any number of single genes. The Lgr6 gene is interfered, and the function of the Lgr6 gene is researched by a genetic means, so that the whole process of bone morphogenesis and the molecular mechanism for regulating the processes are further disclosed, and the Lgr6 gene has very important significance in understanding the pathology of bone diseases and developing new treatment schemes in medicine.)

1. A method for breeding an Lgr6 gene-deleted zebra fish by gene knockout is characterized by comprising the following steps:

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

Inquiring The genomic DNA sequence of zebra fish Lgr6 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 Lgr6 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):

tgTAATACGACTCACTATAgggtcctgtgcccattctcaGTTTTAGAGCTAGAAATAGC

f2 (target site b forward primer):

tgTAATACGACTCACTATAggtcagatcagttggcacgcGTTTTAGAGCTAGAAATAGC

r (common reverse primer): AAGCACCGACTCGGTGCCACT

PCR detection primer

The upstream and downstream primers of the PCR detection primer are both positioned on the No. 1 exon:

F (5’-AGGCACATACACACAGACAAAC-3’)

R (5’-ATCAAAGTGGATGACAGAAGCG-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

Linearizing the plasmid with BsaI restriction endonuclease; the total volume of the enzyme digestion reaction is 20 mu L, and the system is as follows:

p42250 vector 2. mu.L

10× Buffer 2 μL

Bsa I restriction enzyme 1.5. mu.L

ddH2O 14.5 μL

Total volume 20. mu.L

Mixing, performing enzyme digestion in water bath at 37 ℃ for more than 4 h;

c, taking a linearized p42250 vector as a template, and carrying out PCR (polymerase chain reaction) by using the following specific primers to amplify the double-stranded DNA for synthesizing the specific gRNA;

forward specific target site primer F1 or F2: t7 promoter 20pb target sequence 20bp sgRNA upstream backbone, reverse primer R: a downstream framework of 20bp of sgRNA,

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, spotting 1 microliter of sample on 1.8% agarose gel for electrophoresis, and shooting the 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, 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 specific operation is as follows:

in vitro transcription reaction system:

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 all reactants into an EP (advanced peripheral technology) tube of 1.5 mL RNase-Free, uniformly mixing, carrying out water bath at 37 ℃ for 2.5 h, adding 1 mu L of DNase into a transcription system, placing the transcription system in a water bath kettle at 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 transcription product, the successful transcription is indicated; (ii) a

f, purification of specific gRNA

Purifying successfully transcribed gRNA by using RNeasy Mini kit, and storing at-20 ℃; agarose gel electrophoresis was performed to check the purified product, and the concentration of gRNA after purification was determined;

3) microinjection of zebrafish embryos

Within 30min after fertilization, the embryos are sucked by a suction pipe and transferred to a special culture dish for microinjection made of agarose,

before microinjection, Cas9 mRNA and gRNA are prepared into a mixed solution, the mixed solution is fully and evenly mixed, the final concentration of the Cas9 mRNA is 5 ng/mu L, the final concentration of the gRNA is 30-40 ng/mu L, and about 1.8 nL of the mixed solution of the Cas9 mRNA and the gRNA is injected into a fertilized egg at a cell stage; placing the injected fertilized eggs in E3 water, and incubating at 28 ℃; observing embryo phenotype under a body type microscope, and screening the embryo which normally develops for target site mutation analysis;

microinjection systems were as follows:

Cas9 mRNA 5 ng/μL

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 normally-developed early embryos are selected, and whether mutation exists in Lgr6 genes is detected;

a, extracting zebra fish genome

After the zebra fish embryo is fertilized for 60 hours (60 hpf), respectively collecting the wild type and the injected embryo in a 1.5 mL Ep tube, wherein 10-15 embryos in each tube are extracted by the following method, and the specific steps are as follows:

adding 200 mu L of cell lysate and 2 mu L of proteinase K into an Ep tube filled with embryos, and placing the Ep tube in a thermostat at 55 ℃ for cracking overnight;

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

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

adding 60 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, according to CRISPR target site upstream and downstream100-250 bpThe primer sequence is designed by using PrimerPremier 5.0 software to amplify the target DNA fragment;

the PCR reaction system is as follows:

2×Es Taq MasterMix10μL

Primer F (10 μM)0.6μL

Primer R (10 μM)0.6μL

stencil (genome DNA)1μL

ddH2O7.8μL

Total volume20μL

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

performing Sanger sequencing on the PCR result, and preliminarily obtaining the information of insertion or deletion from a sequencing peak map;

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

5) generation F1 to obtain heritable zebrafish mutants

Determining zebra fish mutant F0 generation through a series of previous screens, respectively hybridizing the mutant F0 generation with wild zebra fish to obtain F1 generation embryo, culturing at 28 ℃, and observing the survival rate of F1 generation at the initial stage; after fertilization for two days, 10-15 embryos are respectively taken from each mutant F1 generation for mutation inheritance identification; extracting genome of each embryo independently, performing PCR amplification to obtain a region near a target site of 326bp, observing whether a miniband appears in the PCR amplification, and if the mutation can be inherited to F1 generation, the miniband of less than 326bp appears in the PCR amplification; breeding the zebrafish mutant for up to 2-3 months for F1 generation if the presence of a mutation is detected from the F1 generation embryos; and then respectively carrying out tail shearing on each F1 generation adult zebra fish, and screening F1 generation mutants.

2. The method for breeding the Lgr6 gene-deleted zebra fish by gene knockout 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 selectively breeding the Lgr6 gene-deleted zebra fish by gene knockout according to claim 1, wherein the Tip and EP tubes used in the transcription experiment in the step 2) are DEPC-treated RNase-Free.

4. The method for selectively breeding the Lgr6 gene-deleted zebra fish by gene knockout as claimed in claim 1, wherein the step c) of step 2) is performed by an amplification reaction on a PCR instrument under the following reaction conditions: pre-denaturation at 95 ℃ for 3 min, and repeating the following steps for 32 more times: denaturation at 95 ℃ for 15 s, annealing at 60 ℃ for 15 s, extension at 72 ℃ for 30 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 the Lgr6 gene-deleted zebra fish through gene knockout according to claim 1, wherein the E3 water in the step 3) is a mixture of 5 mmol/L NaCl, 0.33 mmol/L CaCl2, 0.33 mmol/L MgSO4 and 0.17mmol/L KCl.

6. The method for selectively breeding the Lgr6 gene-deleted zebra fish by gene knockout as claimed in 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 30 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 Lgr6 gene-deleted zebra fish through gene knockout.

Background

The Lgr6 (leucoine rich repeat association G protein-associated receptor 6) gene is located on the No. 23 chromosome of zebra fish, comprises 18 exons, has a cDNA full length of 2889bp, and codes 962 amino acids, and meanwhile, through gene differential expression profiling analysis, genome association analysis and the like, the Lgr6 is found to be expressed in a plurality of tissues in the early stage of human embryo, particularly strongly expressed in bones.

The zebra fish has high homology with genes and signal paths in the human skeletal development process, and Lgr6 gene is more conservative in evolution, so that the study finds that the Lgr6 has particularly high early expression level in the zebra fish embryo. 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 an Lgr6 gene of zebra fish, specific sgRNA (the final concentration is 30-40 ng/mu L) and Cas9-mRNA (the final concentration is 5 ng/mu L) synthesized in vitro are injected into a zebra fish cell in a microinjection mode, and after the zebra fish cell is cultured for 60 hours, an embryo is selected for genotype analysis, so that the effectiveness of the targeted site is identified.

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 novel artificial endonuclease clustered regulated short palindromic repeats (CRISPR)/CRISPR-associated (Cas)9 can silence a specific gene in an organism genome more efficiently and more accurately, is simple to manufacture and low in cost, can simultaneously cut multiple sites on a target gene and silence any number of single genes, but 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 an Lgr6 gene-deficient zebra fish by gene knockout, finds a proper targeting site, and breeds the Lgr6 gene-deficient 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 Lgr6 gene is queried on The National Center for Biotechnology Information (NCBI), The functional domain is analyzed on The website SMART (http:// smart.embl-heidelberg. de /), and The target site of Lgr6 gene is designed on The website The ZiFiT target (http:// ZiFiT. 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 gene domain to ensure that the insertion or deletion of the target site base can affect the entire domain of Lgr6 gene, thereby altering gene expression

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

f1 (target site a forward primer):

tgTAATACGACTCACTATAgggtcctgtgcccattctcaGTTTTAGAGCTAGAAATAGC

f2 (target site b forward primer):

tgTAATACGACTCACTATAggtcagatcagttggcacgcGTTTTAGAGCTAGAAATAGC

r (common reverse primer): AAGCACCGACTCGGTGCCACT

PCR detection primer

The upstream and downstream primers of the PCR detection primer are both positioned on the No. 1 exon:

F (5’-AGGCACATACACACAGACAAAC-3’)

R (5’-ATCAAAGTGGATGACAGAAGCG-3’)；

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

a, the gRNA backbone was first cloned into 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 2 μL

Bsa I 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. The reaction conditions are as follows: pre-denaturation at 95 deg.C for 3 min, (denaturation at 95 deg.C for 15 s, annealing at 60 deg.C for 15 s, and extension at 72 deg.C for 30 s) for 30 cycles, and then at 72 deg.C for 5 min. After the reaction is finished, centrifuging the PCR product, taking 1 microliter of sample to sample on 1.5% agarose gel for electrophoresis, and taking a result by a gel imaging system

d, detecting to determine that the band is correct, then carrying out agarose gel DNA recovery, purifying and recovering PCR products

e, measuring the concentration of the purified DNA (as much as 1 mu g), and 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 and EP tubes used in the transcription experiment are all DEPC-treated RNase-Free products, 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 tube of 1.5 mL RNase-Free, uniformly mixing, and carrying out water bath at 37 ℃ for 2.5 h;

after the water bath is finished, adding 1 mu L of DNA enzyme into the transcription system, placing the transcription system in a water bath kettle at 37 ℃ for reaction for 30min to digest the DNA template, taking 1 mu L of sample, and carrying out electrophoresis by using prepared 1.5% agarose gel to detect the transcription result, wherein if the size of the transcription product is consistent with that expected, the successful transcription is indicated;

f) purification of specific gRNA

Successfully transcribed gRNAs were purified using the RNeasy Mini kit and stored at-20 ℃.1 μ L of the purified gRNA solution was aspirated for agarose gel electrophoresis to check the purified product, and the concentration of gRNA after purification was determined

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 mixed, so that the final concentration of Cas9 mRNA is 5 ng/mu L, and the final concentration of gRNA is 30-40 ng/mu L. About 1.8 nL of Cas9 mRNA and gRNA cocktail were injected into fertilized eggs at one cell stage. The injected fertilized eggs were incubated in E3 water (5 mmol/L NaCl, 0.33 mmol/L CaCl2, 0.33 mmol/L MgSO4, 0.17mmol/L KCl) at 28 ℃. Observing embryo phenotype under body type microscope, screening normally developed embryo for target site mutation analysis

Microinjection systems were as follows:

Cas9 mRNA 5 ng/μL

gRNA 30-40 ng/μL

Nuclease free H2O

Total 3 μL

4) sanger sequencing to test the effectiveness of target sites

After microinjection is carried out on zebra fish embryos, partial normally-developed early embryos are selected, whether mutation exists in Lgr6 gene is detected, whether the selected target site has effect or not can be confirmed in advance, and whether microinjection operation is standard or not

a. Extraction of zebra fish genome

After the zebra fish embryo is fertilized for 60 hours (60 hpf), respectively collecting a wild type (serving as a control) and an injected embryo in a 1.5 mLEp tube (10-15 embryos per tube), and extracting genomic DNA according to the following method, wherein the specific steps are as follows:

to Ep tube containing embryo, 200. mu.L of cell lysate and 2. mu.L of proteinase K were added, and the mixture was incubated overnight in a 55 ℃ incubator

After the lysis is finished, the mixture is put on an oscillator to be fully shaken, isopropanol with the same volume (200 mu L) is added into an Ep tube (pre-cooled), the mixture is fully inverted and mixed evenly, and the mixture is heated at 4 ℃,centrifuging at 12000 Xg for 10minPouring out the supernatant;

adding 500 μ L of 75% ethanol, and heating at 4 deg.CIn the following, the first and second parts of the material,centrifuging at 12000 Xg for 5 minRemoving supernatant, and air drying at room temperature for 8-10 min;

adding 60 μ L deionized water, fully beating, mixing, and detecting extraction efficiency by agarose gel electrophoresis

b. PCR amplification of target sequences

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

The PCR reaction (20. mu.L) was as follows:

2×Es Taq MasterMix10μL

Primer F (10 μM)0.6μL

Primer R (10 μM)0.6μL

stencil (genome DNA)1μL

ddH2O7.8μL

Total volume20μL

After shaking and mixing, the mixture was centrifuged at 4 ℃ and subjected to amplification reaction on a PCR instrument. The reaction conditions are as follows: pre-denaturation at 95 deg.C for 5 min, denaturation at 95 deg.C for 30 s, annealing at 60 deg.C for 30 s, and extension at 72 deg.C for 30 s for 30 cycles, and further denaturation at 72 deg.C for 8 min. After the reaction is finished, centrifuging the PCR product, taking 5 microliter of sample to sample on 1.5% agarose gel for electrophoresis, and detecting whether the size of the PCR product is correct

c. If the PCR product is correct, Sanger sequencing is carried out, and the information of the insertion or deletion is obtained primarily from a sequencing peak diagram

4) After two months of injection, the tail-cutting identification is carried out, the identification steps are the same as the above

5) Generation F1 to obtain heritable zebrafish mutants

The zebra fish mutant F0 generation was confirmed by the previous series of screens, and then the F0 generation mutant was respectively crossed with wild zebra fish to obtain F1 generation embryos, which were cultured at 28 ℃ and the survival rate of F1 generation was observed at the early stage. Two days after fertilization, 10-15 embryos are respectively taken from each mutant F1 generation for mutation inheritance identification. Extracting genome of each embryo separately, PCR amplifying 326bp target site area, observing whether PCR amplification will generate small band, if the mutation can be transmitted to F1 generation, PCR amplification will generate small band smaller than 326 bp. The zebrafish mutant was bred for up to 2-3 months for F1 generations if the presence of a mutation was detected from F1 generation embryos. And then, respectively carrying out tail shearing on each F1 generation adult zebra fish, and screening F1 generation mutants (the specific method is as described above).

The invention has the beneficial effects that:

through a CRISPR/Cas9 gene editing technology, a proper targeting site is designed on an Lgr6 gene of zebra fish, specific sgRNA (the final concentration is 30-40 ng/mu L) and Cas9-mRNA (the final concentration is 5 ng/mu L) synthesized in vitro are injected into a zebra fish cell in a microinjection mode, and after the zebra fish cell is cultured for 60 hours, an embryo is selected for genotype analysis, so that the effectiveness of the targeted site is identified. The invention can silence specific gene in organism genome more efficiently and accurately, has simple manufacture and low cost, can cut a plurality of sites on the target gene simultaneously, silence any number of single genes, interfere the Lgr6 gene, and is helpful to further reveal the whole process of bone morphogenesis and the molecular mechanism for regulating and controlling the process by researching the function of the gene by genetics, thereby having very important significance in understanding the pathology of bone diseases and developing new treatment schemes 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 diagram of the structure of the target site on Lgr6 gene;

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

FIG. 4 shows a forward alignment of deletion and WT gene sequences;

FIG. 5 shows a deletion contrast near the target site.

Detailed Description