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Generation of Mutant Pigs by Direct Pronuclear Microinjection of CRISPR/Cas9 Plasmid Vectors
通过直接原核显微注射CRISPR/Cas9质粒载体生成突变株   

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Abstract

A set of Cas9 and single guide CRISPR RNA expression vectors was constructed. Only a very simple procedure was needed to prepare specific single-guide RNA expression vectors with high target accuracy. Since the de novo zygotic transcription had been detected in mouse embryo at the 1-cell stage, the plasmid DNA vectors encoding Cas9 and GGTA1 gene specific single-guide RNAs were micro-injected into zygotic pronuclei to confirm such phenomenon in 1-cell pig embryo. Our results demonstrated that mutations caused by these CRISPR/Cas9 plasmids occurred before and at the 2-cell stage of pig embryos, indicating that besides the cytoplasmic microinjection of in vitro transcribed RNA, the pronuclear microinjection of CRISPR/Cas9 DNA vectors provided an efficient solution to generate gene-knockout pig.

Keywords: CRISPR/Cas9(CRISPR/Cas9), GGTA1(GGTA1), Pronuclear microinjection(原核显微注射)

Background

Since the initial discovery of a highly conserved 29 base pairs (bp) sequence tandemly repeated with a spacing of 32 bp downstream of the iap gene in Escherichia coli genome (Ishino et al., 1987; Nakata et al., 1989), a family of short regularly spaced repeats, varying in size from 25 to 50 bp, were found in about 50% of bacteria and 90% of archaea (Makarova et al., 2015). According to their characteristic structures, the name clustered regularly interspaced short palindromic repeats (CRISPRs) were introduced by Mojica (Mojica et al., 2009) and Jansen (Jansen et al., 2002) and are currently in general use. A set of CRISPR associated genes, cas1 to cas4, was first identified flanking the CRISPR loci by nucleotide sequence alignments (Jansen et al., 2002). New members of cas genes were identified in different bacterial species. According to the cas members within each host, the CRISPR-Cas systems can be classified into three major types based on the hallmark genes (Makarova et al., 2011; Burmistrz and Pyrc, 2015). The function of the CRISPR-Cas system was demonstrated by a seminar experiment. After being challenged by virulent bacteriophages: phage 858 and phage 2,972, new repeat-spacer units were observed on the leading end of the CRISPR array in the surviving Streptococcus thermophlis host cells. The DNA sequences of newly acquired spacers were matched to corresponding fragments, named proto-spacers, in the phage genomes. Streptococcus thermophlis strains with phage spacer(s) were resistant to the phage infection, while strains without phage spacer(s) were sensitive to the phage infection (Barrangou et al., 2007). To distinguish between the spacer in the host bacterial genome and the proto-spacer, which has the same sequence as the spacer in the invader genome, a proto-spacer adjacent motif (PAM) was evolved (Mojica et al., 2009; Shah et al., 2013). The CRISPR-Cas immunity was revealed, it can be divided into three stages (Rath et al., 2015; Wright et al., 2016). The first adaptation or acquisition stage is responsible for the acquisition of spacers into CRISPR array following the exposure to foreign mobile genetic elements, such as phages or plasmids. A Cas2 homodimer was sandwiched by two Cas1 homodimers forming a heterohexameric complex for all three types of CRISPR-Cas systems (Sternberg et al., 2016). In the second stage, the promoter embedded within the AT-rich leader sequence upstream of the CRISPR array transcribed the precursor CRISPR RNAs (pre-crRNAs), these were further processed into short CRISPR RNA (crRNA) guides by Cas proteins. Cas6 was involved in the RNA processing step in both of the type I and type III CRISPR-Cas systems (Charpentier et al., 2015; Hochstrasser and Doudna, 2015). Accompanied by the Cas9 protein, a trans-activating crRNA (tracrRNA) which contains an anti-repeat segment for duplex formation with the repeat compartment of crRNA was involved in the maturation of the crRNAs in the type II system (Deltcheva et al., 2011). In the last interference stage, in cooperation with a mature crRNA and a cascade of Cas proteins, the signature proteins Cas3 and Cas10 were integrated into the RNA guided endonuclease complex in the type I and type III CRISPR-Cas systems, respectively. The type II effector is simply composed of a Cas9 protein, a pair of processed mature crRNA and tracrRNA (Gasiunas et al., 2012). The crRNA and tracrRNA in the ternary complex can be substituted by a fused crRNA-tracrRNA single-guide RNA (sgRNA) (Jinek et al., 2012). Because of extreme simplicity, the Cas9-sgRNA, now commonly termed as CRISPR/Cas9, binary complexes were immediately applied in the field of gene editing (Cong et al., 2013; Mali et al., 2013).

Swine share a number of anatomic and physiologic characteristics with humans. Systems that are mostly cited as suitable models include cardiovascular, urinary, integumentary, and digestive system (Swindle et al., 2012). Therefore, pigs are considered as a good source of organs for xenotransplantation. Two strategies are currently utilized to overcome the interspecies rejection hurdles. The first one is to block donor organs expressing the antigens causing hyper-acute rejection, such as galactose-α1,3-galactose, N-glycolylneuroaminic acid and β1,4-N-acetylgalactosamine, by targeting the GGTA1, CMAH and β4GalNT2 genes, respectively (Estrada et al., 2015; Cooper et al., 2016). The second strategy is to prepare organs which are composed of acceptor’s cells in a surrogate animal by a blastocyst complementation technique (Kobayashi et al., 2010; Usui et al., 2012; Matsunari et al., 2013). To produce gene-knockout pigs, the traditional method uses gene editing in somatic cells, such as fetal fibroblasts, and somatic cell nuclear transfer (SCNT) techniques to create knockout zygotes. The examples of combining CRISPR/Cas9 and SCNT are reported to generate IgM JH (Chen et al., 2015), RUNX3 (Kang et al., 2016b), IL2RG (Kang et al., 2016a), and GGTA1/CMAH/β4GalNT2 triple knockout pigs (Estrada et al., 2015). Direct microinjection of DNA or RNA is another choice to prepare gene knockout pigs. Since the burst of de novo transcription in porcine zygotes was reported at the 4-cell stage (Anderson et al., 1999), RNA is preferred for micro-injection into embryos at the 1-cell stage. Previous studies have demonstrated that cytoplasmic microinjection of Cas9 mRNA with sgRNAs can produce Mitf (Wang et al., 2015) and DJ-1/Parkin/PINK1 triple-gene knockout pigs (Wang et al., 2016). Because de novo zygotic transcription had only been reported in the 1-cell stage mouse embryo (Ram and Schultz, 1993; Bouniol et al., 1995; Aoki et al., 1997), a trial was reported to produce GGTA1 knockout pigs by cytoplasmic microinjection of a CRISPR/Cas9 plasmid. With this strategy, CRISPR/Cas9 was expressed at, or later, than the 2-cell stage, and mosaic mutations on GGTA1 gene were found (Petersen et al., 2016). Pronucleus microinjection is needed to interpret whether zygotic transcription occurs at the 1-cell stage of pig embryo (Chuang et al., 2016).

A series of Cas9 and sgRNA expression vectors was constructed as shown in Figure 1. pCX-Flag2-NLS1-Cas9-NLS2, pCX-HA-NLS1-Cas9-NLS2, and pCX-Myc-NLS1-Cas9-NLS2 can be used to express Cas9 in mammalian cells. Three common tags are available to monitor Cas9 expressions. (Figure 1A) Porcine U6 promoter which could effectively derive short hairpin RNA [Chuang et al., 2009] was used to construct the ppU6-(BsaI)2-gRNA vector (Figure 1B) (Su et al., 2015). A pair of primers containing spacer and part of CRISPR repeat sequences, as shown in Figure 1B, is needed for each target site. Because guanine (G) is favored for U6 promoter as the first transcribed nucleotide, only proto-spacers initiated with G were chosen in our recent works.


Figure 1. Scheme of the Cas9 and single-guide RNA expression vectors. A. pCX-Flag2-NLS1-Cas9-NLS2, pCX-HA-NLS1-Cas9-NLS2, and pCX-Myc-NLS1-Cas9-NLS2; B. ppU6-(BsaI)2-gRNA and primer pair designation.

Materials and Reagents

  1. Preparations of sgRNA expression vectors
    1. BsaI (New England Biolabs, catalog number: R0535 )
    2. T4 DNA ligase (Promega, catalog number: M1801 )
    3. Clean and Gel Extraction Kit (Biokit, catalog number: Bio-C300 )
    4. 2x ligation buffer (Promega, catalog number: C671A )
    5. pGEM-T Easy TA-cloning kit (Promega, catalog number: A1360 )
    6. Plasmid Miniprep Kit (Biokit, catalog number: Bio-P300 )
    7. Genomic DNA isolation Kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: K0721 )
    8. NeonTM Transfection System 100 µl Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: MPK10096 )
    9. Cesium chloride (CsCl) (Avantor® Performance Materials, J.T. Baker®, catalog number: 4042-02 )
    10. Tris-HCl, pH 8.0
    11. EDTA, pH 8.0
    12. Sodium chloride (NaCl)
    13. TE buffer (see Recipes)
    14. TEN buffer (see Recipes)

  2. Collection of pig embryos
    1. Glass tube for embryo flashing (glass tube with outside diameter of 4 mm as shown in Figure 2)
    2. Falcon tube, 50 ml (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 339653 )
    3. Regumate® (containing 0.4% Altrenogest, Intervet, MSD, France, brand name: Regumate®)
    4. PMSG (pregnant mare serum gonadotropin) (ASFK Pharmaceutical, Japan, brand name: PMSG)
    5. hCG (human chorionic gonadotropin) (ASKA Pharmaceutical, Japan, brand name: hCG)
    6. PGF2α (prostaglandin F2α; Estrumate injection) (Intervet Deutschland, Germany)
    7. D-PBS (GE Healthcare, HyCloneTM, catalog number: SH30028.02 )
    8. Fetal bovine serum (FBS) (GE Healthcare, HyCloneTM, catalog number: SH30071.03 )

  3. Embryo transfer
    1. 3M paper tape (3M, catalog number: 200-24mm )
    2. Atropine sulfate (TAI YU CHEMICAL & PHARMACEUTICAL)
    3. Stresnil (azaperona, 40 mg/ml) (Janssen Pharmaceutica N.V., Belgium, brand name: Stresnil)
    4. Citosol (Thiamylal sodium) (Shinlin Sinseng Pharmaceutical, Taiwan, brand name: Citosol)
    5. Amoxicillin (ampicillin, 150 mg/ml) (China Chemical & Pharmaceutical, CCPG, catalog number: E000853 )
    6. Heparin sodium (China Chemical & Pharmaceutical, CCPG, Taiwan, brand name: AGGLUTEX INJECTION)

Equipment

  1. Preparations of sgRNA erxpression vectors
    1. Ultracentrifugator (Beckman Coulter, model: Optima XL-80 ) equipped with NVTi65 rotor
    2. Microcentrifugator (KUBOTA, model: 3740 )
    3. Dry bath incubator (Major Science, model: MD-01N )
    4. Thermocycler (Thermo Fisher Scientific, Applied BiosystemsTM, model: 2720 )

  2. Semen assessment
    1. computer-assisted sperm analysis system (UltiMate CASA system, Hamilton-Thorne Research, Beverly, MA)

  3. Microinjection
    1. Centrifugator (KUBOTA, model: 8920 )
      Note: This product has been discontinued.
    2. Microcentrifugator (KUBOTA, model: 3740 )
    3. Inverted Differential Interference Contrast microscope (Olympus, model: IX71 )
    4. Capillary injection needle (Sutter Instrument, catalog number: BF100-78-10 )
    5. Capillary injection needle puller (Sutter Instrument, model: P-97 )
    6. Micromanipulator (NARISHGE, model: ON3-99D )
    7. Injectors (NARISHGE, model: IM-9B )

  4. Collection of pig embryos and embryo transfer
    1. Operation table (NEWPORT, LW3048B-OPT, VIBRATION ISOLATION TABLE)
    2. Surgery mechanic devises (NARISHIGE, JAPAN)

Procedure

  1. Preparations of sgRNA expression vectors
    1. Take 10 μg of ppU6-(BsaI)2-gRNA and let it be digested with 50 U of BsaI in 100 μl NEB3.1 buffer at 37 °C for 2 h.
    2. Purify the linearized vector DNA with a clean-up kit and elute it with 30 μl of 1/10x TE. Store the purified linearized vector at -20 °C before use.
    3. Dissolve the primers listed in Table 1 in TE and adjust the concentrations to 50 μM.
      Notes:
      1. Add each 10 μl of forward and reverse primers (Table 1, 50 μM, each) to a vial and mix them with 30 μl of TEN. Incubate the mixture at 90 °C for 10 min in a dry block heater.
      2. Transfer the mixture to a 65 °C dry block and incubate for 30 min. Then let the block gradually cool down to room temperature for about 30 min. Put the annealed primer pair on ice until the following ligation reaction.

        Table 1. Primers for ppU6-sgRNA expression vector construction


        The first nucleotide of the last exon of pig GGTA1 gene was numbered as +1, and the last nucleotide of the last intron was numbered as -1 (Su et al., 2015).
    4. For the ligation reaction, mix 1 μl of BsaI digested vector, 3 μl of annealed primer pair, 5 μl of 2x ligation buffer and 1 μl T4 DNA ligase in a vial on ice. Then incubate the vial at 16 °C for 90 min to perform the ligation reaction.
    5. After the transformation, isolate plasmid samples from the individual colonies. Use M13 reverse primer for DNA sequencing (Note 1).
    6. Purify the DNA for pronuclear microinjection by two rounds of CsCl density-gradient ultra-centrifugation. The NVTi65 rotor is used in general and the centrifugation conditions are set at 218,000 x g for 20 h at 20 °C.

  2. Animals and animal care
    1. Raise all animals in a specific pathogen free farm. Feed the animals on a restricted diet (4% body weight) with free access to water.
    2. Raise the recipients normally but treat them with special care particularly during farrowing.
    3. All animals should be managed and treated in accordance to your federal and institutional guidelines.

  3. Synchronization and superovulation
    1. Synchronize the donors and recipients by feeding with a commercial ration supplemented with Regumate® (containing 0.4% Altrenogest; Intervet, MSD, France) for 15 days to synchronize their estrus cycles. Feed the recipients with Regumate® for one more day.
    2. On the last day of feeding Regumate®, inject PGF2 (250 μg Cloprostenol) intramuscularly (i.m.) to lyse any remaining corpus luteum.
    3. For superovulation induction, inject all pigs intramuscularly with PMSG and hCG to induce follicle growth/oocyte maturation and ovulation, respectively. Briefly, on the next day of the last feeding with Regumate®, inject all of the donors with 2,000 i.u. of PMSG. Then inject them with 1,500 i.u. of hCG 78 h later. Inject the recipients by the same regimen with 1,500 i.u. PMSG and 1,250 i.u. hCG to induce follicle growth/oocyte maturation and ovulation, respectively.
    4. For artificial insemination, artificially inseminate the donors with fresh diluted semen containing at least 3 billion viable sperms with 80% mobility as assessed by computer-assisted sperm analysis system at 24 h and 36 h after hCG injection. Induce the synchronized ovulation of recipients by the same methods with a 12 h delay and without insemination.

  4. Pronuclear oocytes recovery
    1. During 54-56 h after hCG injection, sacrifice all of the donors to harvest the upper uterus horns and oviducts.
    2. For the collection of zygotes, insert and connect infundibulum with a glass tube to guide the D-PBS into a 50 ml Falcon tube. Flush out the fertilized eggs from the oviducts by injection of 20 ml D-PBS with 20% fetal calf serum (FCS) into the fallopian tube near the uterotubal junction. (Figure 2)
    3. Keep the embryos in DPBS in a thermal box at 37 °C and carry them to a laboratory for further micromanipulation within 30 min.


      Figure 2. Flushing newly fertilized eggs from porcine fallopian tube. The maturation and ovulation are indicated by the follicle with an ovulation point (arrow head 1). A stainless needle (#19) with a blunt end is used to penetrate the uterus wall, and it is inserted through the uterotubal junction (arrow head 2) into the fallopian tube (arrow head 3). A glass tube (arrow hear 5, outside diameter 4 mm) is connected into the other end of the fallopian tube through the open of infundibulum (arrow head 4). The fertilized eggs within the fallopian tube are flushed out by 20 ml D-PBS with a syringe (arrow head 6) and collected in a 50 ml Falcon tube (arrow head 7).

  5. Microinjection
    1. Centrifuge the recovered fertilized eggs at 15,000 x g for 10 to 15 min at 25 °C to expose their pronuclei (Figure 3).
      Notes:
      1. Then put the embryos into a 20 µl D-PBS micro-drop on an inverted microscope equipped the differential interference contrast system.
      2. Perform the pronuclear microinjection under 300-fold magnification.
      3. Hold the embryos in a proper position to reveal a clear view of pronuclei.
    2. Then microinject the CRISPR/Cas9 DNA mixture into one pronucleus through a capillary needle. The diameter of tip opening for an injection pipette is about 0.5 µm. After applying injection pressure, the continuous flow stream of DNA is shown by moving the zygote away from the tip of the microinjection capillary. Before injection into pronuclei, the pipette tip was placed in the perivitelline space (PVS). A suitable injection flow speed is adjusted by increasing or decreasing the injection pressure according to the swelling velocity of PVS. The concentrations of plasmid DNA were 6 ng/µl for pCX-Flag2-NLS1-Cas9-NLS2 and 2 ng/µl for each of the four ppU6-sgRNA vectors (Note 2).


      Figure 3. Pronuclear microinjection of a pig embryo. Centrifugation is critical to reveal the pronuclei, indicated by arrowheads, from the optically dense contents in the pig embryo. Scale bar = 20 μm.

  6. Embryo transfer
    1. After microinjection of CRISPR/Cas9 plasmid vectors into the pronuclei, transfer 20 to 30 embryos into the oviduct through a transfer pipette by the surgical method:
      1. Before operation, perform a 24 h fasting with free access to water for all of the recipients.
      2. Twenty minutes before surgery, inject 5 mg atropine sulfate and 400 mg Stresnil to decease saliva discharge and sedate the animals, respectively.
      3. Set up an ear vein catheter and fix it by 3M paper tape for i.v. injection of Citosol to maintain anesthesia, with the duration of operations lasting for 40 to 50 min.
      4. Load and fasten the recipients upside down on the operation table. Clean their abdomen skin and shave and sterilize the hair. Then cover the animal with a sterilized drape.
      5. Create an 8-10 cm window on the linea alba among the last 2nd to 3rd nipple, pull a single side of uterus horns, fallopian tubes and ovary outside the abdomen, and then count the number of corpus luteum on the ovary. Put back the largest part of the uterus horn immediately into cavum abdominis. Keep the remaining upper uterus horn, fallopian tube, infundibulum and ovary outside for embryo transfer (Figure 4A).
      6. Load 10-15 embryos in D-PBS in a glass transferring tube controlled by mouth pipet and transfer the embryos into the oviduct isthmus (Figure 4B).
      7. Rinse the uterus horn by saline containing heparin sulfate (50,000 U/L), and then put it back into the cavum abdominis.
      8. Before suturing the cut window, infuse 10 ml of amoxicillin into the cavum abdominis.
      9. After embryos transference, close the peritoneum parietale and muscle of the abdominal wall by continuous suture, and suture the adipose layer and skin by interrupted vertical mattress.
      10. Finally, inject 10 ml of amoxicillin i.m. at the neck during the following three days to prevent infection.


        Figure 4. Embryo transfer. A. Upper uterus horns, uterus, fallopian tubes, infundibula and ovaries are kept outside for embryo transfer. B. The embryos are loaded in a glass tube controlled by mouth pipet, and transferred into the oviductal isthmus (abbreviations: u for the uterus, uh for the uterus horn, ft for the fallopian tube, and inf for the infundibulum).

  7. Farrowing and piglets care
    1. Move the sows to the farrowing pens seven days before the expected delivery day.
    2. Observe the nervous and restless behaviors of the sows; check the lactation further by hand milking.
    3. On the day when sows start to lactate, check the sows intensively to ascertain any one of them need assistance for delivering, and take care of the neonates.
    4. On the next day when farrowing was completed, collect the piglets’ tail tissue samples for DNA extraction and further genomic analysis.

Data analysis

The genomic DNA samples isolated from pig tail tissues were analyzed by PCR with primers listed in Table 2. The PCR products were cloned into the pGEM-T Easy TA-cloning vector. Individual clones of plasmids were sequenced to interpret the mutations. In a typical experiment (Chuang et al., 2016), 41 pronucleus microinjected embryos were transferred into two surrogate sows. One of them was pregnant and 7 piglets were delivered. Two founders: L537-12 and L537-13, were detected carrying GGTA1 mutations in the tail tissues. Wild type DNA sequence and a 4 bp (TTGG) deletion at site 1 were revealed in the L537-12 sample. A 7 bp (TGGTTGG) deletion and another 7 bp (CATGGTT) deletion were identified in the L537-13 sample. After crossing with wild type mate, the 4 bp (TTGG) deletion was measured in 5 of the 14 offspring of the L537-12. The 7 bp (TGGTTGG) and 7 bp (CATGGTT) deletions were detected in 5 and 3, respectively, among the 13 offspring of L537-13. It is noteworthy that among the last 13 offspring, one piglet carried a 149 bp deletion (fragment between site 1 and site 4) which was not detected in the founder’s tail tissue. Theoretically, for a heterologous male, half of the sperms are mutants. For a 50% mosaic male, 25% of the sperms are mutants. As 5 of the 14 offspring of the L537-12 founder carry the 4 bp (GGTT) deletion, it indicates that the mosaic ratio is between 50 to 100%. Since 5 and 3 of the 13 offspring of the L537-13 founder carry the 7 bp (TGGTTGG) and 7 bp (CATGGTT) deletions, respectively, it also supports this conclusion. The other offspring of the L537-13 carrying the 149 bp deletion indicates that the deletion reaction occurred after the 4-cell embryonic stage. It is possible that the majority of the germline was just occupied by the descents of the mutated cells in the L537-12 and L537-13 cases. However, these data also supply a clue that GGTA1 mutations caused by CRISPR/Cas9 plasmids might occur before and at the 2-cell stage of pig zygotes.

Table 2. Primer pairs for PCR

Notes

  1. If PAGE purified primers were used, more than 90% of clones analyzed were composed of correct sequences. E. coli JM109 competent cell was routinely used in our lab.
  2. The gene editing efficiencies of ppU6-GGTA138-60-gRNA, ppU6-GGTA1187-209-gRNA, ppU6-GGTA1181-159-gRNA, and ppU6-GGTA1223-201-gRNA, (site 1 to site 4, respectively) were first checked at cell level with LLC-PK1 cells (Su et al., 2015). Higher efficiencies were found by ppU6-GGTA1187-209-gRNA and ppU6-GGTA1223-201-gRNA. In this pronuclear microinjection experiment, all four ppU6-GGTA1-gRNA vectors were co-injected; coincidently, only indel mutations at site 1 and site 4 were detected.

Recipes

  1. TE buffer
    10 mM Tris-HCl, pH 8.0
    1 mM EDTA, pH 8.0
  2. TEN buffer
    10 mM Tris-HCl, pH 8.0
    1 mM EDTA, pH 8.0
    0.5 M NaCl

Acknowledgments

This protocol is adapted from our recent works (Su et al., 2015; Chuang et al., 2016). We want to thank all the members who have been involved in these works. This work is supported by grants NSC 101-2313-B-059-001 to C.F.T. as well as MOST 104-2321-B-886-002 and MOST 105-2321-B-886-001 to C.K.C. from the Ministry of Science and Technology of Taiwan.

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简介

构建了一套Cas9和单引导CRISPR RNA表达载体。 需要一个非常简单的程序来制备具有高目标精度的特异性单向RNA表达载体。 由于在1细胞期的小鼠胚胎中已经检测到了合并的合子转录,所以将编码Cas9和GGTA1基因特异性单向RNA的质粒DNA载体微注射到合子原核中以确认 1细胞猪胚胎现象。 我们的研究结果表明,这些CRISPR / Cas9质粒引起的突变发生在猪胚胎的2细胞阶段之前和之后,表明除了体外转录的RNA的细胞质显微注射外,CRISPR / Cas9 DNA载体为产生基因敲除猪提供了有效的解决方案。

背景 由于最初发现了大肠杆菌基因组(Ishino)中的大肠杆菌基因组下游的32bp间隔的串联重复的高度保守的29个碱基对(bp)序列,等等,1987; Nakata等人,1989),在约50%至50bp的大小变化的短定期间隔重复序列的家族中发现约50%细菌和90%的古细菌(Makarova等人,2015)。根据它们的特征结构,Mojica(Mojica等人,2009)和Jansen(Jansen等人)引入了定期交织的短回文重复序列(CRISPR)的名称, ,2002),目前普遍使用。首先通过核苷酸序列比对(Jansen等人,2002)在CRISPR基因座的侧翼首先鉴定了一组CRISPR相关基因 cas1 )。在不同的细菌物种中鉴定了新的成虫基因。根据每个主机内的成员,CRISPR-Cas系统可以根据标记基因分类为三种主要类型(Makarova等人,2011; Burmistrz和Pyrc,2015)。通过研讨会实验证明了CRISPR-Cas系统的功能。在被毒力噬菌体攻击后:噬菌体858和噬菌体2,972,在幸存的嗜热链球菌宿主细胞中,在CRISPR阵列的前端观察到新的重复 - 间隔单元。将新获得的间隔物的DNA序列与噬菌体基因组中的相应片段(称为原间隔物)匹配。具有噬菌体间隔物的嗜热链球菌菌株对噬菌体感染具有抗性,而没有噬菌体间隔区的菌株对噬菌体感染敏感(Barrangou等人, 2007)。为了区分宿主细菌基因组中的间隔物和与入侵基因组中与间隔物具有相同序列的原间隔物,进化了原间隔物相邻基序(PAM)(Mojica等人, em>,2009; Shah等人,2013)。 CRISPR-Cas免疫被揭示,它可以分为三个阶段(Rath等人,2015; Wright等人,2016)。第一个适应或收购阶段负责在暴露于外来移动遗传因素(如噬菌体或质粒)后将间隔物采集到CRISPR阵列中。对于所有三种类型的CRISPR-Cas系统(Sternberg等人,2016),Cas2同型二聚体被两个形成异六聚体复合物的Cas1同型二聚体夹持。在第二阶段,嵌入在CRISPR阵列上游的富含AT的前导序列内的启动子转录前体CRISPR RNA(前crRNA),这些被Cas蛋白进一步加工成短CRISPR RNA(crRNA)指导。 Cas6参与了I型和III型CRISPR-Cas系统(Charpentier等人,2015; Hochstrasser和Doudna,2015)中的RNA加工步骤。伴随着Cas9蛋白,包含用于与crRNA的重复区双相形成的抗重复区段的反式激活性crRNA(tracrRNA)涉及II型系统中的crRNA的成熟(Deltcheva等人。,2011)。在最后的干扰阶段,与成熟的crRNA和Cas蛋白级联合作,分别将I型和III型CRISPR-Cas系统的特征蛋白Cas3和Cas10整合到RNA引导的核酸内切酶复合物中。 II型效应物简单地由Cas9蛋白,一对加工的成熟crRNA和tracrRNA(Gasiunas等人,2012)组成。三元复合物中的crRNA和tracrRNA可以被融合的crRNA-tracrRNA单导向RNA(sgRNA)取代(Jinek等人,2012)。由于非常简单,现在通常称为CRISPR / Cas9的二元复合物的Cas9-sgRNA被立即应用于基因编辑领域(Cong等人,2013; Mali等人。,2013)。
 猪与人类分享一些解剖和生理特征。大多数引用为合适模型的系统包括心血管,尿,皮肤和消化系统(Swindle等人,2012)。因此,猪被认为是异种移植器官的良好来源。目前利用两种策略来克服种间排斥障碍。第一种是通过靶向GGTA1,CMAH和β4GalNT2基因来阻断表达引起高急性排斥反应的抗原的供体器官,例如半乳糖-α1,3-半乳糖,N-羟乙酰神经氨酸和β1,4-N-乙酰半乳糖胺, (Estrada等人,2015; Cooper等人,2016)。第二种策略是通过囊胚互补技术(Kobayashi等人,2010; Usui等人,2012),在替代动物中制备由受体细胞组成的器官, ; Matsunari等人,2013)。为了产生基因敲除猪,传统方法使用体细胞如胎儿成纤维细胞和体细胞核转移(SCNT)技术中的基因编辑来产生敲除合子。据报道,组合CRISPR / Cas9和SCNT的实例产生IgM J H(2015),RUNX3(Kang等人, em2,2016b),IL2RG(Kang等人,2016a)和GGTA1 / CMAH /β4GalNT2三重敲除猪(Estrada等人,2015)。 DNA或RNA的直接显微注射是制备基因敲除猪的另一种选择。由于在4细胞阶段(Anderson等人,1999)报道了猪合子中的从头转录的突变,因此优选用于微注射到胚胎中的RNA在1细胞阶段。以前的研究表明,Cas9 mRNA与sgRNA的细胞质显微注射可以产生Mitf(Wang等人,2015)和DJ-1 / Parkin / PINK1三基因敲除猪(Wang等人。,2016)。因为接合子合子转录仅在1细胞阶段的小鼠胚胎中报道(Ram和Schultz,1993; Bouniol等人,1995; Aoki等人[据报道,通过CRISPR / Cas9质粒的细胞质显微注射产生GGTA1敲除猪的试验。通过这种策略,CRISPR / Cas9在2细胞阶段或之后表达,并且发现了GGTA1基因上的镶嵌突变(Petersen等人,2016)。需要原核显微注射来解释合子转录是否发生在猪胚胎的1细胞期(Chuang等人,2016)。
 构建一系列Cas9和sgRNA表达载体,如图1所示。pCX-Flag sub2-NLS 1-sub-Cas9-NLS 2 ,pCX-HA-NLS 1 -Cas9-NLS 2 和pCX-Myc-NLS 1-Cas9-NLS 2 可用于在哺乳动物细胞中表达Cas9。三个常见的标签可用于监控Cas9表达式。 (图1A)可以有效地获得短发夹RNA的猪U6启动子[Chuang等人,2009]用于构建ppU6-(BsaI)2 -gRNA载体(图1B)(Su等人,2015)。每个靶位点需要一对含有间隔区和一部分CRISPR重复序列的引物,如图1B所示。因为鸟嘌呤(G)对于U6启动子是第一转录核苷酸是有利的,所以在我们最近的作品中仅选择用G启动的原间隔物。


图1. Cas9和单向RNA表达载体的方案。 A.pCX-Flag NLS -Cas9-NLS < pCX-HA-NLS 1 -Cas9-NLS 2&gt;和pCX-Myc-NLS 1-Cas9 -NLS <子> 2 ; B.ppU6-(BsaI)2 -gRNA和引物对指定。

关键字:CRISPR/Cas9, GGTA1, 原核显微注射

材料和试剂

  1. sgRNA表达载体的制备
    1. Bsa I(New England Biolabs,目录号:R0535)
    2. T4 DNA连接酶(Promega,目录号:M1801)
    3. 清洁和凝胶提取试剂盒(Biokit,目录号:Bio-C300)
    4. 2x连接缓冲液(Promega,目录号:C671A)
    5. pGEM-T Easy TA克隆试剂盒(Promega,目录号:A1360)
    6. Plasmid Miniprep Kit(Biokit,目录号:Bio-P300)
    7. Genomic DNA islation Kit(Thermo Fisher Scientific,Thermo Scientific TM,目录号:K0721)
    8. Neon TM转染系统100μl试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:MPK10096)
    9. 氯化铯(CsCl)(Avantor Performance Materials,J.T.Baker ,目录号:4042-02)
    10. Tris-HCl,pH 8.0
    11. EDTA,pH 8.0
    12. 氯化钠(NaCl)
    13. TE缓冲(见配方)
    14. TEN缓冲(见配方)

  2. 收集猪胚胎
    1. 用于胚胎闪烁的玻璃管(外径为4mm的玻璃管,如图2所示)
    2. Falcon管,50ml(Thermo Fisher Scientific,Thermo Scientific TM,目录号:339653)
    3. Regumate ®(含有0.4%Altrenogest,Intervet,MSD,France,品牌名称:Regumate ®
    4. PMSG(怀孕母马血清促性腺激素)(ASFK Pharmaceutical,Japan,品牌:PMSG)
    5. hCG(人绒毛膜促性腺激素)(ASKA Pharmaceutical,日本,品牌:hCG)
    6. PGF2α(前列腺素F2α;雌激素注射液)(Intervet Deutschland,德国)
    7. D-PBS(GE Healthcare,HyClone TM,目录号:SH30028.02)
    8. 胎牛血清(FBS)(GE Healthcare,HyClone TM,目录号:SH30071.03)

  3. 胚胎移植
    1. 3M纸带(3M,目录号:200-24mm)
    2. 硫酸阿托品(TAI YU CHEMICAL&amp; PHARMACEUTICAL)
    3. Stresnil(azaperona,40mg / ml)(Janssen Pharmaceutica N.V.,Belgium,品牌:Stresnil)
    4. 柠檬酸钠(Thiamylal sodium)(Shinlin Sinseng Pharmaceutical,Taiwan,品牌:Citosol)
    5. 阿莫西林(氨苄青霉素,150mg / ml)(中国化学药业,CCPG,目录号:E000853)
    6. 肝素钠(中国化学与制药,CCPG,台湾,品牌:AGGLUTEX INJECTION)

设备

  1. sgRNA抑制载体的制备
    1. 配备NVTi65转子的超速离心机(Beckman Coulter,型号:Optima XL-80)
    2. 微量离心机(KUBOTA,型号:3740)
    3. 干浴培养箱(主要科学,型号:MD-01N)
    4. 热循环仪(Thermo Fisher Scientific,Applied Biosystems TM,型号:2720)

  2. 精液评估
    1. 计算机辅助精子分析系统(UltiMate CASA系统,Hamilton-Thorne Research,Beverly,MA)

  3. 显微注射
    1. 离心机(KUBOTA,型号:8920)
      注意:本产品已停产。
    2. 微量离心机(KUBOTA,型号:3740)
    3. 倒置差分干涉对比显微镜(Olympus,型号:IX71)
    4. 毛细管注射针(Sutter Instrument,目录号:BF100-78-10)
    5. 毛细管注射针拔出器(Sutter Instrument,型号:P-97)
    6. 微操纵器(NARISHGE,型号:ON3-99D)
    7. 注射器(NARISHGE,型号:IM-9B)

  4. 收集猪胚胎和胚胎移植
    1. 操作表(NEWPORT,LW3048B-OPT,振动隔离表)
    2. 手术机械设计(NARISHIGE,JAPAN)

程序

  1. sgRNA表达载体的制备
    1. 取10μg的ppU6-(BsaI)2'-gRNA,并将其在50μl的Bsa I中在100μlNEB3.1缓冲液中在37℃下消化2 h。
    2. 用清理试剂盒纯化线性化载体DNA,并用30μl1 / 10x TE洗脱。储存纯化的线性化载体在-20°C使用前。
    3. 将表1中列出的引物溶解在TE中,并将浓度调节至50μM 注意:
      1. 将每个10μl正向和反向引物(表1,每个50μM)加入到小瓶中并与30μlTEN混合。在干燥块加热器中将混合物在90℃下孵育10分钟。
      2. 将混合物转移到65℃的干燥块中并孵育30分钟。然后让块逐渐冷却至室温约30分钟。将退火的引物对置于冰上直到下列连接反应
        表1. ppU6-sgRNA表达载体构建的引物


        猪GGTA1基因的最后一个外显子的第一个核苷酸被编号为+1,最后一个内含子的最后一个核苷酸被编号为-1(Su et al。,2015)。
    4. 对于连接反应,将1μlBsaI消化载体,3μl退火引物对,5μl2x连接缓冲液和1μlT4 DNA连接酶混合在冰上的小瓶中。然后在16℃孵育小瓶90分钟进行连接反应
    5. 转化后,从各个菌落分离质粒样品。使用M13反向引物进行DNA测序(注1)
    6. 通过两轮CsCl密度梯度超离心法纯化DNA进行原核显微注射。通常使用NVTi65转子,在20℃下将离心条件设定为218,000×g 20小时。

  2. 动物和动物护理
    1. 提高特定病原体免费农场的所有动物。喂食有限制饮食(4%体重)的动物免费使用水。
    2. 通常提高收件人,特别是在分娩期间特别小心处理。
    3. 所有动物应根据您的联邦和机构指导进行管理和治疗。

  3. 同步和超排卵
    1. 通过喂养补充有Regumate ®(含有0.4%Altrenogest; Intervet,MSD,France)的商业日粮15天,使供体和受体同步,以使其发情周期同步。向Regumate ®送达收件人一天。
    2. 在喂食Regumate ®的最后一天,肌内注射PGF2(250μgCloprostenol)以溶解任何剩余的黄体。
    3. 对于超排卵诱导,用PMSG和hCG肌内注射所有猪,分别诱导卵泡生长/卵母细胞成熟和排卵。简单来说,在最后一次喂食Regumate ®的第二天,注射所有捐助者2,000 i.u.的PMSG。然后用1,500 i.u注射他们hCG 78 h后用同样的方案注射接受者1,500 i.u. PMSG和1,250 i.u. hCG分别诱导卵泡生长/卵母细胞成熟和排卵。
    4. 对于人工授精,在hCG注射后24小时和36小时通过计算机辅助精子分析系统评估,通过计算机辅助精子分析系统评估含有至少30亿可行精子的新鲜稀释精液,具有80%迁移率。通过相同的方法诱导接受者的同步排卵,延迟12小时,无需授精。

  4. 原核卵母细胞恢复
    1. 在hCG注射后54-56 h内,全部捐献者都要牺牲上部子宫角和输卵管。
    2. 为了收集合子,请用玻璃管插入和连接漏斗,将D-PBS引导入50ml Falcon管。通过将20ml具有20%胎牛血清(FCS)的D-PBS注射到子宫交界处的输卵管中,从输卵管冲洗受精卵。 (图2)
    3. 将DPBS中的胚胎保留在37℃的保温箱中,并在30分钟内将其携带到实验室进行进一步的微操作。


      图2.从猪输卵管冲洗新受精卵。 成熟和排卵由具有排卵点的毛囊(箭头1)指示。使用具有钝端的不锈针(#19)穿透子宫壁,并将其穿过子宫交界处(箭头2)插入输卵管(箭头3)。玻璃管(箭头5号,外径4 mm)通过漏斗开口(箭头4)连接到输卵管的另一端。输卵管内的受精卵用注射器(箭头6)用20ml D-PBS冲洗,并收集在50ml Falcon管(箭头7)中。

  5. 显微注射
    1. 将回收的受精卵以15,000 x g离心10至15分钟,以暴露其原核(图3)。
      注意:
      1. 然后将胚胎放入装有差分干涉对比度系统的倒置显微镜上的20μlD-PBS微滴中。
      2. 在300倍放大倍数下执行原核显微注射。
      3. 将胚胎置于适当的位置,以显示原核的清晰视图。
    2. 然后通过毛细管针将CRISPR / Cas9 DNA混合物显微注射入一个原核。注射吸管的尖端开口直径约为0.5μm。在施加注射压力后,通过将受精卵从显微注射毛细管的尖端移开来显示DNA的连续流动流。在注射到原核中之前,将移液管尖端置于周围空洞(PVS)中。通过根据PVS的溶胀速度增加或减少注射压力来调节合适的注射流速。对于pCX-Flag 2-NLS 1-Sub-Cas9-NLS 2&amp; 2&gt;和2ng /μl,质粒DNA的浓度为6ng /μl,四个ppU6-sgRNA载体中的每一个(注2)

      图3.猪胚胎的原核显微注射。 离心对于从猪胚胎中的光密度含量显示由箭头指示的原核是至关重要的。比例尺=20μm。

  6. 胚胎移植
    1. 将CRISPR / Cas9质粒载体显微注射到原核后,通过手术方法通过移液管将20至30个胚胎转移到输卵管中:
      1. 在运行前,对所有接收者进行24小时禁食,免费使用水。
      2. 手术前20分钟,注射5毫克硫酸阿托品和400毫克Stresnil以分解唾液排泄物并分别镇静动物。
      3. 设置耳静脉导管,并用3M纸带固定i.v。注射Citosol保持麻醉,持续时间为40〜50分钟。
      4. 在操作台上加载并固定收件人。清洁他们的腹部皮肤,剃掉头发。然后用无菌衣服覆盖动物。
      5. 在最后2个 nd 到3 rd 乳头之间的linea alba上创建一个8-10厘米的窗口,拉出单侧的子宫角,输卵管和腹部外的卵巢,然后计算卵巢上的黄体数量。将子宫角的最大部分立即放回腹腔。保留剩余的上部子宫角,输卵管,漏斗和卵巢以进行胚胎移植(图4A)。
      6. 在通过口吸管控制的玻璃转移管中的D-PBS中加载10-15个胚胎并将胚胎转移到输卵管峡部(图4B)。
      7. 用含有硫酸肝素(50,000 U / L)的盐水冲洗子宫角,然后放回腹腔内。
      8. 在缝合切割窗口之前,将10ml的阿莫西林注入腹腔内。
      9. 胚胎移植后,通过连续缝合封闭腹壁腹壁和肌肉,并通过中断的垂直床垫缝合脂肪层和皮肤。
      10. 最后,注射10毫升阿莫西林在接下来的三天颈部,以防止感染。


        图4.胚胎移植 A.上部子宫角,子宫,输卵管,漏斗和卵巢保留在外面用于胚胎移植。 B.将胚胎装载在通过口吸管控制的玻璃管中,并转移到输卵管峡部(缩写:u为子宫,呃为子宫角,ft为输卵管,inf为漏斗)。 />

  7. 饲养和仔猪护理
    1. 在预期交货日期前七天将母猪移至母猪。
    2. 观察母猪的紧张和不安的行为;用手挤奶进一步检查哺乳期。
    3. 在母猪开始哺乳的那一天,仔细检查母猪,确定其中任何一个需要帮助,并照顾新生儿。
    4. 在分娩完成的第二天,收集仔猪的尾部组织样品进行DNA提取和进一步的基因组分析。

数据分析

从猪尾组织分离的基因组DNA样品通过使用表2所示引物的PCR进行分析。将PCR产物克隆到pGEM-T Easy TA克隆载体中。对质粒的单个克隆进行测序以解释突变。在典型的实验(Chuang等人,2016)中,将41个原核微注射的胚胎转移到两个代孕母猪中。其中一只怀孕,7只小猪被送出。检测到两个创始人L537-12和L537-13在尾组织中携带GGTA1突变。 L537-12样品中显示野生型DNA序列和位点1处的4 bp(TTGG)缺失。在L537-13样品中鉴定出7 bp(TGGTTGG)缺失和另外7 bp(CATGGTT)缺失。在与野生型配子交叉后,在L537-12的14个后代中的5个中测量了4bp(TTGG)缺失。在L537-13的13个后代中,分别在5和3中检测到7 bp(TGGTTGG)和7 bp(CATGGTT)缺失。值得注意的是,在最近的13个后代中,一只仔猪携带了149bp的缺失(位点1和位点4之间的片段),这在创始人的尾部组织中没有被检测到。理论上,对于异源雄性,一半的精子是突变体。对于50%的马赛克男性,25%的精子是突变体。由于L537-12创始人14个后代中的5个携带了4 bp(GGTT)缺失,所以表明镶嵌率在50%到100%之间。由于L537-13创始人13个后代的5和3个分别携带7 bp(TGGTTGG)和7 bp(CATGGTT)缺失,因此也支持这一结论。携带149bp缺失的L537-13的其他后代表明缺失反应发生在4细胞胚胎期后。 L537-12和L537-13病例中大部分种系都可能被突变细胞的下降所占据。然而,这些数据也提供了一个线索,即CRISPR / Cas9质粒引起的GGTA1突变可能发生在猪合子的2细胞阶段之前和之中。

表2. PCR引物对

笔记

  1. 如果使用PAGE纯化引物,超过90%的分析克隆由正确序列组成。 电子。大肠杆菌JM109感受态细胞常用于我们的实验室。
  2. ppU6-GGTA1 38-60 -gRNA,ppU6-GGTA1 187-209 -gRNA,ppU6-GGTA1 181-159 的基因编辑效率首先用LLC-PK1细胞在细胞水平检查-gRNA和ppU6-GGTA1 223-201 -gRNA(分别位点1至位点4)(Su等人, em>,2015)。通过ppU6-GGTA1 187-209 -gRNA和ppU6-GGTA1 223-201-gRNA发现更高的效率。在这种原核显微注射实验中,共注射所有四种ppU6-GGTA1-gRNA载体;一致地,仅检测到位点1和位点4的indel突变

食谱

  1. TE缓冲区
    10mM Tris-HCl,pH8.0
    1mM EDTA,pH 8.0
  2. TEN缓冲区
    10mM Tris-HCl,pH8.0
    1mM EDTA,pH 8.0
    0.5 M NaCl

致谢

这个协议是从我们最近的作品(Su <等人,2015; Chuang等人,2016)改编而成。我们要感谢参与这些作品的所有成员。这项工作得到NSC 101-2313-B-059-001授权给C.F.T.的支持。以及MOST 104-2321-B-886-002和MOST 105-2321-B-886-001至C.K.C.来自台湾科技部。

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  • English
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chuang, C., Tu, C. and Chen, C. (2017). Generation of Mutant Pigs by Direct Pronuclear Microinjection of CRISPR/Cas9 Plasmid Vectors. Bio-protocol 7(11): e2321. DOI: 10.21769/BioProtoc.2321.
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