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Jan 2021
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CRISPR-mediated Labeling of Cells in Chick Embryos Based on Selectively Expressed Genes
基于选择性表达基因的CRISPR介导的鸡胚胎细胞标记   

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Abstract

The abilities to mark and manipulate specific cell types are essential for an increasing number of functional, structural, molecular, and developmental analyses in model organisms. In a few species, this can be accomplished by germline transgenesis; in other species, other methods are needed to selectively label somatic cells based on the genes that they express. Here, we describe a method for CRISPR-based somatic integration of reporters or Cre recombinase into specific genes in the chick genome, followed by visualization of cells in the retina and midbrain. Loci are chosen based on an RNA-seq-based cell atlas. Reporters can be soluble to visualize the morphology of individual cells or appended to the encoded protein to assess subcellular localization. We call the method eCHIKIN for electroporation- and CRISPR-mediated Homology-instructed Knock-IN.

Keywords: Chick embryo (小鸡胚胎), Cre (Cre), CRISPR (CRISPR), GFP (绿色荧光蛋白), Homologous recombination (同源重组), Electroporation (电穿孔)

Background

Many functional, structural, molecular, and developmental analyses in model organisms now rely on the abilities to mark and manipulate specific cell types. In a limited number of species – Mus musculus (mice), Danio rerio (zebrafish), C. elegans (worms), and Drosophila melanogaster (flies) – this can be accomplished by germline transgenesis. For example, transgenic animals can be generated in which regulatory elements from a gene selectively expressed by a cell type of interest drive the expression of a reporter.


Alternatively, the transgene can encode an effector such as Cre recombinase, which enables selective expression of a second transgene provided either in the germline or to somatic cells via a viral or other vector (Luo et al., 2018).


For other model organisms in which germline transgenesis is currently infeasible or limited, few methods are available for the selective labeling of somatic cells. One option is to incorporate cell type-specific promoters into viral vectors, but to date, this approach has generally resulted in labeling groups of related cell types (discussed in Domenger and Grimm, 2019; Juttner et al., 2019). Genome editing using CRISPR (clustered regularly interspaced short palindromic repeats) (Jinek et al., 2012; Cong et al., 2013; reviewed in Pickar-Oliver and Gersbach, 2019; Nishizono et al., 2020) provides an alternative approach. The CRISPR-associated endonuclease Cas9 is delivered to specific sites in the genome by a single guide RNA (sgRNAs) to generate double-strand breaks. The breaks can be repaired by non-homologous end-joining (NHEJ) or homology-directed repair (HDR) (Sander and Joung, 2014; Yeh et al., 2019). If cDNAs encoding reporters are flanked by the sequences flanking the cut site, HDR can be harnessed to insert them into the genome at defined sites. Because homologous recombination activity is largely confined to mitotically active cells, methods involving HDR are generally applied to dividing cells (Heidenreich and Zhang, 2016).


The HDR method has primarily been used to generate germline “knock-ins” (Hsu et al., 2014; Nishizono et al., 2020) but can also be used to label somatic cells. By transducing dividing cells in the mouse brain with CRISPR-Cas9-based HDR tools using in utero electroporation, Mikuni et al. (2016) developed a method called SLENDR (single-cell labeling of endogenous proteins by CRISPR-Cas9-mediated homology-directed repair). This method introduces a plasmid encoding an sgRNA and Cas9, along with a single-stranded DNA (ssDNA) encoding a protein tag embedded between sequences that flank the sgRNA recognition site; this results in the HDR-mediated introduction of the tag at the desired genomic location. Related methods have been reported by Uemura et al. (2016), Nishiyama et al. (2017), and Matsuda and Oinuma (2019); Suzuki et al. (2016) devised a method in which homology-independent target integration by NHEJ allows tagging in post-mitotic cells.


Here, we modified these methods for use in chick (Gallus gallus) embryos. Chicks have been used for embryological studies for over a century and remain a well-used model (Stern, 2005). However, while germline transgenesis has been demonstrated in chicks, it remains a difficult and rarely used method (Lee et al., 2017). Moreover, even though the method is likely to improve, few laboratories are equipped with the facilities for the avian husbandry that would be needed to generate or maintain transgenic lines.


Our own studies on the development of the chick retinotectal system have used viral and electroporation methods for gene transfer into chick embryos (e.g., Gray et al., 1988; Galileo et al., 1992; Leber et al., 1996; Yamagata et al., 2002; Yamagata and Sanes, 2008a). However, none of these enabled reliable labeling of specific cell types because neither cell type-specific promoters nor inventories of selectively expressed genes were available. Recently, we addressed the second of these problems by generating a cell atlas of the chick retina using high-throughput single-cell RNA-seq, which provided a set of genes selectively expressed by each of its ≥136 cell types (Yamagata et al., 2021). We therefore attempted to use the CRISPR-based methods described above to insert reporters or tags into some of these genes.


The efficiency of those methods was, unfortunately, unworkably low, leading us to optimize the method in ways detailed here. Innovations include those described next. (A) Whereas previous methods for editing somatic cells introduced Cas9 via a cDNA, sufficient Cas9 protein production may occur too late for efficient editing. For this reason, CRISPR-dependent methods for germline HDR have introduced Cas9 mRNA (Yang et al., 2013; Takahashi et al., 2015), avoiding the need for transcription or translation of the Cas9 protein (Aida et al., 2015; Chen et al., 2016; Troder et al., 2018; Gurumurthy et al., 2019). We adopted this approach using the Cas9 protein to generate a Cas9-ribonucleoprotein complex. (B) In Streptococcus pyogenes, Cas9-based CRISPR uses an RNA that targets a genomic locus (crRNA; 35-36 nt in length) and a sequence that recruits Cas9 (tracrRNA; 67 nt). For most gene editing applications, these are fused to form an sgRNA of 99-100 nt. We used separate crRNA and tracrRNA because they are cheaper to purchase and likely to act more efficiently. (C) Because the brain vesicles of chick embryos are far larger than zygotes, far more ssDNA is required. To produce sufficient quantities, we used asymmetric PCR (Marimuthu et al., 2012). (D) We added reagents to enhance HDR – carrier DNA and a DNA ligase IV inhibitor (Hu et al., 2018). We call the method eCHIKIN (electroporation- and CRISPR-mediated Homology-instructed Knock-IN).


Figure 1 summarizes the key steps of eCHIKIN. The method begins with the design and preparation of CRISPR HDR reagents (Steps 1-3), which must be finished prior to in ovo electroporation. On the day of electroporation, a reagent cocktail including the CRISPR-Cas9 ribonucleoprotein (RNP) complex is prepared (Steps 4-6) and used for injection and electroporation (Steps 7 and 8). At later stages, tissues are dissected, fixed, and analyzed histologically (Step 9).



Figure 1. Overview of the eCHIKIN workflow. The main steps are the design and preparation of RNA and DNA reagents (Steps 1-3), preparation of the CRISPR-Cas9 cocktail (Steps 4-6), in ovo injection and electroporation (Steps 7 and 8), and immunohistochemical analysis (Step 9). Some panels are from https://togotv.dbcls.jp/en/.

Materials and Reagents

  1. 1.5-ml microcentrifuge tubes (DNase, RNase-free low adhesion microcentrifuge tubes) (USA Scientific, catalog number: 1415-2600)

  2. Fertilized chicken eggs: Specific-pathogen-free chicken (SPF) eggs from Charles River Laboratories (Wilmington, MA, USA)

    Upon receipt, eggs should be stored at 16°C and used within 1 week. Embryonic development resumes when the eggs are placed into a humidified incubator at 38°C. Embryos can be staged according to the system of Hamburger and Hamilton (1951).

    Note: The temperature of the eggs is sometimes poorly controlled during shipping, which decreases their viability, and does so differentially during summer and winter.

  3. Alt-R® CRISPR-Cas9 crRNA, 2 nmol, designed as detailed below (Integrated DNA Technologies (IDT))

  4. Alt-R® CRISPR-Cas9 tracrRNA, 20 nmol (IDT, catalog number: 1072533)

  5. Alt-R® S.p. Cas9 Nuclease V3, 100 µg (IDT, catalog number: 1081058)

  6. Alt-R® Cas9 Electroporation Enhancer, 2 nmol (IDT, catalog number: 1075915)

  7. Alt-R® HDR Enhancer, 100 µl (IDT, catalog number: 1081072)

  8. 10× Reaction Buffer (with Cas9 nuclease from GenScript, catalog number: Z03386) (200 mM HEPES, 1 M NaCl, 50 mM MgCl2, 1 mM EDTA, pH 6.5 at 25°C)

  9. Nuclease-free water (tubes with Alt-R® CRISPR-Cas9 RNAs from IDT)

  10. Hanks’ Balanced Salt Solution (HBSS, without calcium, magnesium, or phenol red, Thermo-Fisher Scientific, catalog number: 14175095)

  11. DNA oligo 100 nmol scale, designed as discussed in the protocol (IDT)

  12. Q5 DNA polymerase (NEB, catalog number: M0491)

  13. Deoxynucleotide (dNTP) Solution Mix (NEB, catalog number: N0447S)

  14. Exonuclease I (E. coli) (NEB, catalog number: M0293S)

  15. EconoTaq PLUS GREEN 2× Master Mix (Lucigen, catalog number:30033-1)

  16. QIAquick Gel Extraction Kit (Qiagen, catalog number: 28704)

  17. 2-Propanol BioReagent, for molecular biology, ≥99.5% (Sigma-Aldrich, catalog number: 19516)

  18. Isoamyl alcohol, ≥98%, FG (Sigma-Aldrich, catalog number: W205702)

  19. Ethanol Anhydrous 200 Proof (Koptec, catalog number: V1016)

  20. pXL-BacII-CAG-Venus (plasmid containing piggyBac inverted terminal repeat (ITR) sequences flanking CAG and Venus) (Yamagata and Sanes, 2012)

  21. pXL-BacII-CAG-mCherry (plasmid containing piggyBac ITRs flanking CAG and mCherry)

  22. pXL-BacII-CAG-loxP-STOP-loxP-Venus (plasmid containing piggyBac ITRs flanking CAG, a loxP-STOP-loxP cassette and Venus)

  23. pCAG-mPBorf (plasmid containing piggyBac transposase, codon-optimized)

  24. tPT2A-Venus-pCR21 (plasmid containing tPT2A-Venus sequence) (This plasmid will be available from Addgene, plasmid 170521)

  25. pCAG-Cre:GFP (Addgene, 13776)

  26. Rabbit anti-GFP (Millipore, catalog number: AB3080P)

  27. P-RAN-GFP1 Supernatant (Yamagata and Sanes, 2018a; Addgene plasmid, 106408)

  28. TSA Plus Fluorescein Evaluation kit (PerkinElmer, NEL741E001KIT)Standard lab reagents for molecular biology (e.g., 70% ethanol, agarose for gel electrophoresis)

  29. Agarose (Agarose RA (Amresco); VWR Life Science, catalog number: 97064-258)

  30. Buffer for agarose electrophoresis: 10× Tris-Acetate-EDTA (TAE) (Mediatech, catalog number: SC45001-074)

  31. Kanamycin sulfate (Sigma-Aldrich, catalog number: K1377) (see Recipe 1 below for HBSS-kanamycin)

  32. Fast Green FCF, Dye content ≥85% (Sigma-Aldrich, catalog number: F7252) (see Recipe 2 below for stock solution)

  33. Chloroform, contains 100-200 ppm amylenes as a stabilizer, ≥99.5% (Sigma-Aldrich, catalog number: C2432) (see Recipe 3 below for Chloroform-isoamyl alcohol (25:1))

  34. Phenol, BioUltra, for molecular biology, ≥99.5% (GC) (Sigma-Aldrich, catalog number: 77608) (see Recipe 4 below for Phenol-Chloroform (1:1))

  35. Plasmids (#20, #21, #22) are available from Addgene (Addgene plasmids 170518, 170519, 170520), but current restrictions prevent Addgene from distributing plasmids encoding piggyBac transposase (https://www.addgene.org/terms/1118/). Phenol-chloroform treatment is required for all the plasmids for injection (see Recipe 5 below).

Equipment

  1. Egg incubator

    Many incubators are available commercially; we use a 1550 HATCHER (GQF MFG, Savannah, GA). The incubator should be kept humidified AND set to 38°C. Cartons in which eggs are delivered can be used as holders for incubation and electroporation.

  2. ECM 830 square wave electroporation system (BTX; Holliston, MA)

  3. Tungsten wire (0.1-mm diameter)

  4. Glass capillaries, 3-inch length, 1-mm diameter (World Precision Instruments, 1B100F-3)

  5. Genetrodes, 3-mm L-Shape (GOLD TIP) 45-0116

    Accessories for holding and connecting electrodes to the electroporator are available from several vendors, including BTX and NEPAGENE.

  6. Scotch transparent tape 2592 (25.4-mm width)

    Warning: Chemical adhesives in some tapes from other vendors are toxic to embryos. Suspect tape if the survival rate is low.

  7. 18 ½ G needles

  8. 10-ml syringes

  9. Sharp-edged curved surgical scissors (~4 inches)

  10. #5 Forceps

  11. Mastercycler Pro Thermal Cycler (Eppendorf, 950040015)

  12. Strips of eight tubes, 0.2-ml (Corining PCR-0208-A and PCR-02CP-A)

  13. Dissection stereoscope (for electroporation)

    Carl Zeiss Stemi 2000 (444036-9000) equipped with 10×/23 eye pieces (magnification range: 6.5-50×). The viewing magnification should be adjusted as desired

  14. Fluorescence stereozoom microscope (Olympus, SZX12 equipped with the cubes for observing GFP/fluororescein and mCherry/rhodamine fluorescence

  15. Spectrophotometer (ThermoFisher, model: NanoDrop 2000) to measure OD260

Procedure

  1. Preparation of critical reagents

    1. CRISPR-RNA (crRNA) (IDT)

      Guidelines for designing crRNA are described in Note 1.

    2. ssDNA donor

      Guidelines for designing the ssDNA donor are described in Note 2.

      1. Generate a double-stranded DNA template bearing homology arms.

        Create a double-stranded DNA template (for an example, see Figures 2A and 2B) using two 90-bases ssDNA primers (for an example, see Figure 2C) and a GFP template (for example, pXL-BacII-CAG-Venus). We use a high-fidelity PCR enzyme (Q5 DNA polymerase), perform a 100-µl reaction in a thermocycler, run agarose gels, and purify the appropriately sized major PCR product (Left arm + GFP + right arm: ~850 bp; Figure 2C) using the QIAquick Gel Extraction Kit.



        Figure 2. Designing crRNA and the ssDNA donor: TFAP2A is given as an example. (A) The TFAP2A gene in the UCSC Genome Browser. (B) Genomic sequence of TFAP2A in the targeted region and TFAP2A-crRNA as shown in A. (C) 90 nucleotide primer sequences that anneal to the 5’ (ATG) and 3’ ends of GFP and the structure of the single-strand donor DNA used for inserting GFP into the TFAP2A locus. (D) Primers and single-strand donor DNA for inserting CRE into the TFAP2A locus. See Table 1 for sequences.


        This PCR uses standard methods and equipment. A typical reaction is for 25 cycles (2 min, 95°C; 25 cycles of 94°C, 30 s/60°C, 30 s/72°C, 1 min + 1 s extension at 72°C/cycle; 72°C, 7 min; 4°C). If a clean single PCR band is not obtained, try using buffers optimized for GC-rich templates and/or adding DMSO.


        Table 1. Primer sequences (see Figure 2 and Figure 8)


      2. Optimize PCR for ssDNA generation

        Before carrying out a large-scale preparation of ssDNA by asymmetric PCR, it is helpful to optimize the reaction on a small scale. We use a robust and low-price PCR enzyme mixture for this purpose. First, using the double-stranded DNA template prepared above, we set up two reactions containing different molar ratios of primers: Reaction #1, 1 forward:100 reverse; Reaction #2, 100 forward:1 reverse.


        Reaction #1 and Reaction #2 (2 tubes)

        50 µl EconoTaq PLUS GREEN 2× Master

        47 µl dH2O

        1 µl ~0.01 µg/µl template from Step A2a

        1 µl 0.01 mM primer: (Reaction #1) Forward or (Reaction #2) Reverse

        1 µl 1 mM primer: (Reaction #1) Reverse or (Reaction #2) Forward

        *Note that typical PCR uses 0.1 mM primer stocks. Split into two PCR wells (50 µl each).

        94°C, 2 min

        40 cycles of 94°C, 30 s/60°C, 30 s/72°C, 1 min + 1 s extension at 72°C/cycle

        72°C, 7 min

        4°C


        Run 1%(w/v) agarose gels (with ethidium bromide). Note that ssDNA is less effectively stained with ethidium bromide than is double-stranded DNA.


        A typical result is shown in Figure 3A. In this case (TFAP2A), Reaction #1 gave one intense band (~850 bp) and several faint bands, including one that co-migrated with a ~500 bp double-strand DNA marker. Reaction #2 gave two clean bands: the slow-migrating band (asterisk) corresponds to double-stranded DNA (~850 bp) based on its predicted size. However, electrophoretic migration of ssDNA is often anomalous in regular non-denaturation electrophoresis buffers, making it difficult to be sure which band corresponds to ssDNA species. It is therefore useful to incubate a portion of the PCR product (2 µl) with E. coli exonuclease I, which specifically digests linear ssDNA. As shown in Figure 3B, some bands from the digest, including part of the diffuse band at ~500 bp, were resistant to E. coli exonuclease I digestion, suggesting that some double-strand DNAs were present at ~500 bp. By contrast, in Reaction #2, the corresponding band was less diffuse and was completely digested by E. coli exonuclease I, indicating that it contained only ssDNA (arrow). Therefore, we used the conditions of Reaction 2 for large-scale preparation.



        Figure 3. Assessing the quality of ssDNA. (A) Products generated by asymmetric PCR (molar ratio of primers (forward/reverse): 1/100 in Reaction #1; 100/1 in Reaction #2). (B) PCR products were digested with E. coli Exonuclease I (E. coli Exo I), which specifically digests single-strand DNA (arrow). An additional band (asterisk) corresponds to double-strand DNA. See text for details.


      3. Large-scale preparation of the ssDNA donor

        Scale-up the reaction optimized in b and then purify the ssDNA donor.


        500 µl EconoTaq PLUS GREEN 2× Master

        470 µl dH2O

        10 µl ~0.01 µg/µl template from Step A2a

        10 µl 0.01 mM Reverse primer

        10 µl 1 mM Forward primer

        Split into 20 PCR wells (50 µl each)

        94°C, 2 min

        40 cycles of 94°C, 30 s/60°C, 30 s/72°C, 1 min +1 s extension

        72°C, 7 min

        4°C


        Run a large agarose gel and purify ssDNA using a QIAquick Gel Extraction Kit.


        It is important to use thin wells so that the bands are clearly separated. Typically, we use an 11-cm wide × 13-cm long × ~7-mm thick gel. After solubilizing the agarose in the QG solution (QIAquick Gel Extraction Kit), add 20% volume 2-propanol, and load onto 4 mini spin-columns. Elute each tube with 100 µl EB (QIAquick Gel Extraction Kit) and 50 µl EB, successively. Add 1:10 volume 5 M NaCl, extract twice with phenol-chloroform, then twice with chloroform, and finally ethanol-precipitate by adding 2.5 volumes -20°C ethanol. Rinse the pellets three times with 70% ethanol at -20°C. Spin down, completely remove the residual ethanol, dry, and resuspend in RNase-free water (total 20 µl).

        Estimate the DNA concentration using a conventional or NanoDropTM (Thermo Fisher) spectrophotometer. Note that OD260 of ssDNA is lower due to hyperchromicity (40 µg/ml for OD260 as opposed to 50 µg/ml for double-stranded DNA). Adjust the concentration to 1 µg/µl, freeze, and store at -20°C until use.


  2. Equipment setup

    Figure 4 shows a stereomicroscope setup for injection and electroporation (Figure 4A and 4B).



    Figure 4. Equipment and instruments required for eCHIKIN. A. Setup of the electroporater, dissection microscope, and electrode holder. B-D. Cathode (+) and tungsten anode (-). E. Glass capillary loaded with eCHIKIN cocktail plus Fast Green.


    1. Tungsten needle

      Cut a 0.1-mm tungsten wire to an appropriate length (~2 cm) and sharpen the tip. We sharpen it electrically (Brady, 1965) (see Figure 4C and 4D).

    2. Glass capillaries for injection

      Make glass capillaries for injection. We use home-made micropipettes that are finely drawn from glass capillaries (World Precision Instruments, 1B100F-3 (3-inch, 1-mm) using a puller. Break off the last few mm of the tip to allow facile injection while maintaining sharpness (Figure 4E).


  3. Incubation of eggs

    At appropriate times prior to injection and electroporation (see below), place the eggs in the incubator. Eggs need to be incubated on their side (horizontally; Figure 5A). Mark the top of each egg since the embryo will float on top of the yolk. Incubate more eggs than you plan to inject because some are likely to be infertile or have embryos poorly placed for injection.



    Figure 5. Preparation of chick embryos for injection and electroporation. A. Eggs incubated on their side. B-E. Placing plastic tape on the top of an egg and removing egg albumin from the round side of the egg. F, G. Using surgical scissors with curved blades, a small window (~5-mm diameter) is made on top of the egg by cutting through the tape and shell together.


  4. Injection and in ovo electroporaton

    1. Preparation of injection cocktail

      On the day of in ovo electroporation, prepare the cocktail for injection. All plasticware and reagents must be RNase-free.

      1. crRNA:transcrRNA/Cas9 RNP (Solution “R”)

        To reconstitute the guide RNA duplex, the target-specific crRNA (35-36 nt) and tracrRNA (67 nt) are combined, heat-denatured, and annealed by slowly chilling to room temperature. In a 1.5-ml tube, add these reagents in this order:


        2.5 µl dH2O

        0.5 µl 10× Reaction Buffer

        1 µl crRNA (2 nmol/20 µl dH2O; concentration, 100 µM)

        1 µl tracrRNA (2 nmol/20 µl dH2O; concentration, 100 µM)


        Spin briefly and incubate for 5 min at 95°C in a heat block.

        Leave at room temperature so that cooling is slow, maximizing annealing.

        Leave for 5 min, briefly spin, and incubate for 30 min at room temperature.


        Add 0.8 µl 10 mg/ml Cas9 protein (Streptococcus pyogenes Cas9 Nuclease V3), mix, spin briefly, and incubate for 60 min at room temperature. This solution is Solution “R.”


      2. Denaturation of the ssDNA donor

        The ssDNA donor should be prepared in advance and stored at -20°C until use.

        Thaw the frozen aliquot, heat at 95°C for 5 min, and rapidly cool on ice. This denaturation can be carried out simultaneously with the annealing of crRNA:tracrRNA/Cas9 RNP described in the previous step. However, in contrast to slow annealing of the crRNA:tracrRNA, this tube needs to be cooled quickly by placing on ice immediately. Spin the tube briefly before use.

      3. Dilution buffer with HDR enhancer (Solution “D”)

        In an RNase-free 1.5-ml tube, add these reagents in this order:


        21.5 µl dH2O

        2.5 µl 10× Reaction Buffer

        1 µl Alt-R® HDR Enhancer (available as a solution suspended in DMSO). Mix well and spin briefly.


      4. piggyBac plasmid mixture (Solution “P”)

        For GFP reporter eCHIKIN, mix in a 1.5-ml tube:

        45 µl 1 µg/µl pXL-BacII CAG-mCherry

        5 µl 1 µg/µl pCAG-mPB


        For Cre reporter eCHIKIN, mix in a 1.5-ml tube:

        22.5 µl 1 µg/µl pXL-BacII CAG-mCherry

        22.5 µl 1 µg/µl pXL-BacII CAG-loxP-STOP-loxP-Venus

        5 µl 1 µg/µl pCAG-mPB

        Note that these plasmid DNAs must be RNase-free (see Recipe 5 and Note 3).


      5. Final injection cocktail

        To a tube of Solution “R” (total 5.8 µl), add these reagents in this order (see Note 4):

        11 µl Solution “D”

        3 µl 1 µg/µl ssDNA donor (denatured at 95°C in advance)

        3 µl Alt-R® Cas9 Electroporation Enhancer (2 nmol/50 µl; concentration, 40 µM)

        3 µl Solution “P”

        2 µl 0.1%(w/v) Fast Green


        Mix well and place this solution on ice. Use within 5-6 h.


    2. Preparation of eggs and embryos

      1. Embryos reach stage 9-10 in ~40 h at 38°C. Thus, if eggs are placed in the incubator at 8 pm (Day 0), they should be ready to electroporate at noon on Day 2. However, embryos should be staged according to Hamburger and Hamilton (1951) because the developmental rate is sensitive to small changes in temperature and also varies among eggs. To slow development when planning to inject many embryos, eggs can be removed from the incubator and maintained at room temperature for up to 5-6 h without compromising viability.

      2. Spray eggs with 70% ethanol and allow to dry. Always keep the previously marked top side of the egg facing upward (Figure 5B).

      3. Apply a piece of transparent tape to the top of each egg (Figure 5C).

      4. Insert an 18 ½ G needle on a 10-ml syringe into the rounded side of each egg (Figure 5D); this side contains the air sac.

      5. Remove 1-2 ml egg albumin depending on the size of the eggs (Figure 5E). Some researchers omit this step to obtain better survival.

      6. Make a small window (~5-mm diameter) on top of the egg by cutting through the tape and shell using surgical scissors with curved blades (Figure 5F and 5G).

    3. Injection and in ovo electroporation

      1. If necessary, gently rock the egg so that the embryo sits on top of the yolk. Make the window larger if needed (Figure 6A). In some cases, the translucent membrane beneath the shell needs to be carefully removed using #5 forceps.



        Figure 6. Key steps of injection and in ovo electroporation. (A) Locate the embryo and expand the window. (B) Place the cathode as in G and inject the reagent cocktail (blue) into the embryo. (C) Wet the cathode and embryo with Hanks’ + kanamycin. (D-E) Insert the anode as in G and electroporate. After successful electroporation, small bubbles appear at the anode. (F) Seal the egg with plastic tape. It is important to seal the small hole (green) that was made for removing albumin. (G) Summary.


      2. Place the egg under the dissection microscope. For beginners, it may be challenging to see enough detail in the embryos for accurate injection. One aid is to inject a few µl black India ink beneath the embryo. Images in Hamburger and Hamilton (1951) are useful for orientation.

      3. Place the L-shaped cathode parallel to the embryo (Figure 6A and 6G).

      4. Load the CRISPR cocktail into a micropipette (Figure 4E) and inject ~0.1-0.2 µl using a mouth pipette or microinjector (e.g., FemtoJet, Eppendorf). To transduce the retina, inject to one side of the optic vesicle of stage 9-10 embryos (Figure 6B and 6G). To transduce the optic tectum, injection should be targeted to the midbrain vesicle at stage 11-12.

      5. Wet both the cathode and embryo with 20 µl HBSS + kanamycin using a pipette tip (Figure 6C).

      6. Insert the fine tungsten anode of the electroporator into the head of the embryo (Figure 6D and 6G).

      7. Deliver square wave pulses (Figure 6E and 6G). For optic vesicles, we use 6 pulses (7 V/25 ms) at 1-s intervals. A sign of successful electroporation is the appearance of numerous tiny bubbles at the anode.

      8. Close the window with transparent tape (Figure 6F). It is important to also seal the small hole (green arrowhead in Figure 6F) that was made to remove the albumin.

      9. Place the eggs back in the 38°C incubator.

        This technique (Steps D2 and D3) relies heavily on the performer’s skill and requires considerable practice. Experts can inject one embryo every few minutes or ~120 embryos in a 4-h session.


  5. Histological analysis

    Chick embryos hatch at E21; however, mortality increases greatly from E17. This is a common consequence of manipulations that introduce a window in the eggshell; it is not specific to eCHIKIN. Thus, it is best to retrieve tissue by E16, if possible. Both the retina and optic tectum are fairly mature by that age.

        Although other methods can be used, the main analytical method is immunohistology. Embryos are retrieved, and the appropriate regions (retina or tectum in the cases described here) are dissected. The tissues are fixed with 4% (w/v) paraformaldehyde/PBS for 15 h, sunk in 15% (w/v) and 30% (w/v) sucrose/PBS successively, embedded in Tissue Freezing Medium (Triangle Biomedical Sciences), frozen at -80°C, and sectioned at 20 µm in a cryostat. Sections are then double-stained with anti-GFP and antibodies against cell class- or type-specific markers. Commercially available rabbit polyclonal anti-GFP, mouse monoclonal anti-GFP, and anti-GFP nanobody-reporter conjugates (RANbodies; Yamagata and Sanes, 2018a) can be used. However, commercially available chicken anti-GFP IgY antibodies display high background in chick tissues and should be avoided. Detailed immunohistochemical methods have been described in previous publications (Yamagata and Sanes, 2018b and 2019; Yamagata et al., 2021).

        A problem that we encountered was finding cells marked by eCHIKIN, both because they were rare and because in some cases expression levels were low. To circumvent this limitation, we co-electroporated a second reporter (usually a red fluorescent protein) expressed from a strong, ubiquitous (CAG) promoter. To guarantee maintained expression through multiple cell divisions, we ensured stable genomic integration using a piggyBac transposon/transposase system. In most cases, fluorescence from the CAG-driven fluorescent protein was readily detectable in tissue using a dissection microscope equipped with fluorescence. Regions identified in this way could then be dissected, sectioned, and immunostained.

        A potential confounding factor is that HDR could lead to integration into, and thus inactivation of, both copies of a targeted allele, resulting in alterations in the target cell. We did not encounter this problem: labeled cells that we were able to visualize by alternative methods (e.g., immunohistochemistry) were not detectably abnormal. Nonetheless, we cannot dismiss it and therefore tested two methods to circumvent it. In one, we maintain the function of endogenous protein by targeting the C-terminus to generate a fusion protein. For example, we appended GFP to the C-terminus of the TFAP2A coding region (Figure 7E, Figure 8A, and 8B). This method reveals the subcellular distribution of the tagged protein. In the case shown, the TFAP2A protein is a nuclear transcription factor, so the fusion protein is localized in the nucleus (Figure 7F). Subcellular localization can be either advantageous or disadvantageous depending on the purpose of the experiment. As a second approach, when cell filling is desired, we use the self-cleavable 2A peptide to generate the inserted proteins and, separately, GFP (Figure 7E). We use a tandem fusion of two 2A sequences (tPT2A) comprising P2A (2A from porcine teschovirus-1) and T2A (2A from thosea asigna virus) (Liu et al., 2017) to separate TFAP2A from GFP. In this case, GFP remained soluble and filled the cytoplasm (Figures 7G and 8C).



    Figure 7. eCHIKIN for TFAP2A. (A) Section of an E14 retina transduced with TFAP2A-eCHIKIN-GFP (green) and CAG-mCherry (red). Endogenous TFAP2A protein was stained with an anti-TFAP2A antibody (blue). TFAP2A marks most amacrine cells in the retina and is also weakly expressed by horizontal cells (arrow). (B, C) Sections of an E14 retina transduced with TFAP2A-eCHIKIN-CRE, CAG-loxP-STOP-loxP-GFP (green), and CAG-mCherry (red, shown only in B’). Nuclei were counterstained with NeuroTrace 635 (blue). (D) Similar to C but stained with a RANbody to GFP (P-RAN-GFP1). Note that this method (Yamagata and Sanes, 2018a) results in efficient labeling of horizontal cells (H). (E) Schematic of TFAP2A-Cter-eCHIKIN-GFP and TFAP2A-Cter-tPT2A-GFP-eCHIKIN. (F) Section of an E12 retina transduced with TFAP2A-Cter-eCHIKIN-GFP. Since GFP is fused to the C-terminus of the TFAP2A protein, GFP is localized to the nucleus. Weak staining in the upper INL is bleed-through from the mCherry channel. (G) Section of an E12 retina transduced with TFAP2A-Cter-tPT2A-GFP-eCHIKIN. GFP is rendered soluble in this method, resulting in the cytoplasm being filled with GFP. (H) Section of an E12 optic tectum transduced with TFAP2A-eCHIKIN-GFP (green) and CAG-mCherry (red). (I) Section of an E12 optic tectum stained with an anti-TFAP2A antibody; positive cells are in several laminae of the stratum griseum fibrosum superficiale (SGFS). Laminar distribution matches that of transduced cells in H. ONL, outer nuclear layer, containing photoreceptors. INL, inner nuclear layer, containing interneurons (horizontal, bipolar, and amacrine cells). IPL, inner plexiform layer, containing processes of retinal neurons. GCL, ganglion cell layer, containing retinal ganglion cells and some amacrine cells. Bar in I, 10 µm for B and F-I; Bars in A and C are 10 µm.



    Figure 8. eCHIKIN for TFAP2A: Fusion protein and tPT2A. (A) Genomic sequence of TFAP2A flanking its C-terminus. TGA (red) is a termination codon. The target sequence (opposite strand) of TFAP2A-Cter-crRNA is shown below. (B) To generate a donor DNA template with 70 bases arms, two 90-nt primers were used. One primer (TFAP2A-Cter-left arm-GFP-Forward) links to the 5’end of the GFP sequence (ATG), the other primer (TFAP2A-Cter-right arm-GFP-Reverse) links to the 3’ end of GFP. (C) To insert the tPT2A self-cleavable peptide sequence between TFAP2A and GFP, the donor DNA template was generated with two primers using a tPT2A-GFP plasmid as a template. See Table 1 for sequences.


EXAMPLES

Figure 7 shows examples of TFAP2A-expressing chick cells labeled using the eCHIKIN method. TFAP2A encodes a transcription factor expressed strongly by amacrine cells and weakly by horizontal cells in the inner nuclear layer of the retina. Figure 7A, from a retina transfection with a TFAP2A/GFP knock-in construct, shows that integration events were in some cases quite frequent. It is apparent that eCHIKN-labeled cells are all present in the inner nuclear layer, where amacrine cells are localized, whereas CAG-mCherry labeled cells are distributed among all retinal layers. The number of cells per layer is likely influenced by the number of divisions between the integration of the piggyBac transposon plasmid and later analysis; this varies among cell classes and types. In this image, there are more eCHIKIN-GFP-labeled cells than mCherry-labeled cells, but this is atypical. Figure 7B-D shows sections from retinas labeled by co-electroporation of TFAP2A-cre and a cre-dependent reporter. Cells in Figure 7B and 7C were labeled with anti-GFP, while cells in Figure 7D were labeled with an anti-GFP RANbody (P-RAN-GFP1; Yamagata and Sanes, 2018a). The RANbody provides more intense labeling than conventional indirect immunofluorescence (Yamagata and Sanes, 2018a). Figure 7F and 7G, described above, shows cells labeled with TFAP2A-Cter-eCHIKIN-GFP and TFAP2A-Cter-tPT2A-GFP-eCHIKIN, respectively.

    Application of eCHIKIN to 15 other genes, expressed by all retinal neuronal classes – photoreceptors, horizontal cells, bipolar cells, amacrine cells, and retinal ganglion cells – is illustrated in Yamagata et al. (2021). In our hands, eCHIKIN successfully labels ~90% of genes, although the efficiency of labeling is quite variable. In this fast-moving field, new methods can be added to the eCHIKIN protocol to enhance efficiency (Yeh et al., 2019; Broeders et al., 2020).

    We also applied eCHIKIN to the optic tectum. Figure 7H shows labeling of cells with TFAP2A-GFP in the same laminar distribution as those labeled with anti-TFAP2A (Figure 7I). This result provides encouragement that eCHIKIN could be applied to multiple tissues in chick embryos and possibly to other birds. From our experience to date, we believe that the main key to the success of the technique is to achieve robust electroporation and choose appropriate CRISPR reagents; thus, the protocol may need to be modified for other tissues or species.

Notes

  1. CRISPR-RNA

    This protocol uses the CRISPR/Cas9 system from Streptococcus pyogenes because it is the most commonly used. Moreover, target-specific RNAs and Cas9 protein are readily procured from commercial vendors.

        Genome editing performance of a crRNA is highly dependent on its sequence and secondary structure, as well as the chromatin status of the targeted locus (Doench et al., 2014; Xu et al., 2015). Here, we illustrate the selection process using the design of the TFAP2A-GFP vector (Figure 2 and Figure 8) as an example. One constraint of the CRISPR/Cas9 system is that a protospacer adjacent motif (PAM) must be incorporated into the design of the CRISPR; for Streptococcus pyogenes Cas9, the PAM is the trinucleotide NGG. The targeted CRISPR/Cas9 endonuclease generates blunt ends three nucleotides upstream of NGG. The target sequence needs to be designed such that the intended insertion is close to this double-strand break, preferably within <10 bp. First, sequences are retrieved from the most recent genome assembly (currently Gallus gallus GRCg6a). It may be helpful to confirm the reported sequence because some genes are still incompletely annotated. Ensembl (https://ensembl.org) offers a function, “Export data,” which allows easy retrieval of sequences flanking the searched sequence. The target sequence is then selected based on a panel of specificity and efficiency scores using publicly available tools (e.g., CRISPR 10K Super-track in the UCSC genome server https://genome.ucsc.edu) (Figure 2A). Multiple recent methods are available; however, describing them is beyond the scope of this protocol [see Hanna and Doench (2020) for a comprehensive review]. Targets should not be in repetitive elements spread throughout the genome, but it is not necessary to rigorously rule out potential off-target crRNA cutting sites because HDR greatly enhances the specificity of integration at the targeted locus.

  2. Design and preparation of the donor DNA for HDR

    For HDR, single-stranded DNA (ssDNA) is widely used as a donor. To achieve HDR in high efficiency, ssDNA typically contains flanking sequences around 50-80 bases on each side homologous to the nuclease cleavage site (Chen et al., 2011; Yang et al., 2013; Richardson et al., 2016; Quadros et al., 2017). For eCHIKIN, we use 70 base homology arms because commercial synthesis of 90 bases DNA is available and affordable (see below). It is also possible to use double-strand circular plasmid DNA or double-strand linear DNA as HDR donors. However, they require much longer homology arms (200 bp-2 kbp or longer), which can be prepared by molecular cloning (e.g., Baker et al., 2017). In addition, linearized double-strand DNAs often integrate randomly into the genome rather than specifically at CRISPR-mediated breaks, possibly leading to non-specific patterns of expression.

        Short ssDNA can be obtained as single-strand oligonucleotides (e.g., up to 200 nt; IDT’s Ultramer) that can incorporate short epitope tags with some flanking sequences (e.g., 27 nt for a HA tag) as well as both left and right arms (2 × 70 nt). Chemical synthesis can be used for longer ssDNA, sufficient to encode homology arms plus reporters (e.g., GFP [~700 nt] or Cre [~1,000 nt]); however, these methods are costly due to the large amount of ssDNA needed. Instead, we generate a template using two 90-bp DNA oligonucleotides (70 bases of the left and right arms, and 20 bases complementary to the 5’ and 3’ ends of the reporter cDNA, respectively) (Figure 1, Figure 2D-F). To prepare a large amount of long ssDNA using this template, we use asymmetric PCR, in which one primer is added at a 100-fold greater concentration. The amplified ssDNA can be purified by agarose gel electrophoresis, treated with phenol-chloroform and then chloroform, ethanol-precipitated, and stored at -20°C.

        Although other methods have been used to prepare long ssDNA (e.g., a combination of T7 RNA polymerase/Reverse transcriptase/RNase H, or lambda exonuclease digestion of PCR products produced with one phosphorylated primer), our experience is that the asymmetric PCR protocol is more consistent, robust, and inexpensive.

     It was demonstrated that an ssDNA donor complementary to the nontarget strand is slightly better for HDR than an ssDNA donor complementary to the target strand (Richardson et al., 2016). Thus, the sequence corresponding to the target would be the first choice for ssDNA, although the opposite ssDNA is also effective in our hands. We choose either ssDNA based on the ease of purification from agarose gels.

  3. piggyBac reporters

    To monitor the success of electroporation, we add a piggyBac transposon plasmid equipped with CAG (CMV, beta-actin promoter/enhancer/globin leader)-driven mCherry together with a CAG-driven piggyBac transposase (Yamagata and Sanes, 2012). This transposon system is used to label the electroporated area stably by integration with the aid of the transposase. Randomly integrated mCherry is used to distinguish GFP, which is integrated specifically at the target locus. When Cre is used for HDR, we also use piggyBac to introduce the Cre-dependent GFP reporter (loxP-STOP-loxP-GFP).

  4. HD enhancers

    Two additional reagents are added to enhance HD and electroporation. A DNA ligase IV inhibitor (Alt-R® HDR Enhancer, an SCR7-related proprietary product from IDT) inhibits NHEJ, thereby leading to HDR. SCR7 has been used in several studies to improve HDR (Maruyama et al., 2015; Hu et al., 2018); although, this approach has not yet become common practice (Yeh et al., 2019). For chick embryos, this reagent appears to be non-toxic. We also use the Alt-R® Cas9 Electroporation Enhancer from IDT; this is a mixture of purified carrier DNA fragments that improve the delivery of CRISPR/Cas9 RNP by electroporation. We found that inclusion of Alt-R® HDR Enhancer and Alt-R Cas9 Electroporation Enhancer in the injection cocktail improves the efficiency of eCHIKIN.

Recipes

  1. HBSS with kanamycin

    Add 50 µg kanamycin sulfate per ml HBSS

    Keep at 4°C

  2. 0.1% (w/v) Fast Green FCF

    Dissolve 100 mg Fast Green FCF in 1 ml RNase-free water

    Filter slowly through a 0.45-µm filter

    Keep at 4°C

  3. Chloroform-isoamyl alcohol (25:1)

    25 chloroform:1 isoamyl alcohol

    Keep at room temperature.

  4. Phenol-chloroform (1:1)

    1 phenol:1 chloroform-isoamyl alcohol.

    Phenol needs to be saturated with 10 mM Tris-HCl, 1 mM EDTA, pH 8.0. Keep at 4°C.

  5. Plasmids

    All plasmids need to be RNase-free. After purification using kits (e.g., Qiagen), plasmids should be treated as follows:

    1. Add 1:10 volume of 5 M NaCl and the same volume of phenol-chloroform (1:1) and vortex for 30 s.

    2. Spin for 5 min at the maximum speed of a mini centrifuge (e.g., 16,100 × g) at room temperature. Recover the aqueous phase.

    3. Repeat the phenol-chloroform treatment.

    4. Add chloroform-isoamyl alcohol (25:1), vortex, spin, and recover the aqueous phase.

    5. Repeat the chloroform-isoamyl alcohol treatment.

    6. Add 2.5 volumes of ethanol to precipitate the plasmid DNA.

    7. Pellet and rinse three times with 70% ethanol (prechilled at -20°C).

    8. Dry and resuspend in RNase-free water to make a 1 µg/µl stock solution.

      Keep at 4°C for use within a month or dispense into aliquots and freeze at -20°C for long-term storage.

Acknowledgments

This work was supported by grants R01EY022073 and R37NS029169 from the NIH. The method described here was used in a recent publication:

    Yamagata, M., Yan, W., Sanes, J. R. (2021). A cell atlas of the chick retina based on single-cell transcriptomics. eLife10: e63907. doi: 10.7554/eLife.63907.

Competing interests

The authors report no competing interests.

Ethics

Animals were used in accordance with NIH guidelines and protocols approved by the Institutional Animal Use and Care Committee at Harvard University.

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简介

[摘要]的能力来标记和处理特定细胞类型对于越来越多的功能,结构,分子必要,在模式生物和发育分析。在少数品种,这可以通过生殖细胞移植来完成小号成因; 在其它物种中,其他方法都需要基于选择性标签体细胞的基因即它们表达。这里,W Ë描述了基于CRISPR-的方法记者或Cre重组酶体整合重组进入小鸡基因组的特定基因,然后在细胞中的可视化的视网膜和中脑。基于基于 RNA 序列的细胞图谱选择位点。报告基因可以溶解以显示单个细胞的形态,也可以附加到编码的蛋白质上以评估亚细胞定位。我们调用方法Ë CHIKIN为Ë lectroporation-和ç RISPR介导的^ h omology-指示ķ nock- IN 。


[背景]许多功能,结构,分子,模式生物和发育分析现在靠能力来标记和处理特定的细胞类型。在数量有限的物种中——Mus musculus (小鼠)、Danio rerio (斑马鱼)、C. elegans (蠕虫)和Drosophila melanogaster (苍蝇)——这可以通过种系转基因来实现。例如,可以生成转基因动物,其中从由感兴趣的细胞类型中选择性表达的基因的调节元件驱动的一个报道基因的表达。

可替换地,所述转基因可编码效应,例如Cre重组酶,这使得能够通过在种系或体细胞可能提供了第二转基因的选择性表达一个病毒或其他载体(罗等人。,2018)。

对于生殖系转基因目前不可行或有限的其他模式生物,很少有方法可用于体细胞的选择性标记。一种选择是将细胞类型特异性启动子为病毒载体,但迄今为止,这种方法通常导致编在标记相关的细胞类型的基团(在Domenger和格林,2019中讨论; Juttner等人,2019)。使用 CRISPR(成簇的规则间隔短回文重复序列)进行基因组编辑(Jinek等人,2012 年;Cong等人,2013 年;Pickar-Oliver 和 Gersbach,2019 年回顾;Nishizono等人,2020 年)提供了一种替代方法。CRISPR 相关核酸内切酶 Cas9 由单一引导 RNA (sgRNA) 递送至基因组中的特定位点以产生双链断裂。断裂可以通过非同源末端连接 (NHEJ) 或同源定向修复 (HDR) 修复(Sander 和 Joung,2014 年;Yeh等,2019 年)。如果编码报告基因的 cDNA两侧是切割位点侧翼的序列,则可以利用 HDR 将它们插入到基因组中定义的位点s 。由于同源重组活动主要限于有丝分裂活性细胞,因此涉及 HDR 的方法通常适用于分裂细胞(Heidenreich 和 Zhang,2016)。

HDR 方法主要用于生成种系“敲入” (Hsu等人,2014 年;Nishizono等人,2020 年),但也可用于标记体细胞。Mikuni等人通过使用基于 CRISPR-Cas9 的 HDR 工具在子宫内电穿孔转导小鼠大脑中的分裂细胞。(2016) 开发了一种称为 SLENDR(通过 CRISPR-Cas9 介导的同源定向修复对内源性蛋白质进行单细胞标记)的方法。该方法介绍了一种质粒编码Ñ因组和Cas9,具有单个沿-链DNA(ssDNA)编码嵌入侧翼的因组识别位点序列之间的蛋白质标签; 这导致了在所希望的基因组位置HDR介导引入的标记。Uemura等人已经报道了相关方法。(2016), Nishiyama等人。(2017) ,和松田和Oinuma(2019); 铃木等人。(2016)设计了一种方法,其中通过NHEJ同源性无关的靶整合允许小号标记在后-有丝分裂细胞。

在这里,我们修改了这些方法用于小鸡 ( Gallus gallus ) 胚胎。一个多世纪以来,小鸡一直被用于胚胎学研究,并且仍然是一个被广泛使用的模型(Stern,2005)。然而,虽然生殖系转基因已在雏鸡中得到证实,但它仍然是一种困难且很少使用的方法(Lee等,2017)。此外,即使该方法可能会改进,但很少有实验室配备了产生或维持转基因品系所需的禽类养殖设施。

我们自己对研究的小鸡retinotectal系统的开发使用了病毒和电穿孔法基因转移到鸡胚(例如,灰色等,1988;伽利略等,1992;莱伯等,1996;山形等., 2002; Yamagata 和 Sanes, 2008 a )。然而,这些都不能可靠地标记特定细胞类型,因为既没有细胞类型特异性启动子,也没有选择性表达基因的清单。最近,我们解决这些问题通过产生使用高小鸡视网膜的细胞图谱第二-可以通过单细胞RNA-SEQ,其提供了一组通过其每个≥136细胞类型中选择性表达的基因的(山形等。 , 2021)。因此,我们尝试使用上述基于 CRISPR 的方法将报告基因或标签插入其中一些基因中。

这些方法的效率,不幸的是,unworkably低,导致我们以优化的方式详细的方法编在这里。创新包括下面描述的那些。(甲)w ^ hereas用于编辑的体细胞以前的方法通过的cDNA引入Cas9,足够Cas9蛋白质生产米AY太晚发生高效编辑。因此,用于生殖系 HDR 的 CRISPR 依赖方法引入了 Cas9 mRNA(Yang等人,2013 年;Takahashi等人,2015 年),避免了Cas9 蛋白转录或翻译的需要(Aida等人,2015 年; Chen等人,2016 年;Troder等人,2018 年;Gurumurthy等人,2019 年)。我们通过使用这种方法的Cas9蛋白生成Cas9核糖核蛋白复杂。(乙)我Ñ化脓性链球菌,基于Cas9-CRISPR使用的RNA靶向基因组基因座(crRNA; 35-36个核苷酸的长度),并且募集Cas9序列(tracrRNA; 67个核苷酸)。对于大多数基因编辑应用,它们融合形成 99-100 nt 的 sgRNA。我们使用单独的crRNA 和 tracrRNA,因为它们的购买成本更低,而且可能更有效地发挥作用。( C ) 因为鸡胚的脑囊泡比受精卵大得多,所以需要更多的 ssDNA。为了产生足够的数量,我们使用了非对称 PCR(Marimuthu等,2012)。(d )w ^ È添加的试剂,以提高HDR - (胡载体DNA和连接酶IV抑制剂的DNA等。,2018)。我们称该方法eCHIKIN(Ë lectroporation-和Ç RISPR介导ħ omology-我nstructed ķ nock- IN )。

图1总结了关键步骤的eCHIKIN。该方法开始于所述CRISPR HDR试剂的设计和制备(步骤小号1-3),其必须之前完成卵内电穿孔。上电穿孔的天,试剂鸡尾酒包括所述CRISPR-Cas9核糖核蛋白(RNP)复合物的制备(步骤小号4-6)和用于注射和电穿孔(步骤小号7和8)。在后期阶段,组织被解剖、固定和组织学分析(步骤 9)。



图1 。eCHIKIN 工作流程概述。第m AIN步骤是在设计和制备的RNA和DNA的试剂(步骤1-3) ,制备CRISPR-Cas9鸡尾酒(步骤4-6)的,在卵内注射和电穿孔(步骤7和8) ,和免疫组织化学分析(步骤 9)。一些面板来自https://togotv.dbcls.jp/en/ 。

关键字:小鸡胚胎, Cre, CRISPR, 绿色荧光蛋白, 同源重组, 电穿孔

材料和试剂
 
  1. 1.5 - ml 微量离心管(DNase,无RNase低粘附微量离心管)(USA Scientific,目录号:1415-2600)
  2. 受精鸡蛋:来自查尔斯河实验室(美国马萨诸塞州威尔明顿)的无特定病原体鸡 (SPF) 鸡蛋
    收到后,鸡蛋应储存在 16°C 下,并在 1 周内使用。将卵放入 38 ℃的加湿孵化器中时,胚胎发育恢复° Ç 。胚胎可以根据汉堡和汉密尔顿 (1951) 的系统进行分期。注:温度的鸡蛋,有时不好运输,从而降低自己的生存能力,以及夏季和冬季期间这样做差异时控制。
  3. ALT-R ® CRISPR-Cas9 crRNA,2纳摩尔,设计为以下详述的(集成DNA技术(IDT))
  4. ALT-R ® CRISPR-Cas9 tracrRNA,20纳摩尔(IDT,目录号:1072533)
  5. ALT-R ® SP Cas9核酸V3,100微克(IDT,目录号:1081058)
  6. ALT-R ® Cas9电穿孔增强器,2纳摩尔(IDT,目录号:1075915)
  7. Alt-R ® HDR 增强剂,100 µl(IDT,目录号:1081072)
  8. 10×反应缓冲液(具有Cas9从金斯瑞核酸酶,Ç atalog号:Z03386)(200mM的HEPES,1M NaCl的,50毫摩尔MgCl 2 ,1mM的EDTA,pH为6.5,在25℃)
  9. 不含核酸酶的水(使用Alt-R管® CRISPR-Cas9自RNA的IDT)
  10. Hanks平衡盐溶液(HBSS,不含钙、镁或酚红,Thermo-Fisher Scientific,目录号:14175095)
  11. DNA oligo 100 n mol规模,按照协议(IDT) 中的讨论进行设计
  12. Q5 DNA聚合酶(NEB,目录号:M0491)
  13. 脱氧核苷酸(dNTP)溶液混合物(NEB,目录号:N0447S)
  14. 核酸外切酶 I(大肠杆菌)(NEB,目录号:M0293S)
  15. EconoTaq PLUS GREEN 2 × Master Mix(Lucigen ,目录号:30033-1)
  16. QIAquick Gel Extraction Kit(Qiagen,目录号:28704)
  17. 2-丙醇生物试剂,用于分子生物学,≥99.5%(Sigma-Aldrich,目录号:19516)
  18. 异戊醇,≥98%,FG(Sigma-Aldrich,目录号:W205702)
  19. 无水乙醇 200 Proof(Koptec,目录号:V1016)
  20. pXL-BacII-CAG-Venus(包含位于 CAG 和Venus两侧的 piggyBac 反向末端重复序列 (ITR) 序列的质粒)(Yamagata 和 Sanes,2012)
  21. pXL-BacII-CAG-mCherry(包含位于 CAG 和 mCherry 两侧的 piggyBac ITR 的质粒)
  22. pXL-BacII-CAG-loxP-STOP-loxP-Venus(包含位于 CAG 侧翼的 piggyBac ITR 的质粒、loxP-STOP-loxP 盒和Venus)
  23. pCAG-mPBorf(含有 piggyBac 转座酶的质粒,密码子优化)
  24. tPT2A-Venus-pCR21 (含有 tPT2A-Venus序列的质粒)(该质粒可从 Addgene 获得,质粒 170521 )
  25. pCAG-Cre:GFP (Addgene, 13776)
  26. 兔抗 GFP(Millipore,目录号:AB3080P)
  27. P-RAN-GFP1 上清液(Yamagata 和 Sanes,2018 a ;Addgene 质粒,106408)
  28. TSA Plus 荧光素评估试剂盒(PerkinElmer,NEL741E001KIT)用于分子生物学的标准实验室试剂(例如,70% 乙醇、用于凝胶电泳的琼脂糖)
  29. 琼脂糖(Agarose RA(Amresco);VWR Life Science,目录号:97064-258)
  30. 缓冲液的琼脂糖电泳:10 ×的Tris -乙酸盐- EDTA (TAE)(Mediatech公司,目录号:SC45001 - 074)
  31. 硫酸卡那霉素(Sigma-Aldrich,目录号:K1377)(HBSS-卡那霉素参见下面的配方1 )
  32. Fast Green FCF,染料含量≥85%(Sigma-Aldrich,目录号:F7252)(储备溶液见下面的配方2 )
  33. 氯仿,含有 100-200 ppm 戊烯作为稳定剂,≥99.5%(Sigma-Aldrich,目录号:C2432)(有关氯仿-异戊醇(25:1),请参见下面的配方 3)
  34. 苯酚,BioUltra,用于分子生物学,≥99.5%(GC)(Sigma-Aldrich,目录号:77608)(苯酚-氯仿(1:1)见下文配方 4)
  35. 质粒(#20、#21、#22)可从 Addgene 获得(Addgene 质粒s 170518、170519、170520),但目前的限制阻止 Addgene 分发编码 piggyBac转座酶的质粒( https://www.addgene.org/terms/ 1118/ )。所有注射用质粒都需要苯酚 - 氯仿处理(参见下面的配方 5)。
 
设备
 
1.鸡蛋孵化器      
许多孵化器可在市场上买到;我们使用 1550 HATCHER(GQF MFG,萨凡纳,GA)。培养箱应保持湿润并设置为 38°C。用于运送鸡蛋的纸箱可用作孵化和电穿孔的支架。
2. ECM 830 方波电穿孔系统 (BTX; Holliston, MA)      
3.钨丝(0.1 -毫米直径)      
4.玻璃毛细管,3 -英寸长,1 -毫米直径(世界精密仪器,1B100F-3)      
5. Gentrodes,3 -毫米 L 形(金尖)45-0116      
用于将电极固定和连接到电穿孔器的附件可从多家供应商处获得,包括 BTX 和NEPAGENE 。
6.透明胶带2592(25.4 - mm宽)      
警告:ç从其他供应商的一些录音带hemical粘合剂是有毒的胚胎。可疑磁带,如果该成活率是很低。
7. 18 ½ G针      
8. 10 -毫升注射器      
9.夏普-边缘d弯曲手术剪(约4英寸)      
10. #5 镊子   
11.的Mastercycler P ro的Ť有源冰箱Ç ycler仪(Eppendorf,950040015)   
12.条8管,0.2 -毫升(Corining PCR - 0208 -甲和PCR - 02CP - A)     
13.解剖立体镜(用于电穿孔)   
卡尔蔡司 Stemi 2000 (444036-9000) 配备10 × /23目镜(放大范围:6.5-50 × )。应根据需要调整观察倍率
14.荧光立体显微镜(Olympus,SZX12,配备观察GFP/荧光素和mCherry/罗丹明荧光的立方体     
15.小号pectrophotometer(赛默飞,型号:纳米滴2000)测量OD 260   
 
程序
 
A.关键试剂的制备       
1. CRISPR-RNA (crRNA) (IDT)      
设计crRNA指南中的说明中注释1 。
2. ssDNA 供体      
设计准则的单链DNA的供体是描述了ð在注2 。
a. 产生双-链DNA模板轴承的同源性武器。       
创建一个双-链DNA模板(对于一个实施例,请参阅图小号2A和2乙),使用两个90 -碱基的ssDNA的引物(对于一个实施例,参见图2C )和GFP模板(例如,PXL-BacII-CAG-金星)。我们使用高保真PCR酶(Q5 DNA聚合酶),执行100 -在微升反应一个热循环,琼脂糖凝胶上运行,并纯化适当大小的主要的PCR产物(L EFT臂+ GFP +右臂:〜850bp的;图 2C ) 使用 QIAquick凝胶提取试剂盒。
 
 
图2.设计crRNA和所述单链DNA的供体:TFAP2A给出作为一个例子。(A) UCSC Genome Browser 中的TFAP2A基因。(B)的基因组序列TFAP2A中的目标区域和TFAP2A -crRNA如图A. (C)90个核苷酸的引物序列退火的5 “ (ATG)和3 ”端小号GFP的和所述的结构的单-链用于插入到GFP供体DNA的TFAP2A轨迹。(d)的引物和单-用于插入到CRE链供体DNA的TFAP2A轨迹。小号EE表1的序列。
 
该 PCR 使用标准方法和设备。一个典型的反应是 25 个循环(2分钟,95°C ;25 个循环,94°C,30 秒/60°C,30 秒/72°C,1分钟 + 1 秒,72°C/循环;72 °C,7 分钟;4°C)。如果未获得干净的单一 PCR 条带,请尝试使用针对富含 GC 的模板优化的缓冲液和/或添加 DMSO。
 
 
表 1 引物序列(见图 2 和图 8)
TFAP2A-Nter-left arm-GFP-Forward
 
CGGCTCTGCACAGCTCCGCCGCGCTCCGCAGCACCGCGCCGGGGGGATCGGACCCTCTCCCGCCGACCCATGGTGAGCAAGGGCGAGGAG
 
TFAP2A-Nter-右臂-GFP-反向
 
GGCCCTGGCGGGCGCTTACCTCGCACTCCTCGTACTTGATGTTATCCGTCAGCTTCCAGAGCATTTTTTACTTGTACAGCTCGTCCATGC
 
TFAP2A-Nter-left arm-CRE-Forward
 
CGGCTCTGCACAGCTCCGCCGCGCTCCGCAGCACCGCGCCGGGGGGATCGGACCCTCTCCCGCCGACCCATGGCCAATTTACTGACCGT
 
TFAP2A-Nter-右臂-CRE-Reverse
 
GGCCCTGGCGGGCGCTTACCTCGCACTCCTCGTACTTGATGTTATCCGTCAGCTTCCAGAGCATTTTTTACTTGTACAGCTCGTCCATGC
 
TFAP2A-Cter-左臂-GFP-前进
 
AGCAACAACCCCAACAGCCACACAGACAACAGCACCAAAAGCAGCGACAAAGAGGAGAAGCACCGAAAGATGGTGAGCAAGGGCGAGGAG
 
TFAP2A-Cter-右臂-GFP-反向
 
GGGAGGAGGGAGATGAATGGACTGATGGGCTGCGAGGGGGGGAAGGGAGAGGAGTGGGGGGGAATCCTCACTTGTACAGCTCGTCCATGC
 
TFAP2A-Cter-左臂-tPT2A-GFP-F
 
AGCAACAACCCCAACAGCCACACAGACAACAGCACCAAAAGCAGCGACAAAGAGGAGAAGCACCGAAAGGGCAGCGGCGCCACAAACTTC
 
TFAP2A-Cter-右臂-GFP-反向
 
GGGAGGAGGGAGATGAATGGACTGATGGGCTGCGAGGGGGGGAAGGGAGAGGAGTGGGGGGGAATCCTCACTTGTACAGCTCGTCCATGC
 
 
b. 为 ssDNA生成优化 PCR      
在通过不对称 PCR 进行大规模 ssDNA 制备之前,有助于小规模优化反应。为此,我们使用稳健且价格低廉的 PCR 酶混合物。首先,使用双-链DNA模板上面制备,我们设置了含有的引物不同的摩尔比两个反应:反应#1,1正向:100反向; 反应#2,100 正向:1 反向。
 
反应 #1 和反应 #2 (2 管)
50 µl EconoTaq PLUS GREEN 2 × Master
47 微升 dH 2 O
1微升〜0.01微克/ μ升从模板步骤A2一
1 µl 0.01 mM 引物:(反应 #1)正向或(反应 #2)反向
1 µl 1 mM 引物:(反应 #1)反向或(反应 #2)正向
*请注意,典型的 PCR 使用 0.1 mM 引物储备液。分成两个 PCR 孔(每个孔 50 µl)。
94°C,2 分钟
94°C、30 秒/60°C、30 秒/72°C、1分钟 + 1 秒延伸 72°C/循环 40 个循环
72°C,7 分钟
4°C
 
运行 1% (w/v) 琼脂糖凝胶(使用溴化乙锭)。注意,单链DNA被有效地少用溴化乙锭染色的比是双-链DNA。
 
典型的结果示于图3A 。在这种情况下(TFAP2A ),反应#1,得到一个强谱带(〜850 bp)的和几个弱带,其中包括一个共迁移具有〜500bp的双-链DNA标记物。反应#2给了两个干净频带:在慢- migrat荷兰国际集团带(星号)对应于两倍-链DNA(〜850bp的),根据其预测的大小。然而,ssDNA 的电泳迁移在常规的非变性电泳缓冲液中通常是异常的,因此很难确定哪个条带对应于 ssDNA 物种。因此,将一部分 PCR 产物 (2 µl) 与E一起孵育很有用。大肠杆菌外切酶 I,专门消化线性 ssDNA。如图3B所示,消化中的一些条带,包括约 500 bp 处的部分扩散条带,对E具有抗性。大肠杆菌核酸外切酶I消化,这表明一些双-链DNA的出席〜500bp的。相比之下,在反应#2 中,相应的条带扩散较少,并被E完全消化。大肠杆菌外切核酸酶 I,表明它仅包含 ssDNA(箭头)。因此,我们使用反应 2 的条件进行大规模制备。
 
 
图3 。评估ssDNA的质量。(A)不对称 PCR 产生的产物(引物的摩尔比(正向/反向):反应 #1 中的 1/100;反应#2 中的100/1 )。(B)PCR产物用消化大肠杆菌核酸外切酶I(É 。大肠杆菌外切I) ,其特异性地消化单-链DNA(箭头)。一个额外的带(星号)对应于两倍-链DNA。详情见正文。
 
c. 大-的规模制备的单链DNA的供体       
规模-向上反应b中优化和然后提纯单链DNA供体。
 
500 µl EconoTaq PLUS GREEN 2 × Master
470 微升 dH 2 O
10微升〜0.01微克/ μ升从模板步骤A2A
10 µl 0.01 mM 反向引物
10 µl 1 mM 正向引物
分成 20 个 PCR 孔(每个孔 50 µl )
94°C,2分钟
94°C、30 s/60°C、30 s/72°C、1 min +1 s延伸40个循环
72°C,7分钟
4°C
 
B. 运行大琼脂糖凝胶并使用 QIAquick凝胶提取试剂盒纯化 ssDNA 。
使用细井,所以很重要的是,带被明确分开。通常,我们使用n 11 - cm 宽× 13 - cm 长× ~7 - mm 厚的凝胶。增溶后的在QG溶液(QIA快速琼脂糖凝胶提取试剂盒)中,添加20%体积的2-丙醇,和负载上以4个微型自旋-列。洗脱每个管与100μlEB(QIA快速凝胶提取试剂盒)和50μlEB ,依次。添加1:10体积5M的NaCl,提取两次用苯酚-氯仿,然后两次用氯仿,并通过添加2.5体积-20℃的乙醇最终乙醇沉淀。在-20 °C 下用 70% 乙醇冲洗颗粒3 次。降速,完全除去的残留的乙醇,干燥,并再在无RNA酶的水(总计20μL)暂停。
估计DNA使用常规的或纳米浓度d ROP TM (赛默飞世)分光光度计。需要注意的是OD 260 ssDNA的是低级由于增色(40微克/ ml的OD 260 ,而不是至50μg/ ml的双-链DNA)。将浓度调整至1 µg/ µ l,冷冻,并在 -20°C 下储存直至使用。
 
B.设备设置        
图 4显示了用于注射和电穿孔的立体显微镜设置(图 4A 和 4B )。
 
 
图 4. eCHIKIN 所需的设备和仪器。的A.设置的electroporater,解剖显微镜,和电极保持器。BD。阴极 (+) 和钨阳极 (-)。E. 装有 eCHIKIN 鸡尾酒和 Fast Green 的玻璃毛细管。
 
1.钨针      
切0.1 -毫米的钨丝,以合适的长度(〜2cm)上并锐化尖端。我们以电方式对其进行锐化(Brady,1965 年)(参见图 4C 和 4D )。
2.注射用玻璃毛细管      
制作注射用玻璃毛细管。我们使用从玻璃毛细管(World Precision Instruments,1B100F-3(3 -英寸,1 -毫米)使用拉拔器)精细拉制的自制微量移液器。折断尖端的最后几毫米,以便在保持锋利的同时进行轻松注射(图4E )。
 
C.孵化鸡蛋        
在注射和电穿孔(见下文)之前的适当时间,将所述蛋的孵化器。鸡蛋需要侧身孵化(水平;图 5A )。标记每个鸡蛋的顶部,因为胚胎会漂浮在蛋黄上。孵化比您计划注射更多的卵子,因为有些卵子可能不育或胚胎放置不当以进行注射。
 
 
图 5. 用于注射和电穿孔的鸡胚的制备。A. 侧身孵化的鸡蛋。是。将塑料胶带贴在鸡蛋的顶部,从鸡蛋的圆形一侧去除蛋清蛋白。女,G.使用手术剪具有弯曲叶片,一个小窗口(〜5 -毫米直径)上的蛋的顶部通过带切割制成,壳一起。
 
D.注射和卵内电穿孔       
1.注射鸡尾酒的制备      
在卵内电穿孔当天,准备注射用的鸡尾酒。所有塑料器皿和试剂必须不含 RNase。
a. crRNA:transcrRNA/Cas9 RNP(解决方案“ R ” )       
为了重建引导 RNA 双链体,目标特异性 crRNA (35 - 36 nt) 和 tracrRNA (67 nt) 结合、热变性并通过缓慢冷却至室温退火。在1.5 -毫升管中,以该顺序添加这些试剂:
 
2.5 微升 dH 2 O
0.5 µl 10 ×反应缓冲液
1 µl crRNA(2 n mol /20 µl d H 2 O ;浓度,100 µM )
1 µl tracrRNA(2 n mol /20 µl d H 2 O ;浓度,100 µM )
 
自旋简要并孵育在95℃下在5分钟内一个加热块。
大号在室温下檐,从而该冷却速度慢,最大化退火。
静置 5 分钟,短暂旋转,并在室温下孵育 30 分钟。
 
加入 0.8 µl 10 mg/ml Cas9 蛋白(化脓性链球菌Cas9 Nuclease V3),混合,短暂离心,室温孵育 60 分钟。这个解决方案是解决方案“ R” 。”
 
b. 变性的单链DNA的供体      
ssDNA 供体应提前准备好并储存在 -20°C 直至使用。
解冻冷冻的等分试样,在 95°C 下加热 5 分钟,然后在冰上快速冷却。这种变性可以与上一步中描述的 crRNA:tracrRNA/Cas9 RNP 的退火同时进行。然而,与 crRNA:tracrRNA 的缓慢退火相反,该管需要立即放在冰上快速冷却。使用前短暂旋转试管。
c. 带有 HDR 增强剂的稀释缓冲液(溶液“ D ” )       
在一个不含RNA酶的1.5 -毫升管中,加入这些在此顺序的试剂:
 
21.5 微升 dH 2 O
2.5 µl 10 ×反应缓冲液
1 µl Alt-R ® HDR Enhancer(可作为悬浮在 DMSO 中的溶液提供)。混合均匀并短暂旋转。
 
d. piggyBac 质粒混合物(溶液“ P ” )      
对于GFP报告eCHIKIN,混合在1.5 -毫升管:
45 µl 1 µg/µl pXL-BacII CAG-mCherry
5 µl 1 µg/µl pCAG-mPB
 
对于CRE报道eCHIKIN,混合在1.5 -毫升管:
22.5 µl 1 µg/µl pXL-BacII CAG-mCherry
22.5 µl 1 µg/µl pXL-BacII CAG-loxP-STOP-loxP-Venus
5 µl 1 µg/µl pCAG-mPB
请注意,这些质粒 DNA 必须不含 RNase(参见配方 5 和注释 3)。
 
e. 最后注射鸡尾酒       
向一管溶液“ R ” (共 5.8 µl)中,按以下顺序添加这些试剂(见注 4):
11 µl 溶液“ D ”
3 µl 1 µg/µl ssDNA 供体(提前 95°C 变性)
3 µl Alt-R ® Cas9 电穿孔增强剂(2 n mol /50 µl ;浓度,40 µM )
3 µl 溶液“ P ”
2 µl 0.1%(w/v) 坚牢绿
 
拌匀,和对花边冰这个解决方案。在 5-6 小时内使用。
 
2.卵子和胚胎的制备      
a. 胚胎在 38°C 下在约 40 小时内达到第 9-10 阶段。因此,如果蛋放置在培养箱中在下午8点(第0天)时,它们应该准备中午电穿孔在第2天然而,胚胎应根据汉堡和汉密尔顿(1951),因为上演的发育率是敏感温度变化很小,鸡蛋之间也有差异。为了在计划注射多个胚胎时减缓发育,可以从孵化器中取出卵子并在室温下保持长达 5-6 小时,而不会影响生存能力。       
b.  用 70% 乙醇喷洒鸡蛋并使其干燥。始终保持蛋的先前标记正面面对了病房(图5B )。      
c. 在每个鸡蛋的顶部贴上一块透明胶带(图5C )。       
d. 插入Ñ 18 ½ģ上的10针-毫升注射器到每个蛋(的圆形侧图5D ); 这一面包含气囊。      
e. 除去1-2ml的卵白蛋白取决于大小的鸡蛋(图5E )。一些研究人员省略了这一步以获得更好的生存。       
f. 使一个小窗口(〜5 -毫米直径)上的蛋通过胶带切割的顶部和用手术剪与弯曲叶片(图5F和外壳5G )。        
3.注射和卵内电穿孔      
a. 如果有必要,所以轻轻摇动鸡蛋是胚胎坐落在顶部的蛋黄。如果需要,使窗口更大(图 6A )。在某些情况下,需要使用 #5镊子小心地去除壳下方的半透明膜。       
 
 
图6.关键步骤的注射和卵内电穿孔。(A)定位的胚胎和扩大窗口。(B)将阴极作为G和注入试剂混合物(蓝色)中的胚胎。(C)用汉克斯+卡那霉素润湿阴极和胚胎。(DE)我nsert阳极如在G和电穿孔。成功电穿孔后,小气泡出现在阳极。(F)用塑料胶带密封鸡蛋。它以密封所述小孔(绿色)是重要的是被用于去除由白蛋白。(G)总结。
 
b. 放置的下的蛋的解剖显微镜。对于初学者来说,在胚胎中看到足够的细节以进行准确注射可能具有挑战性。一种帮助是在胚胎下方注入几微升黑色印度墨水。Hamburger 和 Hamilton (1951) 中的图像对于定位很有用。      
c. 将 L 形d阴极平行于胚胎放置(图 6A 和6G )。       
d. 加载CRISPR鸡尾酒成微量(图4E使用口移液管或微量()和注入〜0.1-0.2微升例如,FemtoJet,Eppendorf)中。转导的视网膜,注入到阶段9-10胚胎(的视泡的一侧图6B和图6G )。转导的视顶盖,注射应该在阶段11-12被靶向脑囊泡。      
e. 润湿两者的阴极和胚胎与20μlHBSS +卡那霉素使用移液管尖端(图6C )。       
f.将电穿孔器的细钨阳极插入胚胎的头部 (图 6D 和6G )。        
G。提供方波脉冲(图 6E 和 6G )。视神经囊泡,我们使用6个脉冲(7 V / 25毫秒)在1 -秒的间隔。成功电穿孔的标志是阳极处出现大量微小气泡。      
H。用透明胶带关闭窗口(图 6F )。它也密封所述小孔(绿色箭头是在重要的图6F )这是由以除去白蛋白。      
一世。放置的鸡蛋备份在38℃培养箱中。        
这种技巧(步骤 D2 和D 3)在很大程度上依赖于表演者的技能,需要大量的练习。专家可以每隔几分钟注射一个胚胎或在 4 小时内注射约 120 个胚胎。
 
E.组织学分析        
小鸡胚胎在 E21 孵化;ħ H但是,从E17死亡率大大增加。这是在蛋壳中引入窗口的操作的常见结果;它不是特定于 eCHIKIN。因此,如果可能的话,最好通过 E16 检索组织。两者的视网膜和视顶盖都非常受这个年龄的成熟。
  尽管可以使用其它方法,主要的分析方法是免疫histolog ÿ 。胚胎被检索,并且该适当区域(在视网膜或顶盖的此处描述的情况)被解剖。组织用 4% (w/v) 多聚甲醛/PBS 固定 15 h,依次浸入 15% (w/v) 和 30% (w/v) 蔗糖/PBS 中,包埋于组织冷冻培养基(Triangle Biomedical Sciences)中),在 -80°C 下冷冻,并在低温恒温器中以 20 µm 切片。第然后doubl é染色的抗GFP和抗体对细胞讲座或类型特异性标志物。市售可用的兔多克隆抗GFP ,小鼠单克隆抗GFP ,一个第二抗-GFP纳米抗体-报道缀合物(RANbodies;山形和Sanes,2018一个)都可以使用。然而,市场上可用的鸡抗GFP性IgY抗体显示高的背景我ñ小鸡组织,应予以避免。详细的免疫组织化学方法已在之前的出版物中进行了描述(Yamagata 和 Sanes,2018 b和2019;Yamagata等,2021)。
  一个问题是我们遇到的是找到由eCHIKIN标记的单元格,一方面是因为他们是罕见的,因为在某些情况下表达水平很低。为了规避这一限制,我们共电穿孔了由强大的、无处不在的(CAG) 启动子表达的第二个报告基因(通常是红色荧光蛋白)。为了保证通过多次细胞分裂保持表达,我们使用 piggyBac 转座子/转座酶系统确保稳定的基因组整合。在大多数情况下,使用配备荧光的解剖显微镜可以很容易地在组织中检测到来自 CAG 驱动的荧光蛋白的荧光。然后可以对以这种方式识别的区域进行解剖、切片和免疫染色。
  一个潜在的混淆荷兰国际集团的因素是,HDR可能导致融入,从而失活的,的两个副本的等位基因的目标,因此改变在靶细胞。我们没有遇到这个问题:我们能够通过替代方法(例如,免疫组织化学)可视化的标记细胞未检测到异常。然而,我们不能排除它,并为此Ë测试两种方法来绕过它。一方面,我们通过靶向 C 端产生融合蛋白来维持内源蛋白的功能。例如,我们所附GFP到TFAP2A编码的C-末端区域(图7E,图8A ,及8乙)。该方法揭示了标记蛋白的亚细胞分布。在示出的情况下,所述TFAP2A蛋白是核转录因子,因此所述融合蛋白在细胞核中(局部图7F )。根据实验的目的,亚细胞定位可能是有利的,也可能是不利的。作为第二种方法,当需要填充细胞时,我们使用可自我切割的 2A 肽来生成插入的蛋白质,并单独生成 GFP(图 7E )。我们使用包含 P2A(来自猪 teschovirus-1 的 2A)和 T2A(来自那些 asigna 病毒的 2A)(Liu等,2017)的两个 2A 序列 (tPT2A) 的串联融合将 TFAP2A 与 GFP 分开。在这种情况下,GFP保持可溶并且填充细胞质(图小号7G和图8C )。
 
 
图 7. TFAP2A 的 eCHIKIN。(A)的第一个与TFAP2A-eCHIKIN-GFP(绿色)和CAG-mCherry的(红色)转导的E14视网膜。内源性TFAP2A蛋白用染色的抗-抗体TFAP2A(蓝色)。TFAP2A 标记视网膜中的大多数无长突细胞,并且水平细胞(箭头)也弱表达。(B ,C)的第一个与TFAP2A-eCHIKIN-CRE,转导的E14视网膜CAG-loxP的STOP-loxP的GFP(绿色),和CAG-mCherry的(红色,只在B中所示' )。细胞核用 NeuroTrace 635(蓝色)复染。(D)与 C 相似,但用 RANbody 染色到 GFP (P-RAN-GFP1)。注意,第是方法(山形和Sanes,2018一)导致有效水平的细胞标记(ħ )。(E) TFAP2A-Cter-eCHIKIN-GFP 和TFAP2A-Cter-tPT2A-GFP-eCHIKIN 的示意图。(F)第一个E12视网膜转导TFAP2A-CTER-eCHIKIN-GFP。由于 GFP与TFAP2A 蛋白的 C 端融合,因此 GFP 定位于细胞核。上部 INL 中的弱染色从 mCherry 通道渗出。(G)第一个E12视网膜转导TFAP2A-CTER-tPT2A-GFP-eCHIKIN。GFP在该方法中变得可溶,从而导致在细胞质中填充有GFP。(H)的第一个与TFAP2A-eCHIKIN-GFP(绿色)和CAG-mCherry的转导的E12视顶盖(红色)。(I)第一个与染色E12视顶盖的抗TFAP2A抗体; 阳性细胞位于浅层灰色纤维层 (SGFS) 的几个薄层中。层流分布与包含光感受器的 H. ONL、外核层中的转导细胞的层流分布相匹配。INL,内核层,含有的interneurons(水平,双极,和无长突细胞)。IPL,内丛状层,包含视网膜神经元的突起。GCL,神经节细胞层,含有视网膜神经节细胞和一些无长突细胞。I 中的 Bar,B 和 FI 为 10 µm;A 和 C 中的条形为 10 µm。
 
 
图8 。eCHIKIN为TFAP2A:˚F延髓蛋白和tPT2A。(A) TFAP2A C 端侧翼的基因组序列。TGA(红色)是终止密码子。TFAP2A-Cter-crRNA的靶序列(相反链)如下所示。(B)为了生成具有 70 个碱基臂的供体 DNA 模板,使用了两个 90- nt引物。一个引物(TFAP2A-CTER-左臂-GFP前转)链接到5'端的的GFP序列(ATG) ,牛逼他其他的引物(TFAP2A-CTER-右臂-GFP-反向)链接到3'末端的GFP。(C)要插入TFAP2A和GFP之间的tPT2A自裂解的肽序列,所述用使用tPT2A-GFP质粒为模板的两个引物产生的供体DNA模板。序列见表 1。
 
例子
图 7显示了使用eCHIKIN 方法标记的表达 TFAP2A 的鸡细胞的示例。TFAP2A编码强烈无长突细胞表达的转录因子小号通过水平细胞和弱中所述的内核层视网膜。图 7A来自使用 TFAP2A/GFP 敲入构建体的视网膜转染,显示整合事件在某些情况下非常频繁。很明显,eCHIKN 标记的细胞都存在于无长突细胞所在的内核层,而 CAG-mCherry 标记的细胞分布在所有视网膜层中。每层细胞的数目可能是由间分割数的影响的piggyBac转座子的质粒和以后分析的整合; 这因细胞类别和类型而异。在此图像中,eCHIKIN-GFP 标记的细胞比 mCherry 标记的细胞多,但这是非典型的。图7B-d示出了小号从由TFAP2A-CRE和CRE依赖性报告的共电穿孔标记的视网膜切片。在图细胞7B和7 Ç分别用抗GFP,而在细胞图7D ,用抗GFP RANbody(标记为P -RAN-GFP1;山形和Sanes,2018一)。RANbody 提供比传统间接免疫荧光更强烈的标记(Yamagata 和 Sanes,2018 a )。图7F和图7 ģ ,如上所述,显示小号标记TFAP2A-CTER-eCHIKIN-GFP和TFAP2A-CTER-tPT2A-GFP-eCHIKIN,细胞分别。
  光感受器,水平细胞,双极细胞,无长突细胞- eCHIKIN的到其它15个基因,由所有视网膜神经类表示应用,在山形示出-和视网膜神经节细胞等。(2021)。在我们的手中,eCHIKIN成功标签〜90%的基因,虽然标注的效率我个相当变数。在这个快速发展的领域,可以将新方法添加到 eCHIKIN 协议中以提高效率(Yeh等人,2019 年;Broeders等人,2020 年)。
  我们还将 eCHIKIN 应用于视顶盖。图7H示出了具有TFAP2A-GFP I细胞的标记n中的相同层状分布的那些标记与抗TFAP2A(图7I )。这一结果鼓励了 eCHIKIN 可以应用于鸡胚胎的多种组织,也可能应用于其他鸟类。根据我们迄今为止的经验,我们认为该技术成功的主要关键是实现稳健的电穿孔和选择合适的 CRISPR 试剂;因此,可能需要针对其他组织或物种修改方案。
 
笔记
 
1. CRISPR-RNA      
该协议使用来自化脓性链球菌的 CRISPR/Cas9 系统,因为它是最常用的。此外,目标特异性 RNA 和 Cas9 蛋白很容易从商业供应商处获得。
  crRNA 的基因组编辑性能高度依赖于其序列和二级结构,以及目标基因座的染色质状态(Doench等,2014;Xu等,2015)。在这里,我们以 TFAP2A-GFP 载体(图 2和图8)的设计为例来说明选择过程。所述CRISPR / Cas9系统的一个约束是,一个protospacer相邻基序(PAM)必须被结合到所述CRISPR的设计; 对于化脓性链球菌Cas9,PAM 是三核苷酸 NGG。靶向 CRISPR/Cas9 核酸内切酶在 NGG 上游三个核苷酸处生成平末端。被设计S中的靶序列的需要UCH该预期的插入接近该双-链断裂,preferabl ÿ <10个碱基之内。首先,从最近的基因组组装(目前为Gallus gallus GRCg6a)中检索序列。确认报告的序列可能会有所帮助,因为某些基因仍未完全注释。ENSEMBL(https://ensembl.org)提供了一个功能, “导出数据,”这允许序列便于检索小号侧翼第ë搜索序列。然后使用公开可用的工具(例如,UCSC 基因组服务器 https://genome.ucsc.edu 中的 CRISPR 10K Super-track)基于一组特异性和效率评分选择目标序列(图 2A)。有多种最近的方法可用;然而,对它们的描述超出了本协议的范围[参见 Hanna 和 Doench (2020) 的全面审查] 。目标不应该是重复的元素通过扩散出来的基因组,但没有必要严格地排除潜在的靶crRNA切割网站,因为HDR大大增强了一体化的特异性的靶向的基因座。
2.设计和准备的供体DNA的HDR      
对于HDR,单-链DNA(ssDNA)被广泛用作供体。为了实现HDR在高效率,单链DNA通常包含小号侧翼围绕在每一侧上的同源50-80个碱基的序列的核酸酶切割位点(陈等人,2011;杨等人,2013;理查森等人,2016;德罗斯等,2017)。对于 eCHIKIN,我们使用 70 个碱基的同源臂,因为 90 个碱基的 DNA 的商业合成可用且价格合理(见下文)。另外,也可以使用双-链环状质粒DNA或双-链线性DNA作为HDR供体。然而,它们需要更长的同源臂(200 BP-2 kb的p或更长),这可通过分子克隆来制备(例如,贝克等人。,2017)。此外,线性化的双-链的DNA常常随机地融入的基因组中,而不是专门针对CRISPR介导的断裂,这可能导致非特异性模式表达。
  短单链DNA也可以得到与单-链寡核苷酸(例如,高达200个核苷酸; IDT的Ultramer)其可以结合短的表位标签的一些侧翼序列(例如,27个核苷酸对于HA标签),以及左侧和右侧臂( 2 × 70 nt)。化学合成可用于更长的 ssDNA,足以编码同源臂和报告基因(例如,GFP [~700 nt] 或 Cre [~1 , 000 nt]);ħ H但是,这些方法成本高到由于大量的ssDNA的需要。相反,我们生成使用两个90模板-bp DNA寡核苷酸(70个碱基的左,右手臂,和20个碱基互补的5 “和3 ”的端部的的cDNA,分别记者)(图1,图2D-F )。为了使用此模板制备大量长 ssDNA,我们使用不对称 PCR ,其中以100 倍的浓度添加一个引物。扩增的ssDNA可以通过琼脂糖凝胶电泳纯化,用苯酚-氯仿和氯仿处理,乙醇沉淀,-20°C保存。
  尽管已使用其他方法制备长 ssDNA(例如,T7 RNA 聚合酶/逆转录酶/RNase H 的组合,或使用一种磷酸化引物产生的 PCR 产物的 λ 外切核酸酶消化),但我们的经验是不对称 PCR 方案更多一致、坚固且价格低廉。
  已证实的对非靶链的ssDNA互补施主略好为HDR比的单链DNA的供体互补的靶链(理查森等人。,2016)。因此,对应于目标序列将是单链DNA的第一选择,虽然在相对的ssDNA也是有效的在我们手中。我们根据琼脂糖凝胶纯化的难易程度选择 ssDNA 。
3. piggyBac 记者      
为了监测成功的电穿孔,我们添加一个转座子piggyBac的质粒配备CAG(CMV,β-肌动蛋白启动子/增强子/前导球蛋白)驱动的mCherry连同一个CAG驱动piggyBac转转座(山形和Sanes,2012)。该转系统用于通过集成的帮助下稳定地标记电区域的转座。随机集成的 mCherry 用于区分 GFP,它专门集成在目标基因座上。当 Cre 用于 HDR 时,我们还使用 pigg y B ac来引入依赖于 Cre 的 GFP 报告基因(loxP-STOP-loxP-GFP)。
4.高清增强器      
添加了两种额外的试剂以增强 HD 和电穿孔。甲DNA连接酶IV抑制剂(Alt键-R ® HDR增强器,从IDT的SCR7相关专利产品)抑制NHEJ,由此导致HDR。SCR7有几项研究被用来改善HDR(丸山等人,2015年;胡等人。,2018); 尽管如此,这种方法尚未成为普遍做法(Yeh等,2019)。对于鸡胚胎,这种试剂似乎是无毒的。我们还使用了IDT的 Alt-R ® Cas9 电穿孔增强剂;这是一种纯化载体 DNA 片段的混合物,可通过电穿孔改善 CRISPR/Cas9 RNP 的递送。我们发现在注射液中加入 Alt-R ® HDR Enhancer 和 Alt-R Cas9 Electroporation Enhancer 可以提高 eCHIKIN 的效率。
 
食谱
 
1. HBSS与卡那霉素      
每毫升 HBSS添加50 µg 硫酸卡那霉素
保持在 4°C
2. 0.1% (w/v) 固绿FCF      
将100 mg Fast Green FCF溶解在1 ml 无 RNase 的水中
过滤慢慢通过一个0.45 -微米的过滤器
保持在 4°C
3.氯仿我soamyl醇(25:1)      
25 c氯仿:1 i大豆醇
保持室温。
4.苯酚- Ç hloroform (1:1)      
1个p苯酚的制备:1 Ç hloroform-我soamyl醇。
苯酚需要用 10 mM Tris - HCl、1 mM EDTA、pH 8.0饱和。保持在 4 °C 。
5.质粒      
所有质粒都需要不含 RNase。使用试剂盒(例如Qiagen)纯化后,质粒应如下处理:
一种。加入1 :10体积的5M NaCl和相同体积的p henol- Ç hloroform(1:1)和涡流30秒。       
湾 旋在的最大转速5分钟一微型离心机(例如,16 ,100 ×克在室温下)。回收的水相。      
C。重复的苯酚-氯仿处理。       
d. 添加氯仿-异戊醇(25:1),涡旋,旋转,并恢复所述含水相。      
e. 重复的氯仿-异戊醇处理。       
F。添加2.5体积小号的乙醇,以沉淀的质粒DNA。        
G。P ellet和冲洗用70%乙醇(在-20℃预冷)洗涤三次。      
H。干燥并重新悬浮于不含RNA酶的水,以使一个1微克/微升的库存溶液。      
在 4°C 下保存一个月内使用或分装成等份并在 -20°C 下冷冻以长期储存。
 
致谢
 
这项工作得到了 NIH 的赠款 R01EY022073 和 R37NS029169 的支持。在最近的出版物中使用了此处描述的方法:
Yamagata, M., Yan, W., Sanes, JR (2021)。基于单细胞转录组学的鸡视网膜细胞图谱。eLife10:e6 3907。doi:10.7554/eLife.63907。
 
利益争夺
 
作者报告没有竞争利益。
 
伦理
 
动物按照批准NIH的指导方针和协议所使用的实验动物使用和护理委员会在哈佛大学。
 
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Yamagata, M. and Sanes, J. R. (2021). CRISPR-mediated Labeling of Cells in Chick Embryos Based on Selectively Expressed Genes. Bio-protocol 11(15): e4105. DOI: 10.21769/BioProtoc.4105.
  2. Yamagata, M., Yan, W. and Sanes, J. R. (2021). A cell atlas of the chick retina based on single cell transcriptomics. Elife 10:e63907.
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