参见作者原研究论文

本实验方案简略版
Jul 2020
Advertisement

本文章节


 

CRISPR/Cas9-mediated Precise SNP Editing in Human iPSC Lines
CRISPR/Cas9介导的人iPSC细胞系SNP精确编辑   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

Human induced pluripotent stem cells (hiPSCs) have been extensively used in the fields of developmental biology and disease modeling. CRISPR/Cas9 gene editing in iPSC lines often has a low frequency, which hampers its application in precise allele editing of disease-associated single nucleotide polymorphisms (SNPs), especially those in the noncoding parts of the genome. Here, we present a unique workflow to engineer isogenic iPSC lines by SNP editing from heterozygous to homozygous for disease risk alleles or non-risk alleles using a transient and straightforward transfection-based protocol. This protocol enables us to simultaneously obtain pure and clonal isogenic lines of all three possible genotypes of a SNP site within about 4 to 5 weeks.

Keywords: Induced pluripotent stem cells (诱导多能干细胞), Genome editing (基因组编辑), Disease modeling (疾病模型), CRISPR/Cas9 (CRISPR/Cas9), Homology-directed recombination (同源重组), Allelic specificity (等位基因特异性)

Background

The application of CRISPR/Cas9 gene editing to iPSC cells provides unrivalled potential in the fields of developmental biology, disease modeling, and regenerative medicine. However, since iPSCs are not especially acquiescent to the traditional strategies used in CRISPR/Cas9-gene editing, the editing efficiency is often low, especially for homology-directed repair (HDR)-mediated editing of single nucleotide polymorphisms (SNPs). Special experimental methods must be developed to circumvent this limitation; for instance, using simultaneous CRISPR/Cas9 mutagenesis and reprogramming of iPSCs (Howden et al., 2015; Tidball et al., 2018), advanced cell sorting techniques such as FACS (Forsyth et al., 2006; Miyaoka et al., 2014), or recombination of mutation-carrying cassettes though the presence of long homology arms in targeting plasmids (Hendel et al., 2014). Nevertheless, these highly specialized protocols for gene editing in iPSC lines are time and/or resource-consuming, which persists as a critical limiting factor in generating isogenic iPSC lines via CRISPR/Cas9-mediated precise SNP editing.


Here, we present a demonstrated workflow and detailed procedures that allow direct editing of iPSCs using the CRISPR/Cas9 system with an HDR technique by simple liposome-based transfection, followed by antibiotic selection to enrich the edited iPSCs and single clonal selection to obtain pure iPSC isogenic lines of all three possible genotypes. This method was adapted from Ran et al. (2013) with demonstrated results in our recent publications (Forrest et al., 2017; Zhang et al., 2018 and 2020). Notably, this protocol worked out very well for our unique CRISPR/cas9 SNP editing design from a heterozygous iPSC line to isogenic pairs homozygous for disease risk alleles or non-risk alleles, which makes the functional interpretation of an edited SNP more reliable by directly comparing isogenic lines of all three different genotypes (Forrest et al., 2017; Zhang et al., 2018 and 2020).

Materials and Reagents

  1. Materials

    1. 1.5 ml Eppendorf tubes (VWR, catalog number: 89000-028)

    2. 4-w NunclonTM Delta MultiDishes (Thermo Scientific, catalog number: 62407-068)

    3. 6-well culture plates (Thermo Fisher, catalog number: 140675)

    4. 96-well culture plates (Corning, Falcon®, catalog number: 353072)

    5. Standard 60 × 15 mm dishes w/vent (Fisher Scientific, catalog number: 12-565-95)

    6. 15 ml centrifuge tubes (VWR, catalog number: 21008-216)

    7. 2 ml cryogenic vials with closures, polypropylene (Corning®, catalog number: 89089-764)

    8. 10 μl racked tips, low-retention sterile (VWR, catalog number: 10017-062)

    9. 200 μl racked tips, low-retention sterile (VWR, catalog number: 76322-150)

    10. 1,000 μl low-retention tips (VWR, catalog number: 10017-090)

    11. BioDot 96-well non-skirted PCR plates (Dot Scientific, catalog number: 650-PCR)


  2. Reagents

    1. mTeSR1 (STEMCELL, catalog number: 85850)

    2. mFreSR Cryopreservation Medium (STEMCELL, catalog number: 05855)

    3. ReLeSR (STEMCELL, catalog number: 05872)

    4. Matrigel® hESC-Qualified Matrix (Corning®, catalog number: 354277)

    5. FuGENE® HD Transfection Reagent (Promega, catalog number: E2311)

    6. Accutase (STEMCELL, catalog number: 07920)

    7. Primocin (Invitrogen, ant-pm-1)

    8. Y-27632 dihydrochloride (R&D Systems, catalog number: 1254/1)

    9. QIAprep Spin Miniprep Kit (250) (Qiagen, catalog number: 27106)

    10. Qiaprep EndoFree Plasmid Maxi Kit (10) (Qiagen, catalog number: 12362)

    11. BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, catalog number: 4337455)

    12. DyeEx 2.0 kit (Qiagen, catalog number: 63204)

    13. HotStarTaq DNA Polymerase (250 U) (Qiagen, catalog number: 203203)

    14. PlasmidSafe Exonuclease (Lucigen, catalog number: E3101K)

    15. QuickExtractTM DNA Extraction Solution (VWR, catalog number: 76081-768)

    16. FastDigest BbsI (Thermo Fisher Scientific, catalog number: ER1101)

    17. T7 ligase (NEB, catalog number: M0318S)

    18. ATP solution, 100 mM (NEB, catalog number: N0451)

    19. dNTP solution mix, 10 mM (NEB, catalog number: N0447S)

    20. Opti-MEM I, 100 ml (Thermo Fisher Scientific, catalog number: 31985062)

    21. Shrimp alkaline phosphatase (Thermo Fisher Scientific, catalog number: 783901000UN)

    22. PCRX Enhancer System (Thermo Fisher, catalog number: 11495017)

    23. Isopropanol (Sigma-Aldrich, catalog number: 190764)

    24. Nuclease-free water, PCR grade (Thermo Fisher Scientific, catalog number: AM9937)

    25. Teknova DNA/RNA Resuspension Buffer (TE buffer) (VWR, catalog number: 100216-886)

    26. Invitrogen One Shot® Stbl3TM Chemically Competent E. coli cells (Thermo Fisher Scientific, catalog number: C737303)

    27. 1 kb DNA ladder (Promega, catalog number: G5711)

Equipment

  1. C1000 Touch Thermo Cycler (Bio-Rad, model: 1851197)

  2. Sorvall Legend XTR Centrifuge (Thermo Scientific, catalog number: 75217420)

  3. Benchtop refrigerated centrifuge 5430R (Eppendorf, catalog number: 022620601)

  4. Heracell 150i Tissue Culture Incubator (Thermo Fisher Scientific, catalog number: 51026283)

  5. ABI 3730 DNA Analyzer (Thermo Fisher Scientific, model: 3730S)

  6. Nalgene Mr. Frosty Freezing container (Sigma-Aldrich, catalog number: C1562)

Software

  1. ApE – A plasmid Editor (M Wayne Davis, https://jorgensen.biology.utah.edu/wayned/ape/)

Procedure

  1. Experimental planning

    1. Cell line selection

      Generally, the experiment will be the most convenient if the starting cell line is heterozygous at the desired SNP site to be edited. In this way, we can synthesise two single-stranded DNA oligonucleotides (ssODNs), each carrying a different genotype, and perform two separate SNP editing experiments simultaneously, thereby obtaining edited homozygous cell lines in both allele directions at the end of the experiment (Figure 1A). It is also possible to obtain homozygous cell lines of the opposing genotype by starting from a homozygous cell line when the circumstances do not allow a high degree of freedom, such as using cell lines derived from clinically diagnosed patients (Figure 1B). In such cases, it would be beneficial to increase the number of clones picked in Sections E and F to maximize the chance of detecting a positive one. Whilst there would be a chance that one of the genotypes (heterozygous or homozygous of the opposite genotype) does not appear in the first round of CRISPR/Cas9 editing, obtaining the homozygous cell line of the opposite genotype is usually sufficiently satisfactory for the purpose of the experiment. Alternatively, a heterozygous line obtained from the first round of editing can be used for another round of CRISPR/Cas9 editing with the same ssODN to obtain a homozygous line of the opposite genotype. In all cases, the genotype of the starting cell line at the desired SNP site needs confirmation before proceeding to Section B. To confirm the genotype at the specific SNP site, use 30 ng genomic DNA extracted from the starting cell line and perform Sanger sequencing by proceeding with Steps E4-E5. Alternatively, online databases, next-sequencing results, or cell line-associated patient metadata may also provide genotype information at the SNP site to be edited.



      Figure 1. SNP editing strategies using SNP site rs2027349 as an example. A. SNP editing starting from a heterozygous cell line. Two ssODNs carrying both alleles at the SNP site (A/T) are used to edit the heterozygous line (top) to the opposite direction and generate two homozygous cell lines of different genotypes (middle). Cells that have CRISPR/Cas9-mediated DNA cleavage events but not ssODN integration show the presence of indels in the form of two overlapping peak sets proximal to the target site (bottom). B. SNP editing starting from a homozygous cell line. Only one ssODN carrying the alternative allele (T) is required to generate both the heterozygous (middle-left) and homozygous (middle-right) cell line of the opposite genotype. Note that the chance of obtaining the edited homozygous cell clones is significantly lower than that of the heterozygous one.


    2. Technical requirements. The overall experiment incorporates three main parts: plasmid cloning and preparation; iPSC maintenance, transfection, and subcloning; and Sanger sequencing. Whilst it is desirable that one operator be acquainted with all these skills, this protocol has been designed to work as a collaboration project with several members, each with their own specialization.


  2. gRNA design and vector preparation

    1. Use dbSNP (https://www.ncbi.nlm.nih.gov/snp/) or any genome browser to fetch the DNA sequence proximal to the target SNP for gRNA design. Generally, the sequence will extend 65 bp both upstream and downstream of the target SNP, for a total of 131 bp. Save the sequence as it will be used to synthesise the ssODN with homology arms flanking the target SNP site. This sequence will also be used in a gRNA design tool such as Benchling (https://www.benchling.com) to generate the gRNA sequence (20 bp) for the introduction of a double-strand break (DSB). Pick the gRNA with the highest efficiency score while maintaining a reasonable specificity score. It is also found that gRNA sequences with a cleavage site proximal to the target SNP location may have better recombination efficiency (see Figure 2 for the schematic figure as well as an output example from Benchling for the gRNA and ssODN used to edit SNP rs2027349). In the meantime, design a pair of PCR primers covering the target SNP using Primer3 (http://primer3.ut.ee/) or similar online tools. The PCR product should be 400-600 bp in size, and the SNP is at least 150 bp from either end of the product.



      Figure 2. Schematic of HDR-mediated SNP editing. A. gRNA-introduced double-strand breaking event. The single guide RNA (sgRNA) binds to a specific genomic DNA sequence and introduces a DSB in the genomic DNA double-strand. The ssODN is shown in parallel to highlight the mutation site; B. HDR-mediated DNA repair event using ssODN as the template. After creating the DSB (dashed line), the HDR-mediated DNA repair starts and, during this process in some cells, ssODN carrying the mutant allele is used as the template for the final repaired product; C. Final product after the repair using ssODN as the template, with the point of mutation highlighted; D. Sample gRNA candidate output list from Benchling using human GRCh38 genomic sequence chr1:150,067,554-150,067,687. Here, the highlighted item contains SNP rs2027349 as …AATA[A]CGCC…; E. Sample ssODN output window from Benchling using the same genomic sequence above, showing conversion of the SNP itself from A to G (in red).


    2. Order the following oligonucleotides from a vendor (such as IDT or Sigma-Aldrich):

      1. ssODN, 131 nt in length, single-stranded with the target SNP of the edited allele located at base position 66. Do not order the complementary strand. Two ssODNs carrying both alleles are required if starting from a heterozygous cell line, while one ssODN carrying the opposite allele is required if starting from a homozygous cell line. Order in 4 nmol Ultramer Oligo dry format with standard desalting if from IDT.

      2. sgRNA-top: 5’-[Phos]-CACCgNNNNNNNNNNNNNNNNNNNN;

        sgRNA-bottom: 5’-[Phos]-AAACNNNNNNNNNNNNNNNNNNNNc;

        in which N stands for the gRNA sequence. Note the tailing c at the end of sgRNA-bottom oligo. DO NOT include the PAM sequence in the sgRNA oligos.

        Both oligos can be ordered in 5′-phosphorylated formats (recommended) to avoid the in-house PNK phosphorylation step (the efficiency of which varies). Order in 25 nmol dry DNA Oligo format with standard desalting if from IDT and resuspend to 100 μM with TE buffer upon arrival for long-term storage at -20°C.

      3. Sequencing primers, as mentioned above. The PCR primers can be used as sequencing primers if the SNP is not too near/far from either end. Order in 25 nmol dry DNA Oligo format with standard desalting and resuspend to 100 μM with TE buffer upon arrival for long-term storage at -20°C.

      4. U6-Forward primer (5’-gagggcctatttcccatgatt) for sequencing the gRNA insert of the final plasmid construct. Order in 25 nmol dry DNA Oligo format with standard desalting and resuspend to 100 μM with TE buffer upon arrival for long-term storage at -20°C.

    3. Assemble the pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene, 62988) construct with customized gRNA insert.

      1. Mix well the following components in a PCR tube with a cap at room temperature. Do not use diethyl pyrocarbonate (DEPC)-treated water throughout the experiment as it may inhibit the reaction.

        Component        Amount (μl)
        sgRNA top (100 μM)      1
        sgRNA bottom (100 μM)      1
        T4 ligation buffer, 10× (NEB)      1
        Water, PCR grade      7
        Total      10


      2. Use a thermocycler with a heated lid and set the following parameters to anneal the oligos. Ramp down at the minimum ramp rate (e.g., 0.1°C/s). Throughout this protocol, a thermocycler with a heated lid should be used (with lid heating function turned on) unless otherwise specified.

        Step #      Temperature      Time      Note
        1      95°C      5 min          Minimum ramp rate
        2      25°C      2 min


      3. Dilute the annealed oligos by adding 1 μl to 199 μl PCR-grade water, mix well.

      4. Perform the cut-ligation reaction by mixing well the following components in a PCR tube with cap. We recommend setting up a negative control experiment by substituting the diluted oligos with water, whose function will be introduced later.

        Component          Amount (μl)
        pSpCas9(BB)-2A-Puro (PX459) V2.0, 100 ng/μl          1
        Diluted annealed oligos from step A3c          2
        Tango buffer, 10×          2
        DTT, 10 mM          1
        ATP, 10 mM          1
        FastDigest BbsI          1
        T7 ligase          0.5
        Water, PCR grade          11.5
        Total           20


        Then set up the thermocycler parameters as follows:

        Step #           Temperature        Time             Note
        1           37°C 5 min
        2           21°C 5 min
        3           / /              Return to step #1, 6 cycles

      5. Treat the ligation product and negative control with PlasmidSafe exonuclease to digest any residual linearized DNA by assembling the following components in a PCR tube with cap and mixing well. This heat-inactivated reaction can be stored at -20°C for at least 1 week or -80°C indefinitely if it cannot be used immediately.

        Component          Amount (μl)
        Ligation product from Step A3d          11
        PlasmidSafe buffer, 10×          1.5
        ATP, 10 mM          1.5
        PlasmidSafe exonuclease          1
        Total           15


        Then set up the thermocycler as follows:

        Step #               Temperature             Time Note
        1 37°C             30 min
        2 70°C             30 min
        3 4°C             hold


      6. Transformation of competent cells. We recommend the use of RecA- E. coli strains such as Invitrogen Stbl3 (recA13) or NEB Stable (recA1), since the inserted gRNA and its associated gRNA scaffold may include unstable sequences. As a guideline, use 2 μl product from Step A3e per transformation (include a negative control) following the vendor’s instructions. After heat shock, it is necessary to add recovery media (such as SOC or NEB 10-beta/Stable Outgrowth Medium) at 32°C for 1 h with shaking before spreading on LB/Amp+ agar plates, since the amount of initial DNA input is low.

      7. Incubate at 32°C for 16 h and check for colonies the next day. A large number of colonies on the negative control plate usually suggests that the BbsI digestion did not work. Pick 4-6 colonies from the plate with annealed oligos added and inoculate 4 ml LB/Amp+ media with each colony. Use a new LB/Amp+ agar plate to record all the inoculated colonies and their corresponding tube numbers. Incubate at 37°C overnight.

      8. Isolate plasmid DNA using spin columns such as those from a Qiagen QIAprep Spin Miniprep Kit and send for sequencing using the U6-Forward primer to identify positive clones. In the meantime, keep the record plate from Step A3g at 4°C until all the positive clones have been identified by sequencing.

      9. Inoculate 250 ml LB/Amp+ with the colonies of positive clones from the record plate to prepare transfection-grade plasmid. Culture and extract the plasmid using a Qiagen EndoFree Plasmid Maxi Kit to ensure complete removal of endotoxins according to the manufacturer’s instructions. Dilute the purified plasmid to 1 μg/μl or appropriate in endo-free TE buffer (provided in the maxi-prep kit) as 50-μl aliquots and store at -20°C until use.


  3. Maintenance of human iPSCs for editing

    1. Grow healthy undifferentiated human iPSCs in 6-well plates coated with Matrigel. Generally, for each well of the 6-well plate, use 2 ml mTeSR1 culture media and replace every 24 h. Observe the colony morphology every day and watch for signs of differentiation.

    2. Pass the cultured iPS cells when confluency reaches 60-70% (approximately 4-7 d). Dissociate colonies from the old plates by treating each well with 1 ml ReLeSR and incubating for 3-5 min with the plate lid on according to the technical manual. Dilute the dissociated cell aggregates and transfer 1/10-1/20 of the cells to a new well pre-coated with Matrigel in 2 ml mTeSR1. For details, please refer to Section 3 of the Technical Manual: Maintenance of Human Pluripotent Stem Cells in mTeSRTM1, StemCell Technologies TM (https://www.stemcell.com/media/files/manual/10000005505-Maintenance_of_Human_Pluripotent_Stem_Cells_mTeSR1.pdf).


  4. Transfect human iPSCs with the gRNA-integrated Cas9 vector and ssODN for precise gene editing.

    1. Seed iPSCs in 60-mm Petri dishes for transfection.

      1. As a guideline, each well of the 6-well plate at 60-70% confluence has approximately 1 × 106 iPSCs. On the other hand, each 60-mm Petri dish requires 4 × 105-5 × 105 iPSCs for seeding. Multiply the total number of iPSC wells for seeding by 1.2 to compensate for loss during collection and counting.

      2. Prepare enough 60-mm cell culture grade Petri dishes coated with Matrigel for iPSC seeding. Generally, prepare one Petri dish for each clone that will be generated, plus one dish for transfection efficiency evaluation.

      3. When iPSCs in 6-well plates reach a confluence of 60-70%, calculate the number of iPSCs required for transfection. Check cell morphology and manually scrape off any differentiated colonies under a stereomicroscope before dissociation. Do not use a culture with more than 20% of the colonies with signs of differentiation. Remove culture media from the plates and add 1 ml Accutase per well. Place the plates back into the incubator and wait for 10 min to dissociate cells.

      4. After 10 min, add 1 ml room-temp mTeSR to each well of the 6-well plate. Smack the plate against the palm to dislodge any remaining cells from the bottom gently without spilling media. Collect media from all wells into a 15-ml sterile Falcon tube and gently pipet up and down 5-7 times using a regular 1-ml tip or glass pipette to mix well and break up the clumps.

      5. Determine the cell density using either a hemocytometer or automatic cell counter such as a ThermoFisher Countess 3. The cell density at this stage should be approximately 3 × 105-6 × 105 cells/ml. In the meantime, keep the Falcon tube containing the cells at room temperature; do not place cells on ice. Calculate the total number of cells collected.

      6. Centrifuge the Falcon tube at 200 × g for 5 min at room temperature to precipitate the cells. Carefully remove all media and add an equal volume of fresh mTeSR1. Resuspend the cell pellet using a wide-bore 1-ml tip or glass pipet. Centrifuge the cell suspension again at 200 × g for 5 min at room temperature.

      7. Calculate the final volume of mTeSR1 required for seeding iPSCs into 60-mm Petri dishes, plus one dish for a transfection control. Each dish will need 4-5 × 105 cells suspended in 4 ml mTeSR1. Add the ROCK inhibitor Y27632 to a final concentration of 5 μM in the media to increase viability after replating. If multiple dishes (say, transfections) are required, it may be more convenient to transfer partially resuspended cells into a larger 50-ml Falcon tube and add more mTeSR1 after transfer. Remove all media from Step D1f and replace with mTeSR1 containing Y27632. Dilute the cells to the desired concentration and fill each 60-mm dish with 4 ml fully resuspended iPSCs as single cells. Incubate at 37°C, 5% CO2 for a minimum of 12 h.

    2. Transfect iPSCs with the gRNA-integrated Cas9 vector and ssODN.

      1. 1 h before transfection, check cell growth and replace media with 4 ml fresh antibiotic-free mTeSR1.

      2. For each 60-mm dish, prepare the following mix in a 1.5-ml tube. Pipet up and down to mix well. For the control dish, replace the pSpCas9(BB)-2A-Puro construct with the pEGFP-N2 plasmid.

        Component      Amount (μl)

        pSpCas9(BB)-2A-Puro (PX459) V2.0 with gRNA insert,

        1 μg/μl

             3
        ssODN, 1 μg/μl       3
        Opti-MEM I Reduced Serum Medium      473
        Total      479


      3. Add 21 μl FuGene HD transfection reagent (1:3.5 DNA: reagent ratio, for a total of 500 μl) to the mix from Step D2b and briefly vortex (2-3 s) the tube to mix. Incubate the final mix at room temperature for 6 min.

      4. Add the DNA:FuGENE:Opti-MEM I mix to the 60-mm dish and gently swirl to mix. Do not pipet up and down since this interferes with the transfection process. Incubate at 37°C, 5% CO2 for 24 h.


  5. Select positive clones using puromycin and evaluate the transfection efficiency.

    1. Select positive clones using puromycin

      Note: Also read Step D2.

      1. For all dishes transfected with the pSpCas9(BB)-2A-Puro construct, add 4 ml mTeSR1 supplemented with 0.5 μg/μl puromycin per dish to selectively enrich the cells transfected with the pSpCas9(BB)-2A-Puro construct, which carries a puromycin resistance cassette. The concentration of puromycin used may need optimization on a cell line-specific basis since cell lines may have very different puromycin tolerance levels (ranging from 0.3 to 0.7 μg/μl). Incubate the dishes at 37°C, 5% CO2 for 24 h.

      2. Carefully remove media and any dead cells or cell debris from the dishes. Add 4 ml mTeSR1 supplemented with 0.3 μg/μl (or as determined by the previous assay) puromycin per dish. Incubate the dishes at 37°C, 5% CO2 for another 24 h.

      3. Carefully remove media and any dead cells or cell debris from the dishes. Withdraw puromycin selection from this time (72 h post-transfection). Add 4 ml mTeSR and replace the culture media daily. Incubate the dishes at 37°C, 5% CO2 for 5-10 d until the iPSC colonies reach the proper size for clone selection (1.5-2 mm in diameter, Figure 3).



        Figure 3. Example of an iPSC colony for clone selection


    2. Evaluate the transfection efficiency by EGFP expression. This step should be performed in parallel with Step D1.

      1. For the dish transfected with the pEGFP-N2 plasmid, carefully replace the culture media with 4 ml fresh mTeSR without puromycin. Incubate the dish at 37°C, 5% CO2 for 24 h.

      2. Replace culture media with another 4 ml fresh mTeSR without puromycin. Incubate the dish at 37°C, 5% CO2 for another 24 h.

      3. Count several fields of view to assess the percentage of EGFP+ cells. The transfection efficiency, as calculated by the percentage of EGFP+ cells, should be at least 40-50%.

    3. Isolate individual iPSC colonies for genotype identification of positive clones.

      1. On the day of colony picking, prepare one flat-bottomed 96-well plate coated with Matrigel (referred to as Plate A). Add 80 μl mTeSR1 containing 10 μM ROCK inhibitor Y27632 to each well of Plate A. Prepare another uncoated 96-well plate (referred to as Plate B, flat-bottomed wells).

      2. Replace the media in the 60-mm dish with fresh ROCK inhibitor-free mTeSR1.

      3. Set a P100 or P200 pipette at 50 μl. Attach a P200 tip and use the tip to scrape off a single colony. Aspirate the scraped-off colony or its fragments (if the colony breaks during the process) using the pipet and transfer all its contents (50 μl) to one well of Plate A. Gently pipet up and down 3 times to break the cell clump. Without replacing the pipet tip, transfer 50 μl from Plate A into the corresponding well of Plate B (Say, A1 well to A1 well, etc.) Repeat this step until all the colonies of interest on the 60-mm Petri dish have been picked up.

      4. Spin down both plates at 200 × g for 5 min at room temperature with the lids on.

      5. Keep Plate A at 37°C, 5% CO2 for 24 h to allow iPSCs to reattach to the well. Continue to incubate the plate and replace the media daily (100 μl mTeSR1 per well). Plate A will be used for further clone expansion after genotyping.

    4. Use Plate B for DNA extraction.

      1. Remove Plate B carefully from the centrifuge without shaking and place it on ice. Carefully remove media from the wells without disturbing the cell pellets using either a pipet or a multichannel pipet (set at 200 μl). DO NOT use vacuum aspiration as the cell pellets are not firmly attached to the bottom. Repeat this step to remove media from all wells on the plate.

      2. Add 16 μl quick DNA extraction buffer (Lucigen/VWR) to each well containing the cell pellet. Set a P10 or P50 multichannel pipet at 10 μl. Carefully pipet up and down 5 times to mix; minimize bubble formation. This step resuspends cells and performs cell lysis for subsequent use.

      3. Transfer all (16 μl) the mixed cell suspension to a 96-well PCR plate and seal using either s striped cap or film. Mark the plate as “Extracted DNA” with name and date. The product from this step should be stored at -20°C if not immediately used (1-2 weeks) or -80°C for extended storage. Set up the thermocycler as follows and discard Plate B.

        Step #            Temperature       Time            Note
        1 65°C       15 min
        2 68°C       15 min
        3 98°C       10 min
        4 4°C       hold


    5. Sample preparation for DNA sequencing on a Sanger sequencer.

      1. Prepare the PCR reaction mix using the quick-isolated DNA from the previous step as the template. Assemble the reactions on ice according to the following chart, multiply by the number of samples for each component and further multiply by 1.1 to compensate for potential loss during reagent transfer.

        Component     Amount (μl)
        10× PCR Buffer     1
        10× PCR Enhancer     1
        50 mM MgSO4     0.3
        10 mM dNTP     0.2
        Forward primer (10 μM)     0.2
        Reverse primer (10 μM)     0.2
        Qiagen HotStarTaq DNA polymerase     0.1
        Water, PCR grade     5.5
        Total     8.5


      2. Mix all components WITHOUT the DNA template in a 1.5-ml tube on ice. Use a new 96-well PCR plate and dispense 8.5 μl reaction mix into each well until all the desired wells have been filled. Subsequently, add 1.5 μl extracted DNA to each well (a total of 10 μl). Pipet up and down using a P10 pipet set at 5 μl to mix well while avoiding bubble formation. This operation will be more convenient if performed with a multichannel pipet.

      3. Seal the top of the PCR plate using a striped cap or film and mark the plate as “PCR product”. Set up the thermocycler as follows. The annealing temperature (3) and extension time (4) may require optimization, as the operator sees appropriate.

        Step #             Temperature       Time        Note
        1             95°C       10 min        Taq activation
        2             95°C       30 s
        3             53°C       90 s        Or as appropriate
                    72°C       60 s        Or as appropriate
        5             /        /        Go to 2, 40 cycles
        6             72°C       10 min        Final extension
        7             4°C       hold


      4. While the PCR reaction is running, set up one or more 1.25% TAE/TBE agarose gels with a sufficient number of wells to hold at least 20 samples for PCR efficiency inspection.

      5. Assemble the shrimp alkaline phosphatase (SAP) reaction mix on ice to remove single-stranded DNA and dNTPs from the PCR product according to the following chart. Multiply by the number of samples for each component and further multiply by 1.1 to compensate for potential loss during reagent transfer.

        Component         Amount (μl)
        10× SAP buffer        0.5
        Shrimp alkaline phosphatase (SAP)        0.5
        Exonuclease I        0.1
        Water, PCR grade        3.9
        Total         5


      6. After the PCR reaction has finished, use a new 96-well PCR plate and dispense 5 μl SAP mix into each well until all the desired wells have been filled. Subsequently, transfer 5 μl (1/2 of the total volume) product from the plate marked “PCR product” to the corresponding well in the 96-well plate holding the SAP mix (a total of 10 μl). Pipet up and down to mix well while avoiding bubble formation. Mark the plate as “SAP product” and set up the thermocycler as follows.

        Step #            Temperature              Time          Note
        1 37°C 50 min
        2 95°C 15 min
        3 4°C hold


      7. While the SAP reaction is running, add 1.5 μl 6× loading dye into selected wells of the “PCR product” plate and load their contents onto the TAE/TBE gel prepared in Step E4g. Run the gel at 100 V with an appropriate molecular ladder for at least 25 min. Check the shape, number, and clarity of the bands in each lane. Take images and keep them for the records.



        Figure 4. Sample agarose gel electrophoresis results of PCR products. Lane 3 has too little DNA to be acceptable. Lane 2, albeit of reduced brightness, is still acceptable for DNA content. M: molecular marker, 1 kb, Promega G5711.


      8. After taking gel images of the PCR reactions, count the number of “good” reactions (a single band with the correct molecular size and sufficient DNA amount as evaluated by the band’s brightness, see Figure 4) and prepare the mix for Sanger sequencing. Assemble the Sanger reaction mix on ice according to the following table. Multiply the volume of each component by the count of “good” reactions only and further multiply the results by 1.1 since the BigDye Terminator reagent is expensive.


        Component      Amount (μl)
        BigDye Terminator V3.1      1
        Sequencing primer (10 μM)      1
        Water, PCR grade      3
        Total      5


      9. After the SAP reaction has finished, use a new 96-well PCR plate and dispense 5 μl Sanger sequencing mix into each well until all the desired wells have been filled. Subsequently, transfer 5 μl (1/2 of the total volume) product from the plate marked “SAP product” to the corresponding well of the 96-well plate holding the Sanger sequencing mix (a total of 10 μl). Pipet up and down to mix well while avoiding bubble formation. Mark the plate as “Sequence PCR product” and set up the thermocycler as follows. The annealing temperature (3) may require optimization, as the operator sees appropriate.


        Step #      Temperature      Time      Note
        1 96°C      10 s
        2 55°C      5 s      Or as appropriate
        3 60°C      4 min
        4 /      /      Go to 2, 25 cycles
        5 4°C      hold


      10. Purify the sequencing PCR product using a Qiagen DyeEx 2.0 Spin Kit (best for ≤ 16 samples). If the DyeEx 2.0 Kit is not available or there are more than 16 samples, skip Step E5j and resume at Step E5k.

        1. Loosen the top cap and snap off the bottom closure of the spin column. Insert the spin column into a collection tube and centrifuge at 750 × g for 3 min.

        2. Transfer the spin column to a new 1.5-ml tube labeled with the sample name. Aspirate the sequencing PCR product from the corresponding well and slowly apply the reaction product directly onto the center of the slanted gel bed surface. Centrifuge again at 750 × g for 3 min.

        3. Discard the spin column and place the 1.5-ml tubes in a centrifugal vacuum device (such as a SpeedVac) for 30–60 min until all the liquid has evaporated. Do NOT turn on the heating during the vacuum process. After drying, add 15 μl sequencing grade formamide to the tube and pipet up and down 10 times to dissolve the DNA. Proceed to Step E5kvii.

      11. Purify the sequencing PCR product by isopropanol precipitation.

        Note: (!) Denotes that this step is critical and one should proceed with caution.

        1. Add 40 μl 75% isopropanol to each well and gently pipet up and down 5 times to mix. This operation will be more convenient if performed with a multichannel pipet.

        2. Incubate at room temperature for 20 min, then centrifuge at 3,000 × g for 30 min in a swing-bucket centrifuge at room temperature (20°C). Turn on the cooling function of the centrifuge if available since the air inside the centrifuge may get hot fast.

        3. Carefully remove the plate from the centrifuge without shaking. Invert the plate onto 3-4 layers of paper towel very gently to decant the isopropanol. Repeat the process once with another fresh 3-4 layers of paper towel and keep the plate inverted for 10 s to remove as much isopropanol as possible. Do not use pipetting or a vacuum to remove any remaining isopropanol from the wells.

        4. Flip the 96-well plate back slowly, and carefully add 150 μl 75% isopropanol to each well to wash the pellet.

          Important: Do NOT mix! This operation will be more convenient if performed using a multichannel pipet. Centrifuge the plate again at 3,000 × g for 10 min.

        5. Carefully invert the plate on the paper towel stack as in Step E5kiii to decant the isopropanol. Keep the plate inverted (!), carefully transfer it onto a new paper towel stack, and centrifuge again at 300 × g (!) for 1 min to eliminate the residual isopropanol.

        6. Carefully remove the 96-well plate from the centrifuge and flip it back. Add 15 μl sequencing-grade formamide into each well and pipet up and down 5 times to mix. Avoid the formation of bubbles inside the well.

        7. Seal the plate with an adhesive film and denature the DNA at 95°C for 2 min on a thermocycler. Immediately place the plate on ice and incubate for 2 min.

        8. Centrifuge the plate at 2000 × g for 1 min. The plate is now ready for Sanger sequencing injection on an ABI 310/3730/3730xl/3500 sequencer.


  6. Expand positive clones with the desired genotypes.

    1. Examine the sequencing spectrum files using ApE or ABI GeneMapper to identify positive candidate clones for expansion (see Section A, Figure 1, and Figure 5). The expanded iPSCs will be used for further single-cell subcloning to obtain pure clones.



      Figure 5. Sample sequencing spectrums of good and bad quality. A. A good result, showing clear discrete peaks. B. A bad result, showing multiple overlapping peak sets, implying the presence of multiple PCR products in the sequencing input. C. A bad result, showing a very weak peak signal.


      1. When the colonies reach the proper size (1.5-2 mm in diameter or 60-70% confluence in the wells) in Plate A (from Step E3a), dissociate the iPSC colonies by firstly removing all media from the wells. Gently add 50 μl ReLeSR to each well, aspirate out quickly, and stand for 5 min at room temperature.

      2. Add 100 μl mTeSR1 to each well and carefully dislodge the cell clumps from the bottom of the well by tapping the plate and scraping using a regular P200 pipet tip. Transfer the contents of each well into one well of a Matrigel-coated 4-well or 24-well plate and add mTeSR1 containing 5 μM ROCK inhibitor to a final volume of 500 μl. Continue culture at 37°C, 5% CO2 in an incubator with daily refreshing of mTeSR1 until the cells reach 60-70% confluence in the wells.

    2. Single-cell subcloning to obtain pure isogenic lines.

      1. Carefully remove media from the wells and subsequently add 400 μl Accutase per well for 10 min to dissociate the cells from the bottom. Gently pipet up and down 5 times using a P1000 tip to make a single-cell suspension and then add another 400 μl mTeSR1 to neutralize the Accutase. Take 10 μl suspension to count the cell density using the trypan blue method and centrifuge the remaining cells at 250 × g for 5 min.

      2. Remove the supernatant and resuspend the cells in 1 ml mTeSR1 containing 5 μM ROCK inhibitor Y-27632. Isolate the volume equivalent to 2000 live cells (as determined in the previous step) and replate into a 60-mm Matrigel-coated dish in 4 ml mTeSR1 containing 5 μM ROCK inhibitor Y27632. Continue to culture any remaining cells in 4/24-well plates as backups. Continue culture at 37°C, 5% CO2.

      3. Replace half the media in the 60-mm dish with ROCK inhibitor-free mTeSR1 48 h post replating. Continue culture at 37°C, 5% CO2 in an incubator with daily refreshing of mTeSR1 until the colony size reaches 1.5-2 mm in diameter. Repeat the colony picking procedure as described under Step E3. Pick approximately 30 colonies from each 60-mm dish. Repeat the genotyping procedure for the selected colonies as described under Steps E4-E5.

      4. Once pure clones have been identified by Sanger sequencing, repeat Step F1 to transfer each colony from the 96-well plates to 2 (!) wells of the Matrigel-coated 4/24-well plates until the confluence reaches 60-70%. Continue to monitor the cell growth as different clones may have different growth speed at this stage.

      5. Remove media from the wells and add 400 μl ReLeSR to each well. Remove ReLeSR from the wells and incubate for 5 min at room temperature.

      6. Coat enough 6-well plates with Matrigel and add 2 ml mTeSR1 per well. Add 300 μl mTeSR1 to each well of the 4/24-well plate treated with ReLeSR and gently pipet up and down 5 times using a wide-bore pipet tip to dissociate the cell clumps. Aliquot 100 μl cell clump suspension to each well of the 6-well plate (3 wells in total) and gently shake to mix. Prepare 4-6 wells for each clone. Continue to incubate until the confluence reaches 60-70%.

    3. Freezing cell lines for cryopreservation.

      1. Closely observe the cell growth in 6-well plates until the confluence reaches 60-70%. It is recommended that all wells on the same plate be processed in one run.

      2. Add 1 ml ReLeSR per well of the 6-well plate. Remove ReLeSR from the wells and incubate for 3-5 min at 37°C, 5% CO2 in an incubator.

      3. Add 1 ml room temperature mTeSR1 per well of the 6-well plate treated with ReLeSR and dislodge the cells by tapping the plate firmly. Transfer the dislodged cell suspension to a sterile 1.5-ml Eppendorf tube and centrifuge at 200 × g for 5 min at room temperature.

      4. Carefully remove as much supernatant as possible without touching the cell pellet at the bottom. Add 1 ml room temperature mFreSR to the Eppendorf tube and carefully resuspend the cell pellet using a wide-bore pipet tip by pipetting up and down 5 times. Transfer the tube contents to a pre-labeled cryovial tube. Confirm the tube is firmly sealed and place it in a Mr Frosty Freezing Container. Generally, prepare as many tubes (usually 4-6 tubes as determined in Steps F2d-F2f) as possible for each clone. Repeat this step until all the cells have been transferred to cryovials.

      5. Place the Mr Frosty Freezing Container containing cryovials in a -80°C freezer for at least 24 h (but no longer than 72 h) to freeze the contents. Move all the vials to liquid N2 for long-term storage. Keep detailed records.

Data analysis

  1. Spectrum analysis of Sanger sequencing results

    Successful identification and isolation of precise-edited, pure isogenic clones heavily rely on proper analysis of Sanger sequencing results. As a guideline, firstly remove samples with low signal intensities, aberrant patterns (such as “merged” large peaks rather than individual peaks when nucleotide bases of the same type appear consecutively in a sequence, see Figure 5). Subsequently, positive clones are identified by examining the genotype at the desired SNP site.

  2. Evaluation of gRNA and ssODN efficiencies

    Since the ssODN integration and subsequent precise SNP editing requires CRISPR/Cas9-mediated DNA cleavage to take precedence, the efficiencies of CRISPR/Cas9-mediated DNA cleavage and ssODN integration require independent evaluation. Generally, as most CRISPR/Cas9-mediated DNA breaks are not appropriately repaired and produce indels, the percentage of indel-containing clones (see Figure 1) generated can serve as an indicator of the efficiency of the gRNA sequence used in the experiment. The percentage of indel-containing clones is approximately 60-80% for a successful gRNA sequence design. The integration of ssODN is relatively stable at approximately 5% of CRISPR/Cas9-mediated DNA cleaving events. If the number of positive clones is low, calculate the likelihood of the two events separately from Sanger sequencing results to determine which oligo sequence may be the cause and require redesign.

Acknowledgments

Funding: R01MH106575, R01MH116281, R01AG063175. We acknowledge the concept of origin as developed by Ran et al. (2013) and further methodology demonstration in Zhang et al. (2020) (Doi: 10.1126/science.aay3983).

Competing interests

The authors declare no conflicts of interests.

Ethics

The NorthShore University HealthSystem Institutional Review Board (IRB) approved the study.

References

  1. Forrest, M. P., Zhang, H., Moy, W., McGowan, H., Leites, C., Dionisio, L. E., Xu, Z., Shi, J., Sanders, A. R., Greenleaf, W. J., Cowan, C. A., Pang, Z. P., Gejman, P. V., Penzes, P. and Duan, J. (2017). Open Chromatin Profiling in hiPSC-Derived Neurons Prioritizes Functional Noncoding Psychiatric Risk Variants and Highlights Neurodevelopmental Loci. Cell Stem Cell 21(3): 305-318 e308.
  2. Forsyth, N. R., Musio, A., Vezzoni, P., Simpson, A. H., Noble, B. S. and McWhir, J. (2006). Physiologic oxygen enhances human embryonic stem cell clonal recovery and reduces chromosomal abnormalities. Cloning Stem Cells 8(1): 16-23.
  3. Hendel, A., Kildebeck, E. J., Fine, E. J., Clark, J., Punjya, N., Sebastiano, V., Bao, G. and Porteus, M. H. (2014). Quantifying genome-editing outcomes at endogenous loci with SMRT sequencing. Cell Rep 7(1): 293-305.
  4. Howden, S. E., Maufort, J. P., Duffin, B. M., Elefanty, A. G., Stanley, E. G. and Thomson, J. A. (2015). Simultaneous Reprogramming and Gene Correction of Patient Fibroblasts. Stem Cell Reports 5(6): 1109-1118.
  5. Miyaoka, Y., Chan, A. H., Judge, L. M., Yoo, J., Huang, M., Nguyen, T. D., Lizarraga, P. P., So, P. L. and Conklin, B. R. (2014). Isolation of single-base genome-edited human iPS cells without antibiotic selection.Nat Methods 11(3): 291-293.
  6. Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A. and Zhang, F. (2013). Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8(11): 2281-2308.
  7. Tidball, A. M., Swaminathan, P., Dang, L. T. and Parent, J. M. (2018). Generating Loss-of-function iPSC Lines with Combined CRISPR Indel Formation and Reprogramming from Human Fibroblasts. Bio-protocol 8(7): 2794.
  8. Zhang, S., Moy, W., Zhang, H., Leites, C., McGowan, H., Shi, J., Sanders, A. R., Pang, Z. P., Gejman, P. V. and Duan, J. (2018). Open chromatin dynamics reveals stage-specific transcriptional networks in hiPSC-based neurodevelopmental model. Stem Cell Res 29: 88-98.
  9. Zhang, S., Zhang, H., Zhou, Y., Qiao, M., Zhao, S., Kozlova, A., Shi, J., Sanders, A. R., Wang, G., Luo, K., Sengupta, S., West, S., Qian, S., Streit, M., Avramopoulos, D., Cowan, C. A., Chen, M., Pang, Z. P., Gejman, P. V., He, X. and Duan, J. (2020). Allele-specific open chromatin in human iPSC neurons elucidates functional disease variants.Science 369(6503): 561-565.

简介

[摘要]人类诱导多能干细胞(人iPS细胞)在该领域被广泛地用于小号发育生物学和疾病的模型。在iPSC系CRISPR / Cas9基因编辑往往具有低的频率,这妨碍在精确等位基因编辑疾病相关的单核苷酸多态性(SNP),尤其是那些在非编码部分的其应用小号基因组。在这里,我们提出了一个独特的工作流程来设计的SNP等基因的iPSC系编辑从杂合子到的疾病风险等位基因小号或等位基因非风险小号使用瞬时且直接的基于转染的协议。该协议使我们能够在大约 4 到 5 周内同时获得 SNP 位点所有三种可能基因型的纯和克隆等基因系。


[背景] CRISPR / Cas9基因编辑到iPSC集的应用的细胞提供无与伦比的电位在该领域小号发育生物学,疾病的建模,和再生医学。然而,由于iPSC中没有特别默认在CRISPR / Cas9基因编辑中使用的传统策略,编辑效率通常很低,尤其是对于同源定向修复(HDR)介导的单核苷酸多态性编辑小号(SNP小号)。特别实验人的方法必须被开发来规避这一限制; 例如,使用同时CRISPR / Cas9诱变和重编程的iPSC的(豪顿等人,2015;蒂德博尔等人,2018) ,先进的细胞分选技术,例如FACS (福赛斯等人,2006; Miyaoka 。等人,2014 ) ,或通过靶向质粒中长同源臂的存在重组携带突变的盒(Hendel等人,2014 年)。然而,这些高度SPECIALI ž编基因协议在iPSC系编辑一个重新时间和/或耗费资源的,其持续小号作为在生成经由CRISPR / Cas9介导的精确SNP等基因编辑iPSC系的一个关键限制因素。

在这里,我们提出了一个表现出的工作流程和详细的程序,其允许的iPSC的直接编辑小号使用的CRISPR / Cas9系统与一个由简单的基于脂质体的转染HDR技术,随后抗生素选择以丰富的编辑的iPSC和单一克隆选择以获得纯所有三种可能基因型的 iPSC 同基因系。该方法改编自 Ran等人。(2013 年)在我们最近的出版物中得到了证明(Forrest等人,2017 年;Zhang等人,2018 年和2020 年)。值得注意的是,这个协议的表现还算不错了我们独特的CRISPR / cas9 SNP从杂iPSC系,以基因对纯合子的疾病风险等位基因编辑设计小号或等位基因无风险的小号,这使得经过编辑的SNP的功能解释的更可靠直接比较所有三种不同基因型的等基因系(Forrest等人,2017 年;Zhang等人,2018 年和2020 年)。

关键字:诱导多能干细胞, 基因组编辑, 疾病模型, CRISPR/Cas9, 同源重组, 等位基因特异性



材料和试剂

材料
1.5 ml Eppendorf 管(VWR,目录号:89000-028)
4-w Nunclon TM Delta MultiDishes (Thermo Scientific,目录号:62407-068)
6孔培养板小号(热˚F isher,目录号:140675)
96孔培养板小号(康宁,隼® ,目录号:353072)
小号TANDARD 60 × 15毫米菜瓦特/通气孔(Fisher Scientific公司,目录号:12-565-95)
15毫升Ç entrifuge吨ubes(VWR,目录号:21008-216)
2毫升ç ryogenic v IALS与Ç losures,p聚环氧(康宁® ,目录号:89089-764)
10微升ř ACKED尖端,升流-r etention小号terile(VWR,目录号:10017-062)
200微升- [R ACKED尖端,升流-r etenti ö Ñ小号terile(VWR,目录号:76322-150)
1 ,000微升升流-r etention吨IPS(VWR,目录号:10017-090)
的BioDot 96 -w ELL Ñ上-s kirted PCR p晚小号(打点科学,目录号:650-PCR)
 


试剂
mTeSR1(STEMCELL,目录号:85850)
mFreSR冷冻保存培养基(STEMCELL,目录号:05855)
ReLeSR (STEMCELL,目录号:05872)
Matrigel ® hESC -Qualified Matrix(Corning ® ,目录号:354277)
FuGENE ® HD 转染试剂(Promega,目录号:E2311)
Accutase (STEMCELL,目录号:07920)
Primocin (Invitrogen,ant-pm-1)
Y-27632二盐酸盐(R&D Systems,目录号:1254/1)
QIAprep Spin Miniprep Kit(250)(Qiagen,目录号:27106)
Qiaprep EndoFree Plasmid Maxi Kit(10)(Qiagen,目录号:12362)
BigDye Terminator v3.1 Cycle Sequencing Kit(Thermo Fisher Scientific,目录号:4337455)
DyeEx 2.0 试剂盒(Qiagen,目录号:63204)
HotStarTaq DNA聚合酶(250 U)(Qiagen,目录号:203203)
PlasmidSafe核酸外切酶(Lucigen ,目录号码:E3101K)
QuickExtract TM DNA 提取液(VWR,目录号:76081-768)
FastDigest BbsI (Thermo Fisher Scientific,目录号:ER1101)
T7连接酶(NEB,目录号:M0318S)
ATP 溶液,100 mM(NEB,目录号:N0451)
dNTP 溶液混合物,10 mM(NEB,目录号:N0447S)
Opti-MEM I,100 ml(Thermo Fisher Scientific,目录号:31985062)
虾碱性磷酸酶(Thermo Fisher Scientific,目录号:783901000UN)
PCR X Enhancer System(Thermo Fisher,目录号:11495017)
异丙醇(Sigma-Aldrich,目录号:190764)
无核酸酶水,PCR 级(Thermo Fisher Scientific,目录号:AM9937)
Teknova DNA / RNA ř esuspension乙uffer(TE缓冲液)(VWR,目录号:100216-886)
Invitrogen One Shot ® Stbl3 TM化学感受态大肠杆菌细胞(Thermo Fisher Scientific,目录号:C737303)
1 kb DNA梯(Promega,目录号:G5711)
 


设备

C1000 Touch Thermo Cycler(Bio-Rad,型号:1851197 )
Sorvall Legend XTR Centrifuge(Thermo Scientific,目录号:75217420 )
台式冷冻离心机 5430R(Eppendorf,目录号:022620601)
Heracell 150i 组织培养培养箱(Thermo Fisher Scientific,目录号:51026283)
ABI 3730 DNA 分析仪(Thermo Fisher Scientific,型号:3730S)
Nalgene Mr. Frosty 冷冻容器(Sigma-Aldrich,目录号:C1562)
 


软件

ApE——质粒编辑器(M Wayne Davis,https: //jorgensen.biology.utah.edu/wayned/ape/ )
 


程序

实验人计划
细胞系选择
通常,如果起始细胞系在要编辑的所需 SNP 位点是杂合的,则实验将是最方便的。这样,我们就可以合成两条单链DNA寡核苷酸(ssODNs ),每条携带不同的基因型,同时进行两个单独的SNP编辑实验,从而在实验结束时获得两个等位基因方向的编辑纯合细胞系(图) 1A)。另外,也可以以得到的纯合细胞系的通过从纯合细胞系开始相对基因型时的情况小号不允许自由度高的,例如,使用从临床DIAGN衍生的细胞系osed例(图1B)。在这种情况下,这将是有益的,以增加挑选的克隆的数目在第E和F到maximi ž Ë一种检测阳性一个机会。虽然在第一轮 CRISPR/Cas9 编辑中可能不会出现其中一个基因型(相反基因型的杂合或纯合),但获得相反基因型的纯合细胞系通常足以满足以下目的:该实验。或者,从第一轮编辑中获得的杂合系可以用于具有相同ssODN 的另一轮 CRISPR/Cas9 编辑,以获得相反基因型的纯合系。在所有情况下,的基因型的在期望的SNP位点起始细胞系需要进行到部分B之前确认要确认在该特定SNP位点基因型,使用30纳克来自起始细胞系提取基因组DNA,并通过执行Sanger测序与步骤继续小号E4 - E5。可选地,在线数据库小号,下一代测序结果,或细胞系相关的患者的元数据还可以提供在SNP位点的基因型信息进行编辑。





图1 。以 SNP 位点 rs2027349 为例的 SNP 编辑策略。A.从杂合细胞系开始的 SNP 编辑。两个ssODNs携带荷兰国际集团在SNP位点(A / T)的两个等位基因的用于编辑的杂合线(顶部)到相反的方向,并且产生不同的基因型(中)两种纯合细胞系。具有CRISPR / Cas9介导的DNA cleav细胞年龄事件,但不ssODN集成显示INDEL的中的两个重叠峰集的形式存在近端到目标部位(底部)。B. 从纯合细胞系开始的 SNP 编辑。只有一个ssODN卡尔ÿ我纳克替代等位基因(T)是必需的,以产生两个杂合(中间左)和纯合(中间偏右)细胞的相对的基因型线。请注意,获得编辑过的纯合细胞克隆的机会明显低于杂合细胞克隆。

技术要求。整个实验包括三个主要部分:质粒克隆和制备;iPSC集维护,转染,和亚克隆; 和桑格测序。虽然它是渴望Desir可以是一个运营商,所有相识本身的技能,该协议已被设计为工作与几名成员合作项目,每个都有自己的SPECIALI ž通货膨胀。
 


gRNA 设计和载体制备
使用dbSNP ( https://www.ncbi.nlm.nih.gov/snp/ ) 或任何基因组浏览器获取靠近目标 SNP 的 DNA 序列以进行 gRNA 设计。通常,该序列将在目标 SNP 的上游和下游延伸 65 bp,总共 131 bp。保存序列,因为它将用于合成ssODN ,其同源臂位于目标 SNP 位点两侧。该序列也将在一个gRNA设计工具使用诸如Benchling (https://www.benchling.com)以产生gRNA序列(20碱基对)的introduc一个和灰双链断裂(DSB)。选择具有最高效率分数的 gRNA,同时保持合理的特异性分数。它也发现,gRNA与序列一个切割位点接近所述靶SNP位置可具有更好的重组效率(参见图2的示意图中,以及从输出示例Benchling为gRNA和ssODN用于编辑SNP rs2027349)。在该MEA n时间,设计一对PCR引物覆盖目标SNP美国ING引物3(http://primer3.ut.ee/)或类似的在线工具。PCR 产物的大小应为 400 - 600 bp,并且 SNP 距离产物的任一端至少为 150 bp。
 






图2 。HDR 介导的 SNP 编辑示意图。A. gRNA 引入的双链断裂事件。的单个引导RNA(因组)结合于特定的基因组DNA序列,并引入一个在DSB的基因组DNA的双链。所述ssODN我示出在平行于突出突变位点S; B. 使用ssODN作为模板的HDR 介导的 DNA 修复事件。穿心莲后荷兰国际集团的DSB(虚线)时,HDR介导的DNA修复的开始和期间在一些细胞中这个过程中,ssODN携带突变型等位基因作为模板的最终修复产物; C. 以ssODN为模板修复后的最终产品,突出显示突变点;从D.样品gRNA候选输出列表Benchling 150,067,554-150,067,687:使用人类GRCh38基因组序列CHR1。此处,突出显示的项目包含 SNP rs2027349 作为 …AATA[A]CGCC…;E.样品ssODN从输出窗口Benchling使用上述相同的基因组序列,示出的转换的SNP本身从A到G(红色)。

从供应商(例如 IDT 或 Sigma-Aldrich)订购以下寡核苷酸:
ssODN ,131个核苷酸的长度,单-链版用的靶SNP的编辑等位基因定位d在基部位置66不要为了互补链。如果从杂合细胞系开始,则需要两个携带两个等位基因的ssODN ,而如果从纯合细胞系开始,则需要一个携带相反等位基因的ssODN 。如果从 IDT订购带有标准脱盐的4 nmol Ultramer Oligo 干燥格式。
sgRNA-top: 5 ' -[ Phos ]- CACCgNNNNNNNNNNNNNNNNNNNN ;
sgRNA-bottom: 5 ' -[ Phos ]- AAACNNNNNNNNNNNNNNNNNNNNc ;


i n 其中 N 代表 gRNA 序列。注意sgRNA-bottom oligo 末端的尾部c 。不要在 sgRNA 寡核苷酸中包含 PAM 序列。


两种寡核苷酸都可以以5'-磷酸化形式(推荐)订购,以避免内部 PNK 磷酸化步骤(其效率各不相同)。在25顺序纳摩尔干燥与标准脱盐DNA寡格式,如果从IDT,重悬至100 μM用TE缓冲液后arriv人对于长期储存在-20 ℃下。


测序引物,如上所述。如果 SNP 离任一端都不太近/太远,则 PCR 引物可用作测序引物。在25顺序纳摩尔干燥标准脱盐,重悬至100的DNA寡格式μM用TE缓冲液后arriv人对于长期储存在-20 ℃下。
U6-正向引物 (5'- GAGGGCCTATTTCCCATGATT ) 用于对最终质粒构建体的 gRNA 插入进行测序。在25顺序纳摩尔干燥标准脱盐,重悬至100的DNA寡格式μM用TE缓冲液后arriv人对于长期储存在-20 ℃下。
组装pSpCas9(BB)-2A-迪普罗(PX459)V2.0(Addgene公司,62988)构建体与customi Ž编gRNA插入件。
混合以及在室温下的盖在PCR管中的下列组分。不使用d我Ë THYL p YRO Ç arbonate (DEPC)处理的水在整个实验中,因为它可能抑制反应。
成分


量(微升)


sgRNA 顶部 (100 μM )


1


sgRNA 底部 (100 μM )


1


T4 连接缓冲液,10 × (NEB)


1


水,PCR 级


7


全部的


10

使用带有加热盖的热循环仪并设置以下参数s以对寡核苷酸进行退火。以最小斜率下降(例如0.1°C/s)。贯穿本协议,用加热的盖的热循环应当使用(与盖加热功能接通),除非另有说明。
步 #


温度


时间


笔记


1


95℃


5分钟


最小斜坡率


2


25 ℃


2 分钟

 


向 199 μl PCR 级水中加入 1 μl稀释退火寡核苷酸,混合均匀。
执行由混合切口连接反应以及在PCR管帽以下组件。我们建议通过用水代替稀释的寡核苷酸来设置阴性对照实验,其功能将在后面介绍。
成分


量(微升)


pSpCas9(BB)-2A-Puro (PX459) V2.0,100 ng/ μl


1


来自步骤 A3c 的稀释退火寡核苷酸


2


探戈缓冲器,10 ×


2


DTT,10 毫米


1


ATP,10 毫米


1


FastDigest Bbs I


1


T7连接酶


0.5


水,PCR 级


11.5


全部的


20

然后设置了热循环参数小号如下:


步 #


温度


时间


笔记


1


37℃


5分钟

2


21 ℃


5分钟

3


/


/


返回第 1 步,6 个循环

治疗用连接产物和阴性对照PlasmidSafe核酸外切酶消化的任何残余lineari ž通过组装与帽PCR管下列分量ed DNA并混合荷兰国际集团良好。如果不能立即使用,这种热灭活的反应可以在-20 °C下保存至少 1 周或在 -80°C 下无限期保存。
成分


量(微升)


来自S 步骤A3d 的结扎产品


11


PlasmidSafe缓冲液,10 ×


1.5


ATP,10 毫米


1.5


质粒安全外切酶


1


全部的


15

然后设置了热循环,如下所示:


步 #


温度


时间


笔记


1


37℃


30分钟

2


70°C


30分钟

3


4 ℃


抓住

 


转化感受态t细胞。我们建议使用RecA -大肠杆菌菌株,例如 Invitrogen Stbl3 ( recA13 ) 或 NEB Stable ( recA1 ) ,因为插入的 gRNA 及其相关的 gRNA 支架可能包含不稳定的序列。作为指导原则,使用2微升从产品小号TEP A3E每转化(包括一个阴性对照),按照供应商的说明。热激后,有必要添加回收Ý介质(例如SOC或NEB 10-β/稳定向外生长培养基)在32 ℃下1小时,用扩频之前振摇上琼脂平板LB / AMP + ,因为初始的DNA的量输入低。
在32 °C 下孵育16 小时,并在第二天检查菌落。阴性对照板上的大量菌落通常表明BbsI消化不起作用。选择4 -从板6个菌落与加入退火的寡聚和接种4毫升LB / AMP +媒体与各菌落。使用新的 LB/Amp+ 琼脂平板记录所有接种的菌落及其相应的管号。在 37 °C 下孵育过夜。
使用离心柱(例如来自Qiagen QIAprep Spin Miniprep Kit 的离心柱)分离质粒 DNA,然后使用U6- F正向引物进行测序以鉴定阳性克隆。在此期间,保持从记录板小号TEP A3G在4 ℃下,直到所有的阳性克隆已经鉴定通过测序。
用记录板中的阳性克隆菌落接种250 ml LB/Amp+以制备转染级质粒。文化与提取质粒我们荷兰国际集团一Qiagen公司EndoFree质粒Maxi试剂盒,以确保完全去除内毒素根据制造商的说明。将纯化的质粒稀释至 1 μg / μl或适当的无内源 TE缓冲液(在 maxi-prep 试剂盒中提供)作为 50 - μl 等分试样,并在-20 °C 下储存直至使用。
 


维护人类 iPSC以进行编辑
在涂有 Matrigel 的 6 孔板中培养健康的未分化人类 iPS Cs 。通常,对于 6 孔板的每个孔,使用 2 ml mTeSR1 培养基并每 24 小时更换一次。观察菌落形态,每天和观赏标志小号分化。
通过将培养的iPS细胞汇合时达到60-70%(约4 - 7 d)。通过用1ml处理每个孔从旧板解离的菌落ReLeSR和incubat荷兰国际集团为3 - 5分钟的上根据技术手册板盖。稀释离解的细胞凝集块小号和转让1/10 - 1/20的细胞到新的井预先涂有基质胶在2ml mTeSR1。有关详细信息,请重新FER以小号挠度3的技术手册:人多能性干细胞在维护的mTeSR TM 1 ,干细胞技术TM (https://www.stemcell.com/maintenance-of-人类多能干,干- cell-in-mtesr1.html)。
 


使用集成了gRNA 的Cas9 载体和ssODN转染人类 iPSC,以进行精确的基因编辑。
种子的iPSC小号在60 -毫米培养皿ES用于转染。
作为指导原则,每个孔在6 6孔板的0 - 70%confluenc ë具有大约1 × 10 6的iPSC。另一方面,每个 60 - mm 培养皿需要 4 × 10 5 -5 × 10 5 iPSC 进行播种。将用于播种的 iPSC 井总数乘以 1.2,以补偿收集和计数过程中的损失。
准备足够的 60 - mm 细胞培养级培养皿,涂有 Matrigel,用于 iPSC 接种。通常,为将要生成的每个克隆准备一个培养皿,再加上一个用于评估转染效率的培养皿。
当在6孔平板中的iPSC达到一个confluenc Ë的60-70%,计算转染所需的iPSC的数目。检查细胞形态和手动刮除下的任何区别殖民地一解离前体视显微镜。不要使用超过 20%的菌落有分化迹象的培养物。从除去培养基的板和加入1ml的Accutase每孔。P花边板放回培养箱并等待10分钟以解离细胞。
10 分钟后,将 1 ml 室温mTeSR添加到 6 孔板的每个孔中。将盘子靠在手掌上,轻轻地从底部取出任何剩余的细胞,而不会溢出培养基。从所有孔中收集介质插入15 -毫升无菌˚F爱尔康管,轻轻吸管上下5 - 7倍我们荷兰国际集团一个规则1 -毫升尖或玻璃吸管拌匀分手的团块。
确定细胞密度我们荷兰国际集团无论是血细胞计数器或自动细胞计数器如一个赛默飞伯爵夫人3.在这个阶段的细胞密度应为约3 × 10 5 - 6 × 10 5细胞/ ml。在此期间,保持˚F爱尔康管含有在室温下的细胞; d o 不要将细胞放在冰上。计算收集的细胞总数。
离心机在200 Falcon管×克在室温下5分钟,以沉淀所述细胞。小心地取出所有介质并添加等量的新鲜 mTeSR1。重悬的细胞沉淀我们荷兰国际集团大口径1 -毫升尖或玻璃吸管。在室温下再次以 200 × g离心细胞悬浮液5 分钟。
计算mTeSR1的最终体积需要用于接种的iPSC小号成60 -毫米培养皿ES ,加上一个菜一个转染对照。每道菜将需要 4-5 × 10 5细胞悬浮在 4 ml mTeSR1。将ROCK 抑制剂 Y27632添加到培养基中 5 μM的终浓度,以提高重新接种后的生存能力。如果多个菜(比方说,转染)是必需的,它中号AY更方便的是部分地重悬浮的细胞转移到一大50 -毫升˚F爱尔康管和转印后添加更多mTeSR1。卸下所有媒体小号TEP D1F与mTeSR1替代含Y27632。稀释的细胞至所需浓度和填充每个60 -毫米培养皿用4ml充分再悬浮的iPSC小号作为单细胞。在 37 °C、5% CO 2下孵育至少 12 小时。
转染的iPSC š与所述gRNA集成Cas9矢量和ssODN 。
转染前 1 小时,检查细胞生长并用 4 ml 新鲜无抗生素 mTeSR1 替换培养基。
对于每个60 -毫米培养皿,制备在1.5以下的混合物-毫升管。上下吸管混合均匀。对于对照培养皿,更换pSpCas9(BB)-2A-迪普罗构建体与所述的pEGFP-N2质粒。
成分


量(微升)


pSpCas9(BB)-2A-Puro (PX459) V2.0 与 gRNA 插入,


1微克/微升


3


ssODN ,1微克/微升


3


Opti-MEM I 减血清培养基


473


全部的


479

将 21 μl FuGene HD 转染试剂(DNA:试剂比例为 1:3.5,总共 500 μl )添加到步骤 D2b 的混合物中,并短暂涡旋(2 - 3 秒)管以混合。在室温下孵育最终混合物 6 分钟。
加入DNA:的FuGENE :的Opti-MEM我混合到了60 -毫米菜,轻轻摇动混合。不要吸管向上和向下,因为这会干扰与转染过程。在 37 °C、5% CO 2下孵育24 小时。
 


使用嘌呤霉素选择阳性克隆和评估的转染效率。
使用嘌呤霉素选择阳性克隆
注:一个LSO阅读小号TEP D2 。


对于转染的所有菜肴的pSpCas9(BB)-2A-迪普罗构建体,添加4毫升mTeSR1补充有0.5微克/微升每个培养皿嘌呤霉素选择性富集的与转染的细胞的pSpCas9(BB)-2A-迪普罗构建体,它带有一个嘌呤霉素resistan CE盒。使用可能需要优化解嘌呤霉素的浓度ž细胞上通货膨胀系特异性的基础,因为细胞系可能具有非常不同的嘌呤霉素耐受性水平(范围从0.3至0.7微克/微升)。在 37 °C、5% CO 2下孵育菜肴24 小时。
小心地取出媒体和任何死细胞小号的或细胞碎片的菜肴。每道菜添加 4 ml mTeSR1,并辅以 0.3 μg / μl (或由先前的测定确定)嘌呤霉素。在 37 °C、5% CO 2下孵育菜肴24 小时。
小心地取出媒体和任何死细胞小号的或细胞碎片的菜肴。从此时间(转染后 72 小时)撤回嘌呤霉素选择。加入4毫升的mTeSR并更换了日常培养基。孵育在37碗碟℃,5%CO 2 5-10 d直到所述iPSC集落达到了适当的大小以便克隆选择(1.5 - 2毫米的直径,图3)。
 






图3 。用于克隆选择的 iPSC 集落示例

评估的通过EGFP表达转染效率。该步骤应与并行地执行小号TEP D1。
用于与所述的pEGFP-N2质粒转染的菜,小心地更换的用4ml新鲜培养基的mTeSR不含嘌呤霉素。在 37 °C、5% CO 2下孵育这道菜24 小时。
用另外 4 ml不含嘌呤霉素的新鲜mTeSR替换培养基。在 37 °C、5% CO 2下再培养 24 小时。
计算几个视野以评估EGFP+ 细胞的百分比。转染效率,如由EGFP +细胞的百分比计算的,应至少为40 - 50%。
分离单个iPSC集落的genotyp Ë阳性克隆鉴定。
菌落采摘的当天,制备一个平底ED 96孔板涂布有基质胶(简称到作为板A)。添加80微升mTeSR1含有10 μM ROCK抑制剂Y27632到每个孔板A.制备另一未经涂覆的96孔板(简称到作为板B,平底编孔)。
在60更换介质-毫米培养皿以新鲜ROCK抑制剂成分的mTeSR1。
将 P100 或 P200 移液器设置为 50 μl 。附上P200尖端和使用尖端刮去一个单菌落。吸出刮落的集落或它的片段(如果该过程期间,菌落中断)我们荷兰国际集团的移液管和转移其所有内容(50微升)与一个的孔板A.轻轻吸管上下3次打破细胞团. 不更换移液器吸头,将 50 μl从板 A转移到板 B 的相应孔中(例如,A1 孔到 A1 孔等)重复此步骤直到60 - mm 培养皿上的所有感兴趣的菌落都被挑取向上。
降速在200两个板×克为在室温下用5分钟的上盖子。
保持板甲在37 ℃,5%CO 2中24小时,以允许iPSC集小号重新连接到孔中。继续到孵育板和(100每日更换介质微升每孔mTeSR1)。板 A 将用于基因分型后的进一步克隆扩增。
使用板 B 进行 DNA 提取。
从小心地取出盘B的离心无晃动,并置于冰上。使用移液管或多通道移液管(设置为 200 μl )小心地从孔中取出培养基,而不会干扰细胞沉淀。不要使用真空抽吸作为该细胞沉淀不牢固地附着在底部。重复此步骤以从板上的所有孔中取出介质。
添加16微升快速DNA提取缓冲液(Lucigen / VWR)加入每个孔含有的细胞沉淀。将 P10 或 P50 多通道移液器设置为 10 μl 。小心地上下吸管 5 次混合;米尼米ž ë气泡形成。此步骤重悬细胞并进行细胞裂解以供后续使用。
传输所有(16微升)的混合细胞悬液到96孔PCR板和密封使用任一š条纹帽或薄膜。将板标记为“提取的 DNA”,并带有名称和日期。如果不立即使用(1-2 周)或 -80 °C 以延长储存时间,则此步骤的产品应储存在 -20 °C 。设置了如下的热循环和丢弃盘B 。
步 #


温度


时间


笔记


1


65°C


15分钟

2


68°C


15分钟

3


98 ℃


10分钟

4


4 ℃


抓住

 


在 Sanger 测序仪上进行 DNA 测序的样品制备。
使用上一步中快速分离的 DNA 作为模板准备 PCR 反应混合物。根据以下图表装配在冰上的反应中,含多处ÿ通过为每个组件并进一步含多处的样本数ÿ 1.1以补偿试剂转移期间潜在损失。
成分


量(微升)


10 × PCR 缓冲液


1


10 × PCR 增强剂


1


50 mM 硫酸镁4


0.3


10 mM dNTP


0.2


正向引物 (10 μM )


0.2


反向引物 (10 μM )


0.2


Qiagen HotStarTaq DNA聚合酶


0.1


水,PCR 级


5.5


全部的


8.5

混合所有成分而无需在1.5 DNA模板-在冰上毫升管。使用一个新的96孔PCR板和分配8.5微升,直到所有的反应混合物加入每个孔中的期望的孔被填充。随后,将 1.5 μl提取的 DNA添加到每个孔中(总共 10 μl )。使用设置为 5 μl的 P10 移液管上下移液以充分混合,同时避免气泡形成。如果使用多道移液器进行此操作会更方便。
使用带条纹的盖子或薄膜密封 PCR 板的顶部,并将板标记为“PCR 产品” 。设置了热循环如下。退火温度(3)和延伸时间(4)可能需要优化解Ž通货膨胀,当操作者认为合适的。
步 #


温度


时间


笔记


1


95℃


10分钟


Taq激活


2


95℃


30 秒

3


53°C


90 秒


或视情况而定


4


72 ℃


60 秒


或视情况而定


5


/


/


转至 2, 40 个循环


6


72 ℃


10分钟


最终延期


7


4 ℃


抓住

 


WH ILE PCR反应运行时,设置一个或更多1.25%TAE / TBE琼脂糖凝胶小号具有一个足够数量的井š为保持至少20个样品进行PCR效率检查。
组装该小号hrimp一个lkaline p hosphatase(SAP)的反应混合物在冰上以除去单链编DNA和dNTP从根据下面的图表的PCR产物。乘法由每个组件和进一步的样本数含多处ÿ 1.1以补偿试剂转移期间潜在损失。
成分


量(微升)


10 × SAP 缓冲区


0.5


虾碱性磷酸酶 (SAP)


0.5


核酸外切酶 I


0.1


水,PCR 级


3.9


全部的


5

PCR反应结束后,使用一个新的96孔PCR板和分配5微升SAP混合到每个孔中,直到所有的期望的孔被填充。随后,转印5微升从板的产物(总体积的1/2)标记为相应的井“PCR产物”中的96孔板中保持的SAP混合物(共计10微升)。上下吸管混合均匀,同时避免气泡形成。纪念板作为“SAP的产品”,并设置了热循环如下。
步 #

温度


时间


笔记


1

37℃


50 分钟

2

95℃


15分钟

3

4 ℃


抓住

 


在 SAP 反应运行时,将 1 .5 μl 6 ×加载染料添加到“PCR 产物”板的选定孔中,并将其内容物加载到步骤 E4g 中制备的 TAE/TBE 凝胶上。使用适当的分子梯在 100 V 下运行凝胶至少 25 分钟。检查的形状,数量和清晰的条带在每个通道。拍摄图像,并保持其备案小号。
 






图4 。PCR 产物的琼脂糖凝胶电泳结果样本。泳道 3 的 DNA 太少,无法接受。泳道 2 尽管亮度降低,但 DNA 含量仍然可以接受。M:分子标记,1 kb,Promega G5711。

拍摄PCR 反应的凝胶图像后,计算“良好”反应的数量(具有正确分子大小和足够 DNA 量的单个条带,根据条带的亮度进行评估,参见图 4)并准备用于 Sanger 测序的混合物。根据下表在冰上组装 Sanger 反应混合物。乘法各组分的体积通过的“好”的反应只计数和由1.1进一步乘法结果自的BigDye终止子试剂是昂贵的。
成分


量(微升)


BigDye终结者V3.1


1


测序引物 (10 μM )


1


水,PCR 级


3


全部的


5

所述SAP反应结束后,使用一个新的96孔PCR板和分配5微升桑格测序混合物到每个孔中,直到所有的期望的孔被填充。随后,将 5 μl (总体积的 1/2)产品从标有“SAP 产品”的板转移到装有 Sanger 测序混合物(共 10 μl )的 96 孔板的相应孔中。上下吸管混合均匀,同时避免气泡形成。标记板作为“序列PCR产物”,并设置了热循环如下。退火温度(3)可能需要优化解Ž通货膨胀,当操作者认为合适的。
步 #


温度


时间


笔记


1


96℃


10 秒

2


55℃


5 秒


或视情况而定


3


60℃


4 分钟

4


/


/


转至 2, 25 个循环


5


4°C


抓住

 


纯化使用测序PCR产物一Qiagen公司DyeE X 2.0 Spin试剂盒(最好≤16个样本)。如果染料实施例2.0 ķ它不可用或有16个以上的样品,跳过步骤E5J并在恢复步骤E5K。
松开ñ顶盖和折断离心柱的底部封闭。将离心柱插入收集管并以 750 × g离心3 分钟。
所述旋转柱转移至新的1.5 -毫升管标记的与所述样品名称。抽吸从相应井并缓慢测序PCR产物直接应用的反应产物到CENTE ř倾斜凝胶床的表面。再次以 750 × g离心3 分钟。
丢弃所述旋转柱并放置1.5 -毫升管中的离心式真空装置(如一个SpeedVac中)30-60分钟,直至所有的液体具有蒸发d 。在真空过程中不要打开加热。干燥后,加入15微升测序级甲酰胺向管和吸管上下10次,以溶解该DNA。继续执行步骤 E5kvii。
通过异丙醇沉淀纯化测序 PCR 产物。
注:(!)d enotes,这一步是关键和一个应该谨慎行事。


每孔加入 40 μl 75% 异丙醇,轻轻上下吹打5 次混合。如果使用多道移液器进行此操作会更方便。
在室温下孵育 20 分钟,然后在摆斗式离心机中在室温 (20°C) 下以 3,000 × g离心30 分钟。如果有离心机的冷却功能,请打开离心机的冷却功能,因为离心机内的空气可能会很快变热。
小心地从离心机中取出板,不要摇晃。倒置板到3 -纸的4层毛巾轻轻倾析的异丙醇。重复该过程一次与另一种新鲜3 - 4层纸毛巾,并保持倒10秒的板以除去尽可能多的异丙醇越好。不要用移液或者一个真空并移除任何剩余的异丙醇从井。
翻转96孔板背面缓慢LY,并小心地添加150微升75%的异丙醇到每个孔中,以洗涤沉淀。
重要提示:不要混合!如果使用多通道移液器进行此操作会更方便。将板再次以 3 , 000 × g离心10 分钟。


小心倒置纸在板上巾堆叠如步骤E5kiii倾析的异丙醇。保持反转的板(!) ,小心地将其转移到一个新的纸毛巾叠,再次离心以300 ×克(!),持续1分钟,以消除该残留的异丙醇。
小心地从离心机中取出 96 孔板并将其翻转回来。向每个孔中加入 15 μl测序级甲酰胺,并上下移液 5 次以混合。避免在井内形成气泡。
密封带粘接膜的板和变性的在95℃下的DNA热循环仪上2分钟。立即将板置于冰上并孵育 2 分钟。
将板以 2000 × g离心1 分钟。该板现在准备Sanger测序注射上的ABI3730分之310/ 3730XL / 3500序器。
 
扩大与阳性克隆的期望的基因型。
检查测序光谱文件我们荷兰国际集团猿或ABI的GeneMapper识别用于扩展阳性候选克隆(小号EE部分A,图1和图5)。扩增后的 iPSC 将用于进一步的单细胞亚克隆,以获得纯克隆。
 






图5 。质量好坏的样本测序光谱。A. 良好的结果,显示出清晰的离散峰。B. A B广告结果,显示出多发性overlapp荷兰国际集团峰组,暗示的多个PCR产物在测序输入的存在。C.一个糟糕的结果,显示出非常微弱的峰值信号。

当该菌落到达的适当大小(1.5 -直径2mm或60 - 70%confluenc Ë中的孔)在板A(来自步骤E3A),解离的通过首先祛瘀iPSC集落荷兰国际集团从所有介质的孔中。轻轻加入50微升ReLeSR到每个孔中,迅速吸出,并放置在室温下5分钟。
加入100微升mTeSR1到每个孔中并小心地移去的从细胞团块的底部的井通过轻敲板和使用常规刮P 200吸管尖。传送的内容小号各孔成一个孔的一个基质胶包被的4孔或24孔平板,并添加mTeSR1含有5 μM ROCK抑制剂至500的最终体积微升。继续在37培养℃,5%CO 2中与mTeSR1的每日清爽的培养箱,直到所述细胞达到60 - 70%confluenc Ë在孔中。
单细胞亚克隆获得纯等基因系。
小心地从取出介质的孔中,并随后添加400微升的Accutase每孔10分钟以解离的从底部的细胞。轻轻吸管上下使用P1000尖端,使5倍一个单细胞悬浮液,并然后添加另外400微升mTeSR1到neutrali Ž Ë所述的Accutase 。取 10 μl悬液,用台盼蓝法计算细胞密度,250 × g离心剩余细胞5 分钟。
去除上清液并将细胞重悬在含有5 μM ROCK 抑制剂 Y-27632 的1 ml mTeSR1中。隔离的体积相当于2000活细胞(如在先前步骤中所确定的)和replate成60 -毫米基质胶涂覆的培养皿在4毫升mTeSR1含有5 μM ROCK抑制剂Y27632。继续在 4/24 孔板中培养任何剩余的细胞作为备份。在 37 °C、5% CO 2下继续培养。
一半的介质在60取代-毫米培养皿与ROCK抑制剂成分的mTeSR1 48小时后重新铺板。在37继续培养℃,5%CO 2中与mTeSR1的每日清爽直到菌落大小培养箱达到1.5 -在直径为2毫米。重复步骤 E3 中所述的菌落挑选程序。从每 60 个菌落中挑选大约 30 个菌落—— 毫米菜。对选定的菌落重复基因分型程序,如步骤s E4 - E 5 所述。
一旦纯克隆已经鉴定了由Sanger测序,重复步骤F1至各菌落从转移的(!)96孔板2的孔中的直到Matrigel包被24分之4孔板的confluenc ë达到60 - 70%。继续监测细胞生长,因为在这个阶段不同的克隆可能有不同的生长速度。
从孔中取出培养基,并在每个孔中加入 400 μl ReLeSR 。除去ReLeSR从所述孔中,并孵育在室温下5分钟。
用 Matrigel 涂覆足够的 6 孔板,每孔加入 2 ml mTeSR1。添加300微升mTeSR1到二十四分之四孔板用处理的每个孔ReLeSR并轻轻吸管上下使用宽孔移液管尖端以解离5倍的细胞团块。将 100 μl细胞团悬液分装到 6 孔板的每个孔中(共 3 个孔),轻轻摇晃混合。准备4 - 6口井,每个克隆。继续孵化,直到该confluenc ë达到60 - 70%。
冷冻细胞系用于冷冻保存。
密切观察6孔板中的细胞生长,直到所述confluenc ë达到60 - 70%。建议是在同一块板上所有孔进行加工于一体的运行。
在 6孔板的每孔中加入 1 ml ReLeSR 。除去ReLeSR从所述孔中,并孵育3 -在37℃,5%CO 5分钟2的培养箱中。
加入1ml室温mTeSR1每孔处理的6孔板的ReLeSR和移出所述通过牢固地轻敲板细胞。转移撞出的细胞悬浮到无菌1.5 -毫升Eppendorf管中并离心分离机,在200 ×克为在室温下5分钟。
小心地去除尽可能多的上清液,不要接触底部的细胞沉淀。加入1ml室温mFreSR到Eppendorf管中,并使用大口径吸管尖用移液管小心地重悬细胞沉淀婷上下5次。传送管内容小号到预标记的冷冻管筒。确认试管已牢固密封并将其放入 Mr Frosty 冷冻容器中。通常,为每个克隆准备尽可能多的管(通常为步骤s F2d - F2 f 中确定的4 - 6 管)。重复此步骤,直到所有的细胞已被转移到冷冻管。
放置雾先生冷冻容器含有冷冻管在-80℃冰箱中至少24小时(但没有再高于72小时),以冻结的内容。将所有小瓶移至液态 N 2以进行长期储存。保留详细记录。
 


数据分析

Sanger测序结果的光谱分析
精确编辑的纯同基因克隆的成功鉴定和分离在很大程度上依赖于对 Sanger 测序结果的正确分析。作为指导方针,首先除去样品具有低信号强度,异常模式(例如,当相同类型的核苷酸碱基在连续出现“合并”大的峰而不是单个峰一个序列,参见图5)。随后,通过检查所需 SNP 位点的基因型来鉴定阳性克隆。


gRNA 和ssODN效率的评估
由于ssODN整合和随后的精确 SNP 编辑需要 CRISPR/Cas9 介导的 DNA 切割优先,因此 CRISPR/Cas9 介导的 DNA 切割和ssODN整合的效率需要独立评估。通常,由于大多数 CRISPR/Cas9 介导的 DNA 断裂没有得到适当修复并产生 indel,因此生成的含 indel 克隆(见图 1)的百分比可以作为实验中使用的gRNA 序列效率的指标。含有插入缺失的克隆的百分比为约60 -对于一个成功gRNA序列设计的80%。ssODN的整合在大约 5% 的 CRISPR/Cas9 介导的 DNA 切割事件中相对稳定。如果阳性克隆的数目是低的,从桑格测序结果分别计算这两个事件的可能性,以确定哪些寡聚序列m AY是原因,需要重新设计。

致谢

资金:R01MH106575、R01MH116281、R01AG063175。我们承认由 Ran等人提出的起源概念。(2013)和Zhang等人的进一步方法论证。(2020) ( D oi: 10.1126/ science.aay 3983) 。

利益争夺

作者声明没有利益冲突。

伦理

北岸大学卫生系统机构审查委员会 (IRB) 批准了这项研究。

参考

              Forrest, MP, Zhang, H., Moy, W., McGowan, H., Leites , C., Dionisio, LE, Xu, Z., Shi, J., Sanders, AR, Greenleaf, WJ, Cowan, CA,庞,ZP,Gejman ,PV,彭泽斯,P。和段,J。(2017)。hiPSC 衍生神经元中的开放染色质分析优先考虑功能性非编码精神病风险变异并突出神经发育位点。细胞干细胞21(3):305-318 e308。              
              Forsyth, NR, Musio , A., Vezzoni , P., Simpson, AH, Noble, BS 和McWhir , J. (2006)。生理氧可增强人类胚胎干细胞克隆恢复并减少染色体异常。克隆干细胞8(1): 16-23。              
Hendel, A., Kildebeck , EJ, Fine, EJ, Clark, J., Punjya , N., Sebastiano, V., Bao, G. 和Porteus , MH (2014)。使用 SMRT 测序量化内源基因座的基因组编辑结果。细胞代表7(1):293-305。              
              Howden , SE, Maufort , JP, Duffin, BM, Elefanty , AG, Stanley, EG 和 Thomson, JA (2015)。患者成纤维细胞的同时重编程和基因校正。干细胞报告5(6):1109-1118。              
              Miyaoka, Y., Chan, AH, Judge, LM, Yoo , J., Huang, M., Nguyen, TD, Lizarraga, PP, So, PL 和 Conklin, BR (2014)。无需抗生素选择即可分离单碱基基因组编辑的人类 iPS 细胞。Nat 方法11(3): 291-293。
Ran, FA, Hsu, PD, Wright, J., Agarwala , V., Scott, DA 和 Zhang, F. (2013)。使用 CRISPR-Cas9 系统进行基因组工程。纳特Protoc 8(11):2281至2308年。
              Tidball, AM, Swaminathan, P., Dang, LT 和 Parent, JM (2018)。结合 CRISPR Indel 形成和人成纤维细胞重编程生成功能丧失的 iPSC 系。生物协议8(7)。              
Zhang, S., Moy, W., Zhang, H., Leites , C., McGowan, H., Shi, J., Sanders, AR, Pang, ZP, Gejman , PV 和 Duan, J. (2018)。开放染色质动力学揭示了基于 hiPSC 的神经发育模型中的阶段特异性转录网络。干细胞研究29:88-98。
Zhang, S., Zhang, H., Zhou, Y., Qiao , M., Zhao, S., Kozlova , A., Shi, J., Sanders, AR, Wang, G., Luo, K., Sengupta , S., West, S., Qian, S., Streit , M., Avramopoulos, D., Cowan, CA, Chen, M., Pang, ZP, Gejman , PV, He, X. 和 Duan, J. (2020)。人类 iPSC 神经元中的等位基因特异性开放染色质阐明了功能性疾病变异。科学369(6503):561-565。
登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Zhang, H. and Zhang, S. (2021). CRISPR/Cas9-mediated Precise SNP Editing in Human iPSC Lines. Bio-protocol 11(12): e4051. DOI: 10.21769/BioProtoc.4051.
  2. Zhang, S., Zhang, H., Zhou, Y., Qiao, M., Zhao, S., Kozlova, A., Shi, J., Sanders, A. R., Wang, G., Luo, K., Sengupta, S., West, S., Qian, S., Streit, M., Avramopoulos, D., Cowan, C. A., Chen, M., Pang, Z. P., Gejman, P. V., He, X. and Duan, J. (2020). Allele-specific open chromatin in human iPSC neurons elucidates functional disease variants.Science 369(6503): 561-565.
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。