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Jan 2018
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Generation of Gene Knockout and Gene Replacement with Complete Removal of Full-length Endogenous Transcript Using CRISPR-Trap
通过CRISPR-Trap完全移除内源全长转录本进行基因敲除和基因置换    

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Abstract

This protocol describes the application of the CRISPR-Trap from designing of the gene targeting strategy to validation of successfully edited clones that was validated on various human cell lines, among them human induced pluripotent stem cells (hiPSCs). The advantage of CRISPR-Trap over conventional approaches is the complete removal of any endogenous full-length transcript from the target gene. CRISPR-Trap is applicable for any target gene with no or little coding sequence in its first exon. Several human cell lines and different genes have so far been edited successfully with CRISPR-Trap.

Keywords: CRISPR (CRISPR), CRISPR-Trap (CRISPR-Trap), Gene knockout (基因敲除), Gene replacement (基因置换), Gene editing (基因编辑), hiPSCs (hiPSCs)

Background

The advent of CRISPR/Cas9 technology facilitated the genomic targeting for the generation of gene knockouts and gene editing. The conventional method to perform a knockout relies on the introduction of a frameshift leading to premature termination codons (PTCs), truncating the open reading frame (ORF) and subsequent degradation of the transcript of the targeted gene by nonsense-mediated mRNA decay (NMD). A possible pitfall of this approach is full-length transcripts which may escape NMD and give rise to C-terminal truncated proteins harboring residual or even dominant negative functions. This protocol presents the CRISPR-Trap, a method we recently established (Reber et al., 2018), which upon successful editing will prevent the expression of any full-length transcript from the target gene locus (Figure 1). Simply put, this approach targets the first intron of the gene of interest with CRISPR/Cas9. Using homology-directed repair (HDR) a customizable cassette flanked by a strong 3’-splice site and a strong polyadenylation signal is introduced in the first intron, thereby generating an artificial second and effectively last exon. Since transcription is terminated by the introduced polyadenylation signal, only the first endogenous exon and the inserted cassette is transcribed. The customizable cassette can be used to introduce a selection marker, thereby enabling easy selection for at least heterozygous edited clones. If a gene replacement is wanted, the customizable cassette can be used to introduce the replacement gene, followed by an internal ribosomal entry site (IRES) and a selection marker (Reber et al., 2016 and 2018).


Figure 1. Schematic of the application of the CRISPR-Trap. The first intron of the target gene is cleaved using the CRISPR/Cas9 system and template DNA for homology-directed repair is provided. The template DNA contains a strong 3’ prime splice signal (dark green), a customizable cassette (light green) and a strong polyadenylation signal (turquoise). The customizable cassette can be utilized to either knockout (left) or replace the target gene (right). The cassette contains a selection marker that will be under the control of the endogenous promoter of the target gene upon successful editing. For gene replacements, an IRES is introduced in between the replacement gene and the selection marker. Figure adapted from Reber et al. (2018).

Materials and Reagents

  1. Pipette tips
    1. 10 µl sapphire bulk non-sterile pipette tips (Greiner Bio One International, catalog number: 771250 )
    2. 200 µl polypropylene universal pipette tips with graduation (Greiner Bio One International, catalog number: 739282 )
    3. 1,250 µl sapphire bulk non-sterile pipette tips (Greiner Bio One International, catalog number: 750250 )
  2. TPP® tissue culture plates
    1. 24-well (Sigma-Aldrich, catalog number: Z707791 )
    2. 6-well (Sigma-Aldrich, catalog number: Z707767 )
    3. 15 cm plate (Sigma-Aldrich, catalog number: Z707694 )
  3. TPP cell scraper 20 cm (MIDSCI, catalog number: TP 99010 )
  4. Cloning cylinder sterile, 3/16’’ ID x 5/16’’ H (SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: 37847-0100 )
  5. Cell strainer (EASYstrainer, 70 µm) (Greiner Bio One International, catalog number: 542070 )
  6. Bel-ArtTM SP SciencewareTM BelpenTM black markers (SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: F13374-0000 )
  7. Plasmid of choice, serving as homology directed repair donor template (donor plasmid)
  8. Plasmid of choice for Cas9 and sgRNA expression, e.g., pX330-U6-Chimeric_BB-CBh-hSpCas9 (Addgene, catalog number: 42230 )
  9. Transformation competent E. coli strain of choice for cloning and plasmid amplification (e.g., XL10 Gold) (Agilent, catalog number: 200314 )
  10. Transfection reagent of choice
    1. DogTor (OZ Biosciences, catalog number: DT51000 )
    2. TransIT®-LT1 (Mirus Bio, catalog number: MIR 2300 )
    3. LipofectamineTM 3000 reagent (Thermo Fisher Scientific, catalog number: L3000015 )
  11. Cell detachment solutions
    1. hiPSCs
      1. To generate single cells: StemProTM AccutaseTM Cell Dissociation Reagent (Thermo Fisher Scientific, catalog number: A1110501 )
      2. To split cells as clumps: DPBS (STEMCELL Technologies, catalog number: 37350 ) containing 0.5 mM EDTA (Thermo Fisher Scientific, catalog number: 15575020 )
    2. Other human cell lines: Trypsin-EDTA (0.05%), phenol red (Thermo Fisher Scientific, catalog number: 25300054 )
  12. To grow hiPSCs as single cells: Y-27632, RHO/ROCK pathway inhibitor (STEMCELL Technologies, catalog number: 72302 )
  13. ZeocinTM (InvivoGen, catalog number: ant-zn )
  14. Puromycin dihydrochloride (Santa Cruz Biotechnology, catalog number: sc-108071A )
  15. PBS (Ca2+, Mg2+ free) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
  16. TRIZOL (Sigma-Aldrich, catalog number: T9424 )
  17. Dow Corning® High-Vacuum silicone Grease (Sigma-Aldrich, catalog number: Z273554 )
  18. TOPOTM TA CloningTM Kit, Dual Promoter (Thermo Fisher Scientific, catalog number: 450640 )
  19. Chloroform for analysis EMSURE® ACS, ISO, Reag. Ph Eur (Merck, catalog number: 102445 )
  20. Absolute EtOH (Sigma-Aldrich, catalog number: 59176 )
  21. Trisodium citrate (Sigma-Aldrich, catalog number: W302600 )
  22. 2-propanol for analysis EMSURE® ACS, ISO, Reag. Ph Eur (Merck, catalog number: 109634 )
  23. RNase free glycogen (Thermo Fisher Scientific, catalog number: R0551 )
  24. DNA extraction kit (Quick-DNATM miniprep Kit) (ZYMO RESEARCH, catalog number: D3025 )
  25. NaOH (Sigma-Aldrich, catalog number: 306576 )
  26. Maximo Taq DNA Polymerase 2X-preMix/PCR Master Mix (GeneON, catalog number: S113 )
  27. KAPA Taq ReadyMix PCR Kit (KAPA Biosystems, catalog number: KK1006)

Equipment

  1. Pipettes 
    1. Pipetman P10 (Gilson, catalog number: F144802 )
    2. Pipetman P20 (Gilson, catalog number: F123600 )
    3. Pipetman P200 (Gilson, catalog number: F123601 )
    4. Pipetman P1000 (Gilson, catalog number: F123602 )
  2. Mammalian culture equipment
  3. Heat block (VWR, catalog number: 444-0938 )
  4. Thermocycler (VWR, catalog number: 732-2551 )
  5. NanoDrop (Thermo Fisher Scientific, model: NanoDropTM 2000 , catalog number: ND-2000)
  6. Cooling Centrifuge (Eppendorf, model: 5424 R , catalog number: 5404000010)
  7. Wide-field microscope
  8. Vortex (Scientific Industries, catalog number: SI-0266 )

Software

  1. Clone manager (http://www.scied.com/pr_cmpro.htm) or other cloning software

Procedure

Notes:

  1. One has to be aware that the first exon of the target gene will still be expressed following the CRISPR-Trap strategy. Therefore, the ideal target gene has no or only few amino acids coded in its first exon.
  2. To allow direct selection for genome edited cells, the gene has to be expressed in the cells in which the editing is performed.
  3. As every target behaves differently depending on the specific gene and cell line one might have to adjust various parameters.


  1. Design of sgRNAs and donor plasmids
    sgRNA Design
    Identify a target site within the first intron of your target gene and generate a suitable expression plasmid (hereafter called pCas9/sgRNA)
    1. Most CRISPR/Cas9 expression plasmids use a CMV promoter for the expression of the Cas9 endonuclease. In our hands, this promoter resulted in low Cas9 expression levels in human stem cells. While, this still resulted in successful generation of CRISPR-trap knockouts (Reber et al., 2018) replacement of the CMV promoter with an EF1α promoter could be considered, for specific cell lines in which the CMV promoter displays low activity.
    2. For the intronic sgRNA target site:
      1. Make sure to leave a spacer from the 3’ end of the first exon not to disrupt intronic splicing enhancers, which are typically located within the first 100 nucleotides downstream of the 5’ splice site (Aznarez et al., 2008).
      2. Introduce the cassette upstream of the intronic branch point to ensure proper splicing to the first exon of the target gene. Branch points are typically located < 50 nucleotides upstream of the 3’ splice site, with 90% of all human branch points located 37 nucleotides upstream of the 3’ splice site (Mercer et al., 2015). However, in certain human genes, the branch point can be located further upstream. Hence, we recommend the use of a splice site predictor such as the Human Splicing Finder available at http://www.umd.be/HSF3/ to identify the ideal target region.
    3. If possible, find two or more adjacent sgRNA sequences that can trigger HDR with the same donor plasmid, thereby enhancing the success rate.
      1. Option 1: For many genes, pre-designed and validated sgRNAs in suitable expression plasmids as well as custom sgRNA design are commercially available (e.g., CRISPR/Cas Nuclease RNA-guided Genome Editing from Sigma-Aldrich available at https://www.sigmaaldrich.com/technical-documents/articles/biology/crispr-cas9-genome-editing.html#purified or from Addgene available at https://www.addgene.org/crispr/)
      2. Option 2: Use a dedicated online-tool (we typically used the CRISPR Design Tool by the Feng Zhang group available at http://crispr.mit.edu/) to identify CRISPR/Cas9 target sequences within the first intron of the gene of interest. Subsequently, clone the guide sequence into a suitable CRISPR/Cas9 expression plasmid (e.g., pX330-U6-Chimeric_BB-CBh-hSpCas9, Addgene Plasmid # 42230 ). Follow the Zhang Lab General Cloning protocol available at https://www.addgene.org/crispr/zhang/.

    Donor plasmid design
    Generate donor plasmid containing the customizable cassette either by cloning or by gene synthesis into a plasmid of choice. This donor plasmid is subsequently called pHDR-Don. The donor plasmids used in (Reber et al., 2018) for knockouts and gene replacements (pHDR-FUS KO-Zeo: generation of FUS knockouts & pHDR-TDP43 repl-Puro: gene replacement of TDP-43 with DD-TDP43) are available as Genbank files. Furthermore, pHDR-Don example constructs for gene knockouts are provided for genes with and without coding sequence in the first exon: pHDR-Zeo-Don for genes with a non-coding first exon, pHDR-Zeo-Don_f1 for genes where the splice site after the first exon is located directly after a codon, pHDR-Zeo-Don_f2 for genes where the splice site is located after 1 extra nucleotide following the last codon, and pHDR-Zeo-Don_f3 for genes where the splice site is located after 2 extra nucleotides following the last codon. Furthermore, an example construct for gene replacement with a non-coding first exon is provided (pHDR-IRES-Puro-Don).
    1. We used Zeocin and puromycin as selection markers, however, depending on the targeted cell line alternative resistance markers such as blasticidin could be beneficial.
      In case that the endogenous gene contains some coding sequence in its first exon, make sure that the resistance marker/gene replacement will be spliced in-frame.
    2. Cas9 cuts 3 nucleotides upstream of the PAM motif. Homology arms 5’ and 3’ of the customizable cassette should therefore be identical to the sequences upstream (5’) and downstream (3’) of the Cas9 cleavage site. To allow efficient homology-directed repair mediated by pHDR-Don, we typically use homology arms with a length of 700-800 nucleotides (per homology arm) (see Figure 2).


      Figure 2. Schematic of the pHDR-Don design. Cas9 cuts 3 nucleotide positions upstream of the PAM motif. The homology arms should be identical to the sequences upstream (5’ homology) and downstream (3’ homology) of the cleavage site. Each homology arm should be approximately 750 base pairs long to ensure efficient HDR. In case of short introns, the 750 base pairs of the 5’ homology might contain the first exon and parts of the promoter region of the gene of interest. In that case, make sure not to include promotor sequences in your 5’ homology arm to prevent expression of the cassette directly from the plasmid.

    3. The upstream (5’) homology arm has to be shortened, if it would contain the promoter sequence of the targeted gene. Otherwise, the cassette will be expressed directly from the pHDR-Don plasmid.

  2. Transfect cells
    1. Grow cells in conditions suitable for the respective cell line (growth medium, growth surface, growth conditions). These conditions can be maintained during the whole protocol. Seed cells the day before transfection in all wells of a 6-well plate. In order to seed the cells follow these steps:
      1. Aspirate medium from the plate containing the cells.
      2. Wash the cells once by adding a sufficient amount of PBS to cover the surface of the plate by gently swirling. Subsequently aspirate the PBS.
      3. Add the appropriate reagent to detach the cells (e.g., Trypsin, Accutase) and wait until the cells detach.
      4. Resuspend the cells in appropriate medium and count the cells (e.g., by using a hemocytometer).
      5. Transfer the desired amount of cells into the wells. The optimal amount of cells per well will vary by the cell line and the transfection reagent used. Follow the transfection reagent manufacturer’s protocol for an optimal transfection efficiency using a brightfield microscope to estimate density.
    2. Use the appropriate transfection reagent for your cell line. In our hands, the most efficient transfection reagents were DogTor for HeLa cells, Mirus TransIT®-LT1 for HT1080, and human induced pluripotent stem cells (hiPSCs) and LipofectamineTM 3000 for SH-SY5Y neuroblastoma cells. Transfect each well with pCas9/sgRNA and pHDR-Don. Table 1 gives empirical values for successful edited cell lines in our hands (values each for the transfection of one well of a 6-well plate).

      Table 1. Empirical values for amounts of transfected plasmids. Empirical values for transfected pCas9/sgRNA and pHDR-Don plasmids in successful editings of various cell lines.


      Note: When sgRNAs for two adjacent targets were cloned, transfect 3 wells with one sgRNA and the other 3 wells with the other sgRNA. The 6-wells can also be used to vary either the total amount of transfected plasmids or the ratio between pCas9/sgRNA and pHDR-Don. A good starting point is to try molarity ratios of pCas9/sgRNA:pHDR-Don of 1:6 to 2:1. If 6-wells are not sufficient for all variation, more wells can be transfected. Since CRISPR-Trap relies on homology-directed repair, small molecules increasing the likelihood of these events such as L755507 (Yu et al., 2015; Li et al., 2017) can be added at this stage.
    3. Two to four days after transfection, detach cells using the appropriate cell detachment solution and pool the cells from all wells in one 15 cm dish. Let cells attach, and then start with selection using the selection marker introduced with the cassette. Table 2 gives empirical values for successful edited cell lines in our hands.

      Table 2. Empirical values for antibiotic selection. Empirical values for antibiotic concentrations and duration of selection in successful editings of various cell lines.


  3. Pick selected clones
    It is best to pick colonies while looking through a microscope, as this gives better control and reduces the risk of mixing multiple colonies. Here, we describe a method that allows manual picking of single colonies when picking under a microscope under sterile conditions is not possible. If a microscope for manual picking under sterile conditions is not available, follow this protocol. Otherwise, pick colonies directly under the microscope under sterile conditions and continue from Step C10.
    1. Upon finishing the selection process, allow single cells to grow into colonies. It is important however to not let the colonies grow into to close proximity of one another, to ensure that each colony picked grew from one single cell. If the colonies grow to close to each other, split the cells to a new plate (use a cell strainer to ensure new colonies emerge from single cells).
      Note: As rule of thumb, pick colonies consisting of at least 10 cells and 1 cm of space from the edge to the edge of the next colonies.
    2. Count and note the number of colonies which are good to pick (i.e., consisting of at least 10 cells and 1 cm of space from the edge to the edge of the next colonies). Use a pen to mark all colonies which are good to pick by circling them on the underside of the 15 cm plate (see Figure 3).
      Note: If two colonies are too close to each other, forego to pick them to avoid picking of mixed colonies.
    3. Apply vacuum grease on a fresh sterile dish and use a sterile cell scraper to generate a grease film of approximately 5 mm thickness (Figures 3A and 3B).
    4. Prepare cloning cylinders in the number assessed in Step C2. Put cloning cylinders with their narrow side onto the grease. Try to avoid any horizontal movement of the cloning cylinders once they are sticking to the grease (Figure 3C).
    5. Sterilize 20 min under UV light.
    6. Aspirate medium from the 15 cm plate containing cells and wash once with PBS by adding 10 ml of PBS, distributing the PBS among the plate by gently swirling and subsequently aspirating the PBS.
    7. Remove cloning cylinders one by one from the grease film and put them on the colonies with the greased side facing the plate. If you are picking without concurrent usage of a microscope, use the marks from Step C2 as a guide to where to put the cloning cylinders (Figure 3D).
      Note: Apply a little pressure for a watertight sealing.


      Figure 3. Preparation of vacuum grease and cloning cylinders for manual clone picking. A. Apply grease on sterile dish. B. Generate an approximately 5 mm thick grease film using a sterile cell scraper. C. Put cloning cylinders with their narrow side onto the grease. D. Put the cloning cylinders with the greased side first onto the colonies.

    8. Add 20 μl of the appropriate reagent to detach the cells (e.g., Trypsin, Accutase) into each cloning cylinder and wait until the cells detach.
    9. As soon as the cells detach, add 80 μl of appropriate medium into each cloning cylinder and directly transfer all liquid into a separate well of a 24-well plate for each colony. Subsequently top up medium in each well to 500 μl.
    10. Let the colonies grow over the next couple of days in the 24-well plate and transfer the cells into a well of a 6-well plate as soon as they reach confluence.
      Note: It is also possible to harvest clones directly from 24-wells following the same protocol from Procedure D.

  4. Validation of successful edited clones
    1. As soon as the clones reach confluence on the 6-well plates (or 24-well plates), detach the cells and split them into two parts. Freeze half the cells to continue with in case of a successful genome editing, while extracting the DNA and RNA from the other half to assess the genotype. Hereto we re-suspend the cells in TRIZOL to isolate gDNA as well as RNA.
      Note: A DNA extraction kit can be used as an alternative to the extraction using TRIZOL. The advantage of the TRIZOL extraction, however, is the possibility to simultaneously extract RNA. Using RT-qPCR allows for a quick check if any RNA from the target gene downstream from the introduced gene trap is expressed. Low traces of such RNA can indicate a mixed population of cells (picked clone originates not from a single cell).
      Furthermore, samples in TRIZOL can be stored for long-term at -20 °C allowing batch analysis of a high number of clones simultaneously.
    2. Isolation of gDNA from TRIZOL (protocol modified from the manufacturer's protocol)
      1. Add 0.5 ml TRIZOL per well (6-well or 24-well) and pipette up and down in order to lyse the cells.
      2. Transfer the cell lysate into a 1.5 ml Eppendorf tube.
      3. Add 0.1 ml chloroform, close the lid tightly and shake vigorously for 15 sec.
      4. Incubate for 2-15 min at RT.
      5. Centrifuge for 15 min at 12,000 x g at 2-8 °C.
      6. Remove upper phase (contains RNA, store at -20 °C).
      7. Add 0.3 ml 100% EtOH and 1.5 μl glycogen, mix by inversion.
        Note: Adding the glycogen is essential if you work with a low amount of cells–without it, you won’t see a pellet in the later steps.
      8. Incubate for 2-3 min at RT.
      9. Centrifuge for 5 min at 2,000 x g at 2-8 °C.
      10. Remove supernatant.
      11. Wash DNA solution twice:
        1. Add 1 ml 0.1 M trisodium citrate, 10% EtOH.
        2. Incubate (with occasional mixing, inversion) for at least 30 min at RT.
        3. Centrifuge for 5 min at 2,000 x g at 2-8 °C.
        4. Remove supernatant.
          Note: The DNA pellet is typically not attaching strongly to the tube, be careful not to aspirate it along with the supernatant; better leave a small amount of supernatant.
      12. Resuspend pellet in 1 ml 75% EtOH.
      13. Incubate for 10-20 min at RT.
      14. Centrifuge for 5 min at 2,000 x g at 2-8 °C.
      15. Air-dry pellet by opening the lid of the Eppendorf and placing it with the opening facing down on a clean tissue at room temperature for 15 min.
      16. Dissolve the pellet in 50-100 μl 8 mM NaOH with repeated pipetting.
      17. Use a Spectrophotometer (e.g., NanoDrop) to assess DNA concentration.
        Note: DNA isolated from a low amount of cells following this protocol usually results in a high ethanol/phenol peak measuring DNA concentration using a Spectrophotometer (e.g., NanoDrop). These contaminants however did not interfere with downstream PCR reactions in our hands (using Maximo Taq DNA Polymerase 2x-preMix [ S113 , GeneON] or KAPA Taq ReadyMix [KK1006, KAPA Biosystems]).
      18. Store at -20 °C.
    3. Isolation of RNA from TRIZOL (Protocol modified from the manufacturer's protocol)
      1. Continue with upper phase from Step D2f from DNA isolation.
      2. Add 600 μl isopropanol + 1.5 μl glycogen, invert to mix multiple times.
      3. Spin for 30 min 16,000 x g 4 °C.
      4. Wash pellet twice in 70% EtOH.
        1. Vortex.
        2. 15 min at 7,500 x g, 4 °C.
        3. Remove supernatant.
      5. Air-dry pellet by opening the lid of the Eppendorf and placing it with the opening facing down on a clean tissue at room temperature for 10 min.
      6. Resuspend in 100 μl RNase free water.
      7. Use a Spectrophotometer (e.g., NanoDrop) to assess RNA concentration.
      8. Store at -80 °C.
    4. Use PCR to amplify the purified DNA. Use PCR product for Sanger sequencing
      Note: If possible, use primers annealing outside the homology arms of the donor plasmid to avoid amplifying possible traces of pHDR-Don plasmids.
    5. If the reads are ambiguous/not clean (e.g., overlap of multiple signals in the sequencing chromatogram after a certain point in the sequence), use TOPO-TA cloning to assess if you have a heterozygous clone or a mixed population. In case of a heterozygous clone, the TOPO-TA cloning will yield E.coli colonies containing the allelic sequences in a 1:1 ratio.

Notes

Homozygous clones should then finally be analyzed by Western Blotting to demonstrate the absence of the targeted endogenous protein. An example for cell harvesting, extract preparation and immunoblotting is described in the original CRISPR-trap publication (Reber et al., 2018). In our hands, CRISPR-trap yielded no homozygous clones when targeting essential genes (lethal knockout). In this case, a gene replacement with a degron-tagged cDNA (see pHDR-TDP43 repl-Puro as example) should be considered to allow an inducible depletion of the protein of interest.
  The success rate of CRISPR-trap will depend on cell line and target gene. It is therefore difficult to predict the number of clones that need to be sequenced before a homozygous clone is detected. In our hands, the success rate for homozygous clones ranged from 10 % to 90 % of the analyzed clones.

Acknowledgments

This work was made possible by the support of the NOMIS Foundation, the National Centre of Competence in Research (NCCR) RNA & Disease funded by the Swiss National Science Foundation, and the support of the UK Dementia Research Institute. The protocol is based on the publications (Reber et al., 2016 and 2018) in The EMBO Journal and Molecular Biology of the Cell.

Competing interests

The authors declare that they have no competing or conflicting interests.

References

  1. Aznarez, I., Barash, Y., Shai, O., He, D., Zielenski, J., Tsui, L. C., Parkinson, J., Frey, B. J., Rommens, J. M. and Blencowe, B. J. (2008). A systematic analysis of intronic sequences downstream of 5' splice sites reveals a widespread role for U-rich motifs and TIA1/TIAL1 proteins in alternative splicing regulation. Genome Res 18(8): 1247-1258.
  2. Li, G., Zhang, X., Zhong, C., Mo, J., Quan, R., Yang, J., Liu, D., Li, Z., Yang, H. and Wu, Z. (2017). Small molecules enhance CRISPR/Cas9-mediated homology-directed genome editing in primary cells. Sci Rep 7(1): 8943.
  3. Mercer, T. R., Clark, M. B., Andersen, S. B., Brunck, M. E., Haerty, W., Crawford, J., Taft, R. J., Nielsen, L. K., Dinger, M. E. and Mattick, J. S. (2015). Genome-wide discovery of human splicing branchpoints. Genome Res 25(2): 290-303.
  4. Reber, S., Stettler, J., Filosa, G., Colombo, M., Jutzi, D., Lenzken, S. C., Schweingruber, C., Bruggmann, R., Bachi, A., Barabino, S. M., Muhlemann, O. and Ruepp, M. D. (2016). Minor intron splicing is regulated by FUS and affected by ALS-associated FUS mutants. EMBO J 35(14): 1504-1521.
  5. Reber, S., Mechtersheimer, J., Nasif, S., Benitez, J. A., Colombo, M., Domanski, M., Jutzi, D., Hedlund, E. and Ruepp, M. D. (2018). CRISPR-Trap: a clean approach for the generation of gene knockouts and gene replacements in human cells. Mol Biol Cell 29(2): 75-83.
  6. Yu, C., Liu, Y., Ma, T., Liu, K., Xu, S., Zhang, Y., Liu, H., La Russa, M., Xie, M., Ding, S. and Qi, L. S. (2015). Small molecules enhance CRISPR genome editing in pluripotent stem cells. Cell Stem Cell 16(2): 142-147.

简介

该协议描述了CRISPR-Trap从设计基因靶向策略到验证成功编辑的克隆的应用,所述克隆在各种人细胞系上得到验证,其中人类诱导的多能干细胞(hiPSC)。 CRISPR-Trap优于常规方法的优点是从靶基因完全去除任何内源全长转录物。 CRISPR-Trap适用于在其第一个外显子中没有编码序列或编码序列很少的任何靶基因。 到目前为止,已经使用CRISPR-Trap成功编辑了几种人细胞系和不同基因。

【背景】CRISPR / Cas9技术的出现促进了基因敲除和基因编辑的基因组靶向。执行敲除的常规方法依赖于引入移码导致过早终止密码子(PTC),截短开放阅读框(ORF)以及随后通过无义介导的mRNA衰变(NMD)降解靶基因的转录物。 。这种方法的一个可能的缺陷是全长转录物,其可以逃避NMD并产生具有残余或甚至显性负功能的C末端截短蛋白。该协议提出了CRISPR-Trap,这是我们最近建立的一种方法(Reber et al。>,2018),成功编辑后将阻止从靶基因位点表达任何全长转录本(图1)。简而言之,这种方法针对CRISPR / Cas9感兴趣基因的第一个内含子。使用同源定向修复(HDR),在第一个内含子中引入侧翼为强3'-剪接位点和强聚腺苷酸化信号的可定制盒,从而产生人工第二个并且有效地持续外显子。由于通过引入的多腺苷酸化信号终止转录,仅转录第一内源外显子和插入的盒。可定制的盒可用于引入选择标记,从而能够容易地选择至少杂合编辑的克隆。如果需要基因替换,可定制的盒可用于引入替代基因,然后是内部核糖体进入位点(IRES)和选择标记(Reber 等。>,2016和2018) 。


图1. CRISPR-Trap的应用示意图使用CRISPR / Cas9系统切割靶基因的第一个内含子,并提供用于同源定向修复的模板DNA。模板DNA含有强烈的3'主要剪接信号(深绿色),可定制的盒(浅绿色)和强聚腺苷酸化信号(绿松石)。可定制的盒可用于敲除(左)或替换靶基因(右)。该盒含有选择标记,该选择标记在成功编辑后将受靶基因的内源启动子的控制。对于基因置换,在置换基因和选择标记之间引入IRES。图改编自(Reber et al。>,2018)。

关键字:CRISPR, CRISPR-Trap, 基因敲除, 基因置换, 基因编辑, hiPSCs

材料和试剂

  1. 移液器吸头
    1. 10μl蓝宝石散装非无菌移液器吸头(Greiner Bio One International,目录号:771250)
    2. 带分度的200μl聚丙烯通用移液器吸头(Greiner Bio One International,目录号:739282)
    3. 1,250μl蓝宝石散装非无菌移液器吸头(Greiner Bio One International,目录号:750250)
  2. TPP ®组织培养板
    1. 24孔(Sigma-Aldrich,目录号:Z707791)
    2. 6孔(Sigma-Aldrich,目录号:Z707767)
    3. 15 cm板(Sigma-Aldrich,目录号:Z707694)
  3. TPP细胞刮刀20厘米(MIDSCI,目录号:TP99010)
  4. 克隆圆筒无菌,3 / 16''ID x 5 / 16''H(SP Scienceware - Bel-Art Products - H-B Instrument,目录号:37847-0100)
  5. 细胞过滤器(EASYstrainer,70μm)(Greiner Bio One International,目录号:542070)
  6. Bel-Art TM SP Scienceware TM Belpen TM 黑色标记(SP Scienceware - Bel-Art Products - HB Instrument,目录号:F13374-0000 )
  7. 选择的质粒,作为同源定向修复供体模板(供体质粒)
  8. Cas9和sgRNA表达的首选质粒,例如>,pX330-U6-Chimeric_BB-CBh-hSpCas9(Addgene,目录号:42230)
  9. 转型胜任者 E.用于克隆和质粒扩增的大肠杆菌>菌株(例如>。,XL10 Gold)(安捷伦,产品目录号:200314)
  10. 选择转染试剂
    1. DogTor(OZ Biosciences,目录号:DT51000)
    2. TransI > T ® -LT1(Mirus Bio,目录号:MIR 2300)
    3. Lipofectamine TM 3000试剂(赛默飞世尔科技,目录号:L3000015)
  11. 细胞分离解决方案
    1. hiPSCs
      1. 产生单个细胞:StemPro TM Accutase TM 细胞解离试剂(Thermo Fisher Scientific,目录号:A1110501)
      2. 将细胞分裂为团块:含有0.5mM EDTA的DPBS(STEMCELL Technologies,目录号:37350)(Thermo Fisher Scientific,目录号:15575020)
    2. 其他人类细胞系:胰蛋白酶-EDTA(0.05%),酚红(Thermo Fisher Scientific,目录号:25300054)
  12. 将hiPSCs培养为单细胞:Y-27632,RHO / ROCK途径抑制剂(STEMCELL Technologies,目录号:72302)
  13. Zeocin TM (InvivoGen,目录号:ant-zn)
  14. 嘌呤霉素二盐酸盐(Santa Cruz Biotechnology,目录号:sc-108071A)
  15. PBS(Ca 2 + ,Mg 2 + free)(Thermo Fisher Scientific,Gibco TM ,目录号:10010023)
  16. TRIZOL(西格玛奥德里奇,目录号:T9424)
  17. 道康宁®高真空硅油脂(Sigma-Aldrich,目录号:Z273554)
  18. TOPO TM TA克隆 TM 试剂盒,双启动子(Thermo Fisher Scientific,目录号:450640)
  19. 用于分析的氯仿EMSURE ® ACS,ISO,Reag。 Ph Eur(默克,目录号:102445)
  20. 绝对EtOH(西格玛奥德里奇,目录号:59176)
  21. 柠檬酸三钠(Sigma-Aldrich,目录号:W302600)
  22. 用于分析的2-丙醇EMSURE ® ACS,ISO,Reag。 Ph Eur(默克,目录号:109634)
  23. 无RNase糖原(赛默飞世尔科技,目录号:R0551)
  24. DNA提取试剂盒( Quick > -DNA TM miniprep Kit)(ZYMO RESEARCH,目录号:D3025)
  25. NaOH(Sigma-Aldrich,目录号:306576)
  26. Maximo Taq DNA聚合酶2X-preMix / PCR Master Mix(GeneON,目录号:S113)
  27. KAPA Taq ReadyMix PCR Kit(KAPA Biosystems,目录号:KK1006)

设备

  1. 移液器&nbsp;
    1. Pipetman P10(吉尔森,产品目录号:F144802)
    2. Pipetman P20(吉尔森,产品目录号:F123600)
    3. Pipetman P200(吉尔森,产品目录号:F123601)
    4. Pipetman P1000(吉尔森,产品目录号:F123602)
  2. 哺乳动物培养设备
  3. 加热块(VWR,目录号:444-0938)
  4. 热循环仪(VWR,目录号:732-2551)
  5. NanoDrop(Thermo Fisher Scientific,型号:NanoDrop TM 2000,目录号:ND-2000)
  6. 冷却离心机(Eppendorf,型号:5424 R,目录号:5404000010)
  7. 宽视野显微镜
  8. Vortex(科学工业,目录号:SI-0266)

软件

  1. 克隆管理器( http://www.scied.com/pr_cmpro.htm )或其他克隆软件

程序

注意:>

  1. 必须注意的是,靶基因的第一个外显子仍然会在CRISPR-Trap策略后表达。因此,理想的靶基因在其第一个外显子中没有或只有很少的氨基酸编码。>
  2. 为了允许直接选择基因组编辑的细胞,必须在进行编辑的细胞中表达基因。>
  3. 由于每个目标的行为都不同,具体取决于具体的基因和细胞系,因此可能需要调整各种参数。>


  1. sgRNA和供体质粒的设计
    sgRNA设计>
    在目标基因的第一个内含子中识别目标位点并生成合适的表达质粒(以下称为pCas9 / sgRNA)
    1. 大多数CRISPR / Cas9表达质粒使用CMV启动子来表达Cas9内切核酸酶。在我们的手中,该启动子导致人干细胞中低Cas9表达水平。然而,这仍然导致成功产生CRISPR捕获子敲除(Reber et al。>,2018),对于CMV启动子的特定细胞系,可以考虑用EF1α启动子替换CMV启动子。显示低活动。
    2. 对于内含子sgRNA靶位点:
      1. 确保从第一个外显子的3'末端留下间隔区不破坏内含子剪接增强子,这些增强子通常位于5'剪接位点下游的前100个核苷酸内(Aznarez 等。> ,2008)。
      2. 在内含子分支点的上游引入盒子以确保正确剪接到靶基因的第一个外显子。分支点通常位于&lt;在3'剪接位点上游50个核苷酸,其中90%的所有人分支点位于3'剪接位点上游37个核苷酸处(Mercer 等人,>,2015)。然而,在某些人类基因中,分支点可位于更上游。因此,我们建议使用剪接网站预测器,例如 http://www.umd上提供的人体拼接查找器.be / HSF3 / 确定理想的目标区域。
    3. 如果可能,找到两个或多个相邻的sgRNA序列,这些序列可以用相同的供体质粒触发HDR,从而提高成功率。
      1. 选项1:对于许多基因,在合适的表达质粒中预先设计和验证的sgRNA以及定制的sgRNA设计是可商购的(例如>,来自Sigma-Aldrich的CRISPR / Cas核酸酶RNA指导的基因组编辑可用在 https://www.sigmaaldrich。 com / technical-documents / articles / biology / crispr-cas9-genome-editing.html #cimified 或来自Addgene的 https://www.addgene.org/crispr/ )
      2. 选项2:使用专用的在线工具(我们通常使用Feng Zhang组的CRISPR设计工具,可从 http://获得) crispr.mit.edu/ )在感兴趣的基因的第一个内含子中鉴定CRISPR / Cas9靶序列。随后,将指导序列克隆到合适的CRISPR / Cas9表达质粒(例如>,pX330-U6-Chimeric_BB-CBh-hSpCas9,Addgene Plasmid#42230)中。按照 https://www.addgene.org/crispr/zhang/ <上提供的Zhang Lab General Cloning协议进行操作/ A>。

    供体质粒设计>
    通过克隆或通过基因合成将含有可定制盒的供体质粒产生到所选择的质粒中。该供体质粒随后称为pHDR-Don。 (Reber et al。>,2018)用于敲除和基因置换的供体质粒(pHDR-FUS KO-Zeo:FUS敲除的产生和pHDR-TDP43 repl-Puro:TDP的基因替代) -43与DD-TDP43)可用作 Genbank文件 。此外,为第一外显子中具有和不具有编码序列的基因提供用于基因敲除的pHDR-Don实例构建体:pHDR-Zeo-Don用于具有非编码第一外显子的基因,pHDR-Zeo-Don_f1用于剪接位点的基因在第一个外显子直接位于密码子之后,pHDR-Zeo-Don_f2用于剪接位点位于最后一个密码子后1个额外核苷酸之后的基因,pHDR-Zeo-Don_f3用于剪接位点位于2个额外之后的基因最后一个密码子后的核苷酸。此外,提供了用非编码第一外显子进行基因置换的示例构建体(pHDR-IRES-Puro-Don)。
    1. 我们使用Zeocin和嘌呤霉素作为选择标记,但是,取决于靶细胞系,替代抗性标记物如杀稻瘟素可能是有益的。
      如果内源基因在其第一个外显子中含有一些编码序列,请确保抗体标记/基因替换将在框内拼接。
    2. Cas9在PAM基序上游切割3个核苷酸。因此,可定制盒的同源臂5'和3'应与Cas9切割位点的上游(5')和下游(3')序列相同。为了实现pHDR-Don介导的有效同源定向修复,我们通常使用长度为700-800个核苷酸的同源臂(每个同源臂)(见图2)。


      图2. pHDR-Don设计的示意图。 Cas9切割PAM基序上游的3个核苷酸位置。同源臂应该与切割位点的上游序列(5'同源性)和下游序列(3'同源性)相同。每个同源臂应长约750碱基对,以确保有效的HDR。在短内含子的情况下,5'同源性的750个碱基对可能含有目的基因的第一个外显子和启动子区域的部分。在这种情况下,请确保不要在5'同源臂中包含启动子序列,以防止质粒直接从质粒中表达。

    3. 如果它含有靶基因的启动子序列,则必须缩短上游(5')同源臂。否则,将直接从pHDR-Don质粒表达盒。

  2. 转染细胞
    1. 在适合各细胞系的条件下培养细胞(生长培养基,生长表面,生长条件)。在整个方案期间可以保持这些条件。在转染前一天在6孔板的所有孔中接种细胞。为了使细胞接种,请按照以下步骤操作:
      1. 从含有细胞的平板中吸出培养基。
      2. 通过轻轻旋转加入足够量的PBS以覆盖板的表面来洗涤细胞一次。随后吸出PBS。
      3. 添加适当的试剂以分离细胞(例如>,胰蛋白酶,Accutase)并等待细胞分离。
      4. 将细胞重悬于适当的培养基中并计数细胞(例如>,使用血细胞计数器)。
      5. 将所需量的细胞转移到孔中。每孔的最佳细胞量将根据细胞系和所用的转染试剂而变化。遵循转染试剂制造商的方案,使用明场显微镜估算密度,以获得最佳转染效率。
    2. 为您的细胞系使用适当的转染试剂。在我们的手中,最有效的转染试剂是用于HeLa细胞的DogTor,用于HT1080的Mirus TransI > T ® -LT1,以及人诱导的多能干细胞(hiPSC)和Lipofectamine < sup> TM 3000用于SH-SY5Y神经母细胞瘤细胞。用pCas9 / sgRNA和pHDR-Don转染每个孔。表1给出了我们手中成功编辑的细胞系的经验值(每个转染6孔板的一个孔的值)。

      表1.转染质粒量的经验值。成功编辑各种细胞系后转染的pCas9 / sgRNA和pHDR-Don质粒的经验值。


      注意:当克隆两个相邻靶标的sgRNA时,用一个sgRNA转染3个孔,用另一个sgRNA转染另外3个孔。 6孔也可用于改变转染质粒的总量或pCas9 / sgRNA与pHDR-Don之间的比例。一个好的起点是尝试pCas9 / sgRNA:pHDR-Don的摩尔比为1:6至2:1。如果6孔不足以进行所有变异,则可以转染更多的孔。由于CRISPR-Trap依赖于同源定向修复,因此可以在此阶段添加增加这些事件可能性的小分子,如L755507(Yu等,2015; Li等,2017)。>
    3. 转染后2至4天,使用适当的细胞分离溶液分离细胞,并将来自所有孔的细胞汇集在一个15cm培养皿中。让细胞附着,然后使用盒带引入的选择标记开始选择。表2给出了我们手中成功编辑的细胞系的经验值。

      表2.抗生素选择的经验值。抗生素浓度的经验值和成功编辑各种细胞系的选择持续时间。


  3. 选择选定的克隆
    最好在通过显微镜观察时选择菌落,因为这样可以更好地控制并降低混合多个菌落的风险。在这里,我们描述了一种方法,允许在无菌条件下在显微镜下采摘时手动挑选单菌落。如果无法在无菌条件下进行手动拣选的显微镜,请遵循此协议。否则,在无菌条件下直接在显微镜下挑取菌落,并从步骤C10继续。
    1. 完成选择过程后,允许单个细胞生长成菌落。然而,重要的是不要让菌落生长到彼此非常接近的位置,以确保每个菌落从一个单细胞生长。如果菌落长得彼此靠近,将细胞分裂成新的平板(使用细胞过滤器确保从单个细胞中出现新的菌落)。
      注意:根据经验,从边缘到下一个菌落的边缘挑选由至少10个细胞和1厘米空间组成的菌落。>
    2. 计算并记下好的挑选菌落数(即>,由边缘到下一个菌落边缘至少10个细胞和1厘米的空间组成)。用一支笔在15厘米板的下面盘旋它们,标记所有可以挑选的菌落(见图3)。
      注意:如果两个菌落彼此太靠近,请放弃它们以避免采摘混合菌落。>
    3. 在新鲜的无菌培养皿上涂抹真空油脂,并使用无菌细胞刮刀生成厚度约为5毫米的油脂膜(图3A和3B)。
    4. 按步骤C2中评估的数量准备克隆柱。将带有窄边的克隆圆筒放在润滑脂上。克隆油缸粘在油脂上后尽量避免水平移动(图3C)。
    5. 在紫外线下灭菌20分钟。
    6. 从含有细胞的15cm平板吸出培养基,并通过加入10ml PBS用PBS洗涤一次,通过轻轻旋转将PBS分配到平板中,随后吸出PBS。
    7. 从油脂薄膜中逐一取出克隆圆筒,然后将它们放在菌落上,使润滑面朝向平板。如果您在没有同时使用显微镜的情况下进行拾取,请使用步骤C2中的标记作为克隆圆柱放置位置的指南(图3D)。
      注意:施加一点压力以防止密封。
      >

      图3.用于手动克隆采摘的真空润滑脂和克隆圆筒的制备 A.在无菌培养皿上涂抹润滑脂。 B.使用无菌细胞刮刀产生约5mm厚的油脂膜。 C.将克隆圆筒的窄边放在润滑脂上。 D.将带有润滑侧的克隆缸首先放在菌落上。

    8. 加入20μl适当的试剂,将细胞(例如>,胰蛋白酶,Accutase)分离到每个克隆圆筒中,等待细胞分离
    9. 一旦细胞分离,将80μl适当的培养基加入每个克隆圆筒中,并将所有液体直接转移到每个菌落的24孔板的单独孔中。随后在每个孔中补充培养基至500μl。
    10. 让菌落在接下来的几天内在24孔板中生长,并在它们达到汇合时将细胞转移到6孔板的孔中。
      注意:也可以按照程序D中的相同方案从24孔中直接收获克隆。>

  4. 验证成功编辑的克隆
    1. 一旦克隆在6孔板(或24孔板)上达到汇合,就将细胞分离并分成两部分。在成功进行基因组编辑的情况下冻结一半细胞继续进行,同时从另一半提取DNA和RNA以评估基因型。为此,我们将细胞重新悬浮在TRIZOL中以分离gDNA和RNA。>
      注意:DNA提取试剂盒可用作TRIZOL提取的替代方法。然而,TRIZOL提取的优点是可以同时提取RNA。使用RT-qPCR可以快速检查是否表达了来自引入基因阱下游的靶基因的任何RNA。这种RNA的低痕量可以表明混合的细胞群(挑选的克隆不是来自单个细胞)。>
      此外,TRIZOL中的样品可以长期保存在-20°C,允许同时批量分析大量克隆。>
    2. 从TRIZOL中分离gDNA(从制造商协议修改的协议)
      1. 每孔加入0.5ml TRIZOL(6孔或24孔)并上下移液以裂解细胞。
      2. 将细胞裂解物转移到1.5ml Eppendorf管中。
      3. 加入0.1毫升氯仿,盖紧盖子,剧烈摇晃15秒。
      4. 在室温下孵育2-15分钟。
      5. 在2-8℃下以12,000 x g >离心15分钟。
      6. 去除上相(含有RNA,保存在-20°C)。
      7. 加入0.3 ml 100%EtOH和1.5μl糖原,通过倒置混合。
        注意:如果您使用少量细胞,添加糖原是必不可少的 - 如果没有它,您将不会在后面的步骤中看到颗粒。>
      8. 在室温下孵育2-3分钟。
      9. 在2-8℃下在2,000 x g >下离心5分钟。
      10. 去除上清液。
      11. 洗涤DNA溶液两次:
        1. 加入1ml 0.1M柠檬酸三钠,10%EtOH。
        2. 在室温下孵育(偶尔混合,倒置)至少30分钟。
        3. 在2-8℃下在2,000 x g >下离心5分钟。
        4. 去除上清液。
          注意:DNA沉淀通常不会强烈附着在管上,注意不要将其与上清液一起吸出;最好留下少量上清液。>
      12. 将沉淀重悬于1ml 75%EtOH中。
      13. 在室温下孵育10-20分钟。
      14. 在2-8℃下在2,000 x g >下离心5分钟。
      15. 通过打开Eppendorf的盖子并在室温下将开口朝下放置在干净的纸巾上15分钟来风干颗粒。
      16. 将沉淀溶解在50-100μl8mMNaOH中,重复移液。
      17. 使用分光光度计(例如>,NanoDrop)评估DNA浓度。
        注意:按照该方案从少量细胞中分离的DNA通常使用分光光度计(例如NanoDrop)测量高乙醇/苯酚峰测量DNA浓度。然而,这些污染物不会干扰我们手中的下游PCR反应(使用Maximo Taq DNA Polymerase 2x-preMix [S113,GeneON]或KAPA Taq ReadyMix [KK1006,KAPA Biosystems])。>
      18. 储存在-20°C。
    3. 从TRIZOL中分离RNA(从制造商的协议修改的协议)
      1. 从DNA分离继续步骤D2f的上部阶段。
      2. 加入600μl异丙醇+1.5μl糖原,颠倒混合多次。
      3. 旋转30分钟16,000 x g > 4℃。
      4. 在70%EtOH中洗涤沉淀两次。
        1. 涡流。
        2. 在7,500 x g >,4℃下15分钟。
        3. 去除上清液。
      5. 通过打开Eppendorf的盖子并将其开口朝下放置在室温下干净的纸巾上10分钟来风干颗粒。
      6. 重悬于100μl无RNase的水中。
      7. 使用分光光度计(例如>,NanoDrop)评估RNA浓度。
      8. 储存在-80°C。
    4. 使用PCR扩增纯化的DNA。使用PCR产物进行Sanger测序
      注意:如果可能,使用在供体质粒同源臂外退火的引物,以避免扩增可能的pHDR-Don质粒的痕迹。>
    5. 如果读数不明确/不清洁(例如>,序列中某一点后序列色谱图中多个信号的重叠),使用TOPO-TA克隆来评估您是否有杂合克隆或混合人口。在杂合克隆的情况下,TOPO-TA克隆将产生含有1:1比例的等位基因序列的大肠杆菌>菌落。

笔记

然后最终应通过Western印迹分析纯合克隆以证明不存在靶向内源蛋白。细胞收集,提取物制备和免疫印迹的实例描述于原始CRISPR-trap出版物(Reber 等人,2018)中。在我们的手中,CRISPR-trap在靶向必需基因(致死基因敲除)时不产生纯合克隆。在这种情况下,应考虑使用带有degron标记的cDNA的基因替换(参见pHDR-TDP43 repl-Puro作为实例),以允许感兴趣的蛋白质耗尽。
&NBSP; CRISPR陷阱的成功率取决于细胞系和靶基因。因此,难以预测在检测到纯合克隆之前需要测序的克隆的数量。在我们的手中,纯合克隆的成功率为分析克隆的10%至90%。

致谢

这项工作得到了NOMIS基金会,国家研究能力中心(NCCR)RNA&amp; amp;疾病由瑞士国家科学基金会资助,并得到英国痴呆症研究所的支持。该方案基于 EMBO期刊>和细胞分子生物学>中的出版物(Reber 等人,2016和2018)。

利益争夺

作者声明他们没有竞争或冲突的利益。

参考

  1. Aznarez,I.,Barash,Y.,Shai,O.,He,D.,Zielenski,J.,Tsui,L.C.,Parkinson,J.,Frey,B.J.,Rommens,J.M。和Blencowe,B.J。(2008)。 对5'剪接位点下游的内含子序列进行系统分析,揭示了富含U的基序的广泛作用和替代剪接调节中的TIA1 / TIAL1蛋白。 Genome Res > 18(8):1247-1258。
  2. Li,G.,Zhang,X.,Zhong,C.,Mo,J.,Quan,R.,Yang,J.,Liu,D.,Li,Z.,Yang,H。和Wu,Z。( 2017年)。 小分子增强原代细胞中CRISPR / Cas9介导的同源定向基因组编辑。 Sci Rep > 7(1):8943。
  3. Mercer,T.R.,Clark,M.B.,Andersen,S.B.,Brunck,M.E.,Haerty,W.,Crawford,J.,Taft,R.J.,Nielsen,L.K.,Dinger,M.E。和Mattick,J。S.(2015)。 全基因组发现人类剪接分支点。 Genome Res > 25(2):290-303。
  4. Reber,S.,Stettler,J.,Filosa,G.,Colombo,M.,Jutzi,D.,Lenzken,SC,Schweingruber,C.,Bruggmann,R.,Bachi,A.,Barabino,SM,Muhlemann, O.和Ruepp,MD(2016年)。 轻微内含子剪接受FUS调节,受ALS相关FUS突变体的影响。 EMBO J > 35(14):1504-1521。
  5. Reber,S.,Mechtersheimer,J.,Nasif,S.,Benitez,J.A.,Colombo,M.,Domanski,M.,Jutzi,D.,Hedlund,E。和Ruepp,M。D.(2018)。 CRISPR-Trap:用于在人体细胞中产生基因敲除和基因替换的简洁方法。< / a> Mol Biol Cell > 29(2):75-83。
  6. Yu,C.,Liu,Y.,Ma,T.,Liu,K.,Xu,S.,Zhang,Y.,Liu,H.,La Russa,M.,Xie,M.,Ding,S。和Qi,LS(2015)。 小分子增强多能干细胞中的CRISPR基因组编辑。 细胞干细胞> 16(2):142-147。
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引用:Mechtersheimer, J., Reber, S. and Ruepp, M. (2018). Generation of Gene Knockout and Gene Replacement with Complete Removal of Full-length Endogenous Transcript Using CRISPR-Trap. Bio-protocol 8(20): e3052. DOI: 10.21769/BioProtoc.3052.
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