Generation of Targeted Knockout Mutants in Arabidopsis thaliana Using CRISPR/Cas9
拟南芥中利用CRISPR/Cas9技术生成定向基因敲除突变体   

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Frontiers in Plant Science
0 2017

 

Abstract

The CRISPR/Cas9 system has emerged as a powerful tool for gene editing in plants and beyond. We have developed a plant vector system for targeted Cas9-dependent mutagenesis of genes in up to two different target sites in Arabidopsis thaliana. This protocol describes a simple 1-week cloning procedure for a single T-DNA vector containing the genes for Cas9 and sgRNAs, as well as the detection of induced mutations in planta. The procedure can likely be adapted for other transformable plant species.

Keywords: CRISPR/Cas9 (CRISPR/Cas9), Genome editing (基因组编辑), Arabidopsis thaliana (拟南芥), Plants (植物), Knockout (基因敲除)

Background

The CRISPR/Cas9 system (Cas9) provides a simple and widely applicable approach to modify genomic regions of interest and has therefore become the tool of choice for genome editing in plants and other organisms (Schiml and Puchta, 2016). The system relies on the bacterial Cas9 nuclease from Streptococcus pyogenes (Cas9), which can be directed by a short artificial single guide RNA molecule (sgRNA) towards a genomic DNA sequence (Jinek et al., 2012), where it creates a double strand break (DSB). These DSBs are then repaired by the innate DNA repair mechanism of the plant cell. Here, two main pathways can be distinguished (Salomon and Puchta, 1998). (i) DNA molecules with high homology to the DSB site can be used as repair template. This homology directed repair (HDR) approach can be exploited to introduce specific sequences at the site of the DSB (Schiml et al., 2014; Baltes and Voytas, 2015). However, due to low integration rates of these sequences, HDR mediated gene editing in plants remains challenging. (ii) An easier and more efficient approach is the use of the non-homologous end joining (NHEJ) repair pathway of the plant, which is the dominant repair pathway in most plants, such as Arabidopsis thaliana (Arabidopsis). Since NHEJ is error-prone, small insertions or deletions (indels) of a few base pairs (bp) occur often at the DSB site, leading to frameshift mutations and gene knockouts (Pacher and Puchta, 2016). Here, we provide a detailed protocol for targeted gene knockout in the model plant Arabidopsis including a simple 1-week cloning protocol for a plant vector system containing the Cas9 and sgRNA, and then Arabidopsis transformation and detection of mutations.

Materials and Reagents

  1. 1.5 ml microcentrifuge tubes (SARSTEDT, catalog number: 72.690.001 )
  2. 200 µl PCR tubes (Labomedic, catalog number: 2081644AA )
  3. Petri dishes (SARSTEDT, catalog number: 82.1472 )
  4. 2 ml microcentrifuge tubes (SARSTEDT, catalog number: 72.691 )
  5. 20 µl pipette tips (SARSTEDT, catalog number: 70.1116 )
  6. 200 µl pipette tips (SARSTEDT, catalog number: 70.760.012 )
  7. 1,000 µl pipette tips (SARSTEDT, catalog number: 70.762.010 )
  8. Agrobacterium tumefaciens (A. tumefaciens) strain GV3101::pMP90
  9. Arabidopsis thaliana seeds (Col-0)
  10. Vectors (see Figure 1)


    Figure 1. Vector maps of pUB-Cas9 (A) and pFH6 (B). pFH6 is used to integrate the 20 bp target sequence upstream of the sgRNA scaffold and under the control of the Arabidopsis U6-26 RNA polymerase III promoter. The whole sgRNA cassette is then transferred via Gibson cloning into the binary T-DNA vector pUB-Cas9 (contains the Cas9 gene under the control of the Ubiquitin10 promoter) for plant transformation. Maps were generated using SnapGene Viewer (http://www.snapgene.com/products/snapgene_viewer/).

    1. Plant T-DNA Cas9 vector pUB-Cas9 (GenBank accession number KY080691), containing a Chlamydomonas reinhardtii codon-optimized ubiquitously (UBIQUITIN10 promoter) expressed Cas9 gene, a kanamycin resistance cassette for bacterial selection and a hygromycin resistance cassette as plant selection marker (Hahn et al., [2017]; available at Addgene, catalog number: 86556 )
    2. sgRNA subcloning vector pFH6 (GenBank accession number KY080689) containing the Arabidopsis U6-26 promoter, the integration site for the 20 bp protospacer sequence, the sgRNA scaffold and an ampicillin resistance cassette (Hahn et al. [2017]; available at Addgene, catalog number: 86555 )
      Note: pFH6 contains an additional 9 bp fragment (GTCCCTTCG) between the 3’ end of the U6-26 promoter and the protospacer integration site. In several experiments, we could show that this does not affect gene editing activity. However, we have also cloned a new version of the subcloning vector without the additional fragment (pFH6_new), which shows high cleavage activity in preliminary experiments and can be obtained from us. This version only contains an additional guanine (G) between the 3’ end of the U6-26 promoter and the protospacer integration site, which allows the integration of any 20 bp protospacer without restriction of G as first bp (compare e.g., Fauser et al. [2014]). The cloning strategy for pFH6_new is analogous to the one described in this protocol, the only difference is that the forward primer for cloning your 20 bp protospacer sequence contains a different overlap and lacks the need for an initial G at the beginning (ATTG-N20, compare procedure section).
  11. Competent Escherichia coli (E. coli) cells (e.g., Mach1TM competent cells, Thermo Fisher Scientific, InvitrogenTM, catalog number: C862003 )
  12. BbsI-HF + CutSmart buffer (New England Biolabs, catalog number: R3539S )
  13. Distilled H2O
  14. Plasmid mini prep kit and agarose gel extraction kit (e.g., GeneMATRIX 3 in 1–Basic DNA Purification Kit, Roboklon, catalog number: E3545 )
  15. T4 DNA ligase with 10x ligation buffer (New England Biolabs, catalog number: M0202S )
  16. Ampicillin (Amp) (Carl Roth, catalog number: K029.2 )
  17. Primers (see Table 1)

    Table 1. List of oligonucleotides


  18. 5x Green GoTaq Reaction buffer (Promega, catalog number: M791A )
  19. dNTPs (10 mM each) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0192 )
  20. GoTaq G2 polymerase (Promega, catalog number: M7841 ) or other standard PCR polymerase
  21. HindIII-HF + CutSmart buffer (New England Biolabs, catalog number: R3104S )
  22. KpnI-HF + CutSmart buffer (New England Biolabs, catalog number: R3142S )
  23. Phusion High-Fidelity polymerase (New England Biolabs, catalog number: M0530S ) or other proofreading polymerases
  24. Gibson Assembly Cloning Kit (New England Biolabs, catalog number: E5510S )
  25. Kanamycin sulfate (Kan) (Carl Roth, catalog number: T832.4 )
  26. Rifampicin (Rif) (Molekula, catalog number: 32609202 )
  27. Gentamycin sulfate (Gent) (Carl Roth, catalog number: 0233.3 )
  28. Hygromycin B (Hyg) (Carl Roth, catalog number: CP12.2 )
  29. T7 Endonuclease I (optional; New England Biolabs, catalog number: M0302S )
  30. LB medium (agar plates and liquid), supplemented with 200 μg/ml ampicillin (see Recipes)
  31. LB medium (agar plates and liquid), supplemented with 30 μg/ml kanamycin sulfate (see Recipes)
  32. YEP medium (agar plates and liquid), supplemented with 150 μg/ml rifampicin, 50 μg/ml gentamycin sulfate, 50 μg/ml kanamycin (see Recipes)
  33. ½ MS medium agar plates, supplemented with 33.3 μg/ml hygromycin B (see Recipes)

Equipment

  1. Agarose gel electrophoresis equipment (e.g., VWR, Peqlab, model: PerfectBlueTM Gel System Mini M, catalog number: 700-0434 )
  2. Bacteria plate incubators (28 °C, 37 °C, e.g., Memmert, model: IN55 ) and shaker (28 °C, 37 °C; e.g., Eppendorf, New BrunswickTM, model: Innova® 44 , catalog number: M1282-0002)
  3. Heating blocks (e.g., Eppendorf, model: Thermomixer Compact , catalog number: T1317-1EA)
  4. PCR cycler (e.g., Thermo Fisher Scientific, Applied BiosytemsTM, model: VeritiTM 96-well Thermal Cycler, catalog number: 4375786 )
  5. 10 µl pipette (e.g., Pipetman Neo P10N, Gilson, catalog number: F144562 )
  6. 20 µl pipette (e.g., P20N, Gilson, catalog number: F144563 )
  7. 200 µl pipette (e.g., P200N, Gilson, catalog number: F144565 )
  8. 1,000 µl pipette (e.g., P1000N, Gilson, catalog number: F144566 )
  9. Plant growth chamber (e.g., CLF Plant Climatics, Percival Scientifici, model: AR-66L )

Software

  1. SerialCloner
  2. Cas-OFFinder
  3. 4Peaks software
  4. MUSCLE (Optional)

Procedure

  1. Design of sgRNA sequence for gene knockout
    The design of the sgRNA is possibly the most critical step of Cas9-mediated gene editing. Thus, the Cas9 target site should be carefully selected. Several online tools (Heigwer et al., 2014; Lei et al., 2014; Xie et al., 2014) assist with finding suitable target sites, however, we still choose most target sites manually in standard molecular biology software (e.g., SerialCloner, http://serialbasics.free.fr/Serial_Cloner.html). When choosing the target site, the following aspects should be taken into account:
    1. Search your gene of interest (GOI) for a GN19NGG sequence on both strands to find putative target sites. The NGG is the Streptococcus pyogenes Cas9 protospacer adjacent motive (PAM), which has to be present in the target locus for efficient binding of the Cas9 protein (Jinek et al., 2012). The GN19 represents the 20 bp long protospacer motif, defining the target site where you want to create mutations. This 20 bp sequence, which marks the 5’ end of your sgRNA molecule, should start with a G, since this is a prerequisite for transcription of the sgRNA under the control of the U6-26 promoter of Arabidopsis.
      Note: If there is no suitable target sequence, you might just add the G as 21st bp, even if it does not have a complementary base in the target locus. These sgRNAs might still lead to cleavage at the target site (e.g., Hahn et al., 2017). However, we recommend using the canonical 20 bp protospacer sequences if possible.
    2. Choose a target site in an exon of your GOI, which is preferably close to the 5’ end of the coding sequence, since mutations in this region are more likely to disrupt the protein’s function. If you know highly important domains of the protein, such as nucleic acid binding sites, you might want to create mutations in these regions.
    3. Consider choosing several different target sites, since not all sgRNAs work equally well (Liang et al., 2016). Additionally, you might want to use two sgRNAs at once (see cloning strategy) to create large deletions, which might cause more drastic effects.
    4. Search for putative off-target sites to avoid knocking out genes that share the 20 bp target site or highly similar sites. For this purpose, we use the Cas-OFFinder program (Bae et al., 2014), which is available at http://www.rgenome.net/cas-offinder/. Here, we use the following settings:
      PAM-Type: SpCas9 from Streptococcus pyogenes: 5’-NRG-3’ (R = A or G)
      Target genome: Arabidopsis thaliana (TAIR 10)
      Mismatch number: 3
      After searching for your 20 bp target site in the reference genome, the program displays all similar sites with up to 3 mismatches. If you have more than one hit with zero mismatches (one is always your GOI), you should choose a different target site. If you have 1-3 mismatches, you should take into account that Cas9 might also mutagenize these sites, especially if these mismatches are at the 5’ end of the 20 bp sequence (Semenova et al., 2011). Even though the affinity for Cas9 will be lower than to your GOI and plants seem to be in general less affected by off targeting (Feng et al., 2014), we recommend careful sequencing of these off target site in the knockout plants to ensure the absence of mutations in these genes.
    5. Consider using a 20 bp target site that also contains a recognition site of a commercially available restriction enzyme. The target site of the restriction enzyme should span the site of Cas9 cleavage (3 bp in front of the PAM). This will allow you to use the restriction enzyme length polymorphism assay to detect mutations in your GOI.

  2. Subcloning of 20 bp protospacer sequence into pFH6
    1. An overview how to perform the subcloning of the protospacer sequence into pFH6 is given in Figure 2.
    2. Order forward and reverse primers for your candidate target sequence N20 with overlaps for cloning and dilute them to a final concentration (f.c.) of 10 μM.
      Forward primer: TTCG-GN19
      Reverse primer: AAAC-N19 (reverse complement) C (see Figure 2)


      Figure 2. Subcloning of protospacer sequence in pFH6. The 20 bp protospacer sequence can be integrated between the sgRNA scaffold and the U6-26 promoter (U6p) by ligation of dimerized primers into BbsI-digested pFH6 vector.

    3. Pipette 10 μl of each primer dilution into a 1.5 ml microcentrifuge tube and mix.
    4. Heat primer mix to 98 °C, 10 min, then 55 °C, 10 min, afterwards slowly cool down to RT (e.g., by switching off the heating block). By doing so, a double stranded DNA including your protospacer sequence (= annealed primers) will be generated.
    5. Digest pFH6 with BbsI-HF for 3 h at 37 °C.


    6. Run the restriction digest on an agarose gel and extract the vector backbone (3,612 bp) from the gel using the GeneMATRIX 3 in 1–Basic DNA Purification Kit.
    7. Ligate annealed primers with digested pFH6 at 4 °C overnight.


    8. Transform competent MachI E. coli cells with the ligation reaction and spread the transformed cells on LB agar plates supplemented with Amp.
      Note: Usually, plating 100 μl of the transformation mixture is sufficient. Protocols for transformation of E. coli cells are provided by the authors upon request.
    9. Verify eight E. coli colonies by colony PCR using primer M13 R and your protospacer primer TTCG-N20 with the following setup:


      PCR program:


      Note: We recommend using pFH6 as negative control. We have sometimes observed fragments at the expected band size (281 bp) in the negative control since the protospacer primer might partly bind to the BbsI-sites. However, positive colonies usually have a much stronger PCR product signal and can therefore be distinguished from negative clones.
    10. Inoculate positive colony in LB medium supplemented with Amp and grow overnight.
    11. Purify the plasmid using the GeneMATRIX 3 in 1–Basic DNA Purification Kit.
    12. Sequence the plasmid and verify the sequence of the protospacer insertion using primer M13R (Table 1).

  3. Cloning of final plant T-DNA Cas9 vector using Gibson Assembly
    1. An overview how to clone the final plant T-DNA Cas9 vector is given in Figure 3.


      Figure 3. Generation of a plant T-DNA vector with all necessary components for Cas9 induced mutagenesis. The sgRNA cassette including the 20 bp target site is amplified from pFH6 and integrated into KpnI/HindIII-digested pUB-Cas9 via Gibson Assembly. The resulting vector contains the sgRNA cassette as well as the Cas9 gene under the control of the UBIQUITIN10 promoter (UB10p), and the hygromycin (Hyg) resistance gene as selection marker. RB, right T-DNA border; PolyT, polythymidine transcription terminator; NLS, nuclear localization signal; NosTm, nopaline synthase terminator; CaMV35S, Cauliflower Mosaic Virus 2x35S promoter; 35STm, 35S terminator; LB, left T-DNA border.

    2. Digest vector pUB-Cas9 with HindIII-HF and KpnI-HF for 3 h at 37 °C.


      Note: Efficient digestion of the pUB-Cas9 plasmid is critical for further cloning steps. In case you have problems in the subsequent cloning steps, try to digest pUB-Cas9 with a fresh batch of enzymes and longer incubation times.
    3. Run out the restriction digest reaction on an agarose gel and extract the vector backbone (14,097 bp) from the gel using the GeneMATRIX 3 in 1–Basic DNA Purification Kit.
    4. Amplify the sgRNA cassette (expected size: 771 bp) from pFH6 with target sequence using primer FH41 and FH42 (Table 1) and Phusion High-Fidelity polymerase and the following setup:


      PCR program:


    5. Run out the PCR reaction on an agarose gel and extract the sgRNA cassette (771 bp) from the gel using the GeneMATRIX 3 in 1–Basic DNA Purification Kit.
    6. Assemble the pUB-Cas9 backbone and the sgRNA cassette to generate the final vector pUB-Cas9-@GOI using Gibson Assembly Master Mix according to the manufacturer’s instruction with an incubation time of 1 h.
      Note: We propose a nomenclature for the final constructs, where the target gene is marked by the @-sign. A vector designed against the GL1 gene of Arabidopsis would therefore be called pUB-Cas9-@GL1 (Hahn et al., 2017).
    7. Transform competent E. coli cells with the Gibson reaction mixture and spread the transformed cells on LB agar plates supplemented with Kan.
      Note: New England Biolabs provides highly competent E. coli cells in the Gibson Assembly Cloning Kit. However, we also used competent E. coli cells produced in our lab. Since these are usually less competent, we recommend plating all transformed cells on the LB agar plate. A protocol for generation of competent MachI E. coli can be found in Sambrook and Russell (2006).
    8. Verify eight positive colonies by colony PCR using primer M13F and FH179 (Table 1) with the following setup:


      PCR program:


      Note: Expected product size is 779 bp, pUB-Cas9 vector can be used as negative control.
    9. Inoculate positive colony into LB medium supplemented with Kan and let grow overnight.
    10. Purify the plasmid using the GeneMATRIX 3 in 1–Basic DNA Purification Kit.
    11. Sequence the plasmid for verification of protospacer sequence insertion using primer FH179 and M13F (Table 1).
      Note: The vector system also allows cloning of two different sgRNAs into the pUB-Cas9 vector backbone to induce large deletions in the target genome or to destroy two genes at once. Therefore, both protospacer sequences have to be cloned separately into pFH6 as described. Then, the first sgRNA cassette has to be amplified from its subcloning pFH6-construct using the primers FH41 and FH254 (Table 1). The second sgRNA cassette has to be amplified with FH42 and FH255 (Table 1), using the PCR conditions described above. Using the Gibson Assembly Master Mix, both cassettes can be integrated into KpnI-HF/HindIII-HF-digested pUB-Cas9 in a single step. As two fragments are integrated into the digested vector, the amount of colonies is usually lower compared to the integration of one fragment. However, the proportion of correctly assembled vectors is comparable. The colony PCR can be performed with the same primers but using a longer elongation time (1.5 min), since the PCR reaction amplifies both sgRNA cassettes (expected product size: 1,512 bp). Sequencing should be performed as described above.

      All cloning steps are summarized in Table 2:

      Table 2. Timetable for cloning of plant T-DNA Cas9 vector for targeted gene knockout


  4. Arabidopsis transformation with pUB-Cas9-@GOI
    1. Transform competent A. tumefaciens cells with 1 μg of pUB-Cas9-@GOI and spread the transformed cells on YEP agar plates supplemented with Rif/Gent/Kan.
      Note: Protocols for generation of competent A. tumefaciens cells and transformation of A. tumefaciens cells are described elsewhere (Hofgen and Willmitzer, 1988).
    2. After two days of growth at 30 °C, verify plasmid presence in at least three colonies by colony PCR using primer FH61 and FH201 (Table 1). In order to verify colony, pick half of a colony with a pipette tip and suspend cells in a 1.5 ml microcentrifuge tube containing 100 μl ddH2O. Heat to 98 °C for 10 min and use 2 μl as DNA template for the PCR. Streak the other half of the colony on a fresh YEP agar plate supplemented with Rif/Gent/Kan. Grow cells at 30 °C for one day and then store at 4 °C. For the colony PCR, use the following setup:


      PCR program:


      Note: Expected product size is 1,003 bp.
    3. Transform Arabidopsis plants using Agrobacterium-mediated T-DNA transfer with the floral dipping method (Clough and Bent, 1998).
      Note: Per construct, we usually transform four pots with five Arabidopsis plants, each. We recommend removing of all present siliques before transformation to avoid excessive screening for transformation events afterwards.
    4. Select the T1 generation plants for successful T-DNA insertion events on ½ strength MS agar plates supplemented with Hyg. Verify the integration of the T-DNA by isolating leaf genomic DNA (gDNA) and subsequent PCR using primers FH61/FH201with the parameters described before.
      Note: A detailed protocol for plant gDNA extraction by isopropanol precipitation can be found elsewhere (Weigel and Glazebrook, 2009).

  5. Detection of Cas9 induced mutations
    The Cas9 protein in our vector system is transcribed under the control of the UBIQUITIN10 promoter of Arabidopsis, which allows Cas9 expression in nearly all tissues of the plant (Norris et al., 1993). Since the editing mechanisms of the Cas9 system work independently in each plant cell, T1 generation plants are usually chimeras with distinct genotypes in different cells. Only if mutations occur in germline cells, can progeny plants arise that have a uniform genotype. This is usually the prerequisite for further analyses. Therefore (and due to the limited amount of plants available in the T1 generation), screening approaches for detection of mutations should be applied to T2 or following generations. We recommend screening descendants of three to four independent transformed lines, since mutation efficiencies might vary between different lines due to the integration site of the T-DNA cassette. To choose promising T1 lines for further screening, we also searched for mutational events in the T1 generation. We suggest to screen the plants for mutation in this way:
    1. Amplify the genomic region around the Cas9 cutting site in your GOI (± 300-500 bp upstream and downstream) with gene specific primers from leaf gDNA using Phusion polymerase.


      PCR program:


    2. Run out the PCR products on an agarose gel and extract your GOI from the gel using the GeneMATRIX 3 in 1–Basic DNA Purification Kit.
    3. Sequence PCR product with Sanger sequencing. When Cas9 was active and mutations occurred at least in a part of the leaf cells, the sequencing histogram will show a mixture of WT sequence and mutated sequences (Figure 4).


      Figure 4. Exemplary sequencing result histogram of amplified target gene from chimeric leaf gDNA. Double peaks appear at the site of Cas9 cleavage (3 bp in front of the PAM motive NGG) pointing to mutation events in the GOI in a part of the leaf cells.

      The efficiency of the Cas9 system varies considerably as its mutagenic effect relies on various aspects, such as: the plant species, Cas9 and sgRNA expression levels, and the sequence of the target loci (Bortesi and Fischer, 2015; Paul and Qi, 2016). Therefore, it is difficult to predict how many plants have to be screened to identify a heritable mutation. We recommend starting screening 30 plants per line. Screening for mutations in the T2 generation can be performed by amplification and Sanger sequencing of your GOI as described before. As this can become costly, several methods enable detecting mutations on genomic level without sequencing.
    4. If you expect a specific phenotype due to the knockout of your gene, the easiest way to screen for mutations is a visual screen in the first place. These phenotypes can be changes in anatomy or survival of plants on selection medium. Plants that show a phenotype are then analyzed on genomic level as described above.
    5. If you target two sites in your GOI at the same time, a simultaneous cleavage with two sgRNAs can result in large deletions, which can already be detected during the PCR amplification of your GOI (Figure 5A). Larger deletions can in theory also occur using one sgRNA, however, we usually discovered small indels in the range of a few bp, which usually cannot be detected by PCR product size difference using agarose gel electrophoresis.
      Note: If you expect that only few cells contain the expected mutation (e.g., in chimeric plants), you can improve the PCR amplification efficiency of the mutant alleles by digesting unmutated gDNA with a restriction enzyme that cuts between the two sgRNA target sites before PCR amplification of your GOI.


      Figure 5. Summary of methods that allow the detection of induced mutations after amplification of the GOI with a forward (FW) and reverse (RV) primer (green arrows). A. Usage of two sgRNAs can induce larger deletions in the GOI, which can be detected by PCR amplicon size difference between WT and mutated GOI (goi). B. If a restriction site (red letters) is present in the mutagenic region 3 bp in front of the PAM site (bold, underlined), mutations (blue X) lead to cleavage resistant PCR amplicons. C. The T7 Endonuclease assay allows cleavage of heteroduplexes, which are produced by imperfect DNA pairing due to mutations on one strand of the DNA amplicon (red asterisk).

    6. The restriction fragment length polymorphism (RFLP) assay is an elegant method to detect bp exchanges or small indels in your GOI. The advantage of this method is that heterozygous, biallelic and homozygous mutations can be detected (Figure 5B). The RFLP assay relies on the disruption of a restriction enzyme target site by Cas9 induced mutations. Therefore, the restriction enzyme target site should span the region at 3 bp in front of the PAM motive. For detection of mutations, the GOI is amplified with gene specific primers as described above. The PCR amplicon is then digested with the restriction enzyme of choice according to the manufacturer’s instructions. The sizes of the restriction fragments are analyzed by agarose gel electrophoresis. For the RFLP assay, amplify your GOI as described before. Separate the PCR products on an agarose gel and extract the PCR product from the gel using the GeneMATRIX 3 in 1–Basic DNA Purification Kit. Digest the PCR product with the suitable restriction enzyme according to the manufacturer’s instruction.
      Notes:
      1. WT gDNA should be used as negative control.
      2. In case you have a homozygous or a heterozygous mutation in your GOI, you either get only the undigested PCR amplicon or a mixture of undigested and digested PCR bands.
      3. It can be favorable if your PCR product contains more than one cleavage site of your restriction enzyme (which are not targeted by the Cas9) as it provides you with an internal control that the digest itself worked.
      4. If you have already detected mutations in your T1 plants by sequencing, you can use the sequenced PCR product as positive control for this assay.
      5. If you expect only small amounts of mutant alleles (e.g., in chimeric plants), you can enrich these amplicons by digesting the gDNA with the restriction enzyme before PCR amplification of your GOI. In this case, no other cut sites besides the one in the sgRNA target site should be present between your primer annealing sites.
    7. The T7 endonuclease assay and the Surveyor assay allow detection of small indels and bp exchanges in your target site (Figure 5C). First, the GOI is PCR amplified using a proofreading DNA polymerase. The PCR amplicon is then denatured and reannealed. In this step, ssDNA strands containing a mutation will anneal to DNA strands that do not contain a mutation, leading to non-perfectly matching dsDNA strands. These heteroduplexes are then recognized and cleaved by the T7 Endonuclease I or the Surveyor Nuclease. The restriction fragment pattern is analyzed by agarose gel electrophoresis. The advantage of these methods is that they do not require a restriction site within your gRNA target site. For details on this method, we refer to detailed protocols of the Endonuclease suppliers (e.g., https://www.neb.com/protocols/2014/08/11/determining-genome-targeting-efficiency-using-t7-endonuclease-i) and the experimental procedures of other research groups (Mao et al., 2013; Xie and Yang, 2013; Qiu et al., 2004).
      Note: These assays are not able to detect homozygous mutations as no heteroduplexes occur.

Data analysis

  1. There is no specific requirement for large-scale data analysis to generate Cas9-mediated knockout mutants. The mutagenic effect of the Cas9 system varies considerably depending on the plant species, Cas9 and sgRNA expression levels, and the sequence of the target loci (Bortesi and Fischer, 2015; Paul and Qi, 2016). Additionally, the Cas9 system produces chimeric plants due to the possibility of independent mutation events in each cell. Therefore, statistical analyses of mutation efficiencies are complicated and the results of one experiment might not be transferable to another one, especially if a different sgRNA is used.
  2. Sequencing data obtained from transformed plants can be compared to sequences from WT plants using alignment tools like MUSCLE with default settings (Edgar, 2004). Sequencing histograms can be analyzed using 4Peaks software.

Notes

  1. All analyses on gDNA level (sequencing, RFLP-assay, T7 assay) rely on the gDNA of a single leaf. If your knockout mutant does not display a visible phenotype and the Cas9 cassette is still integrated into the plant genome, it is difficult to tell whether you are analyzing a chimeric mutation only present in the leaf that you used for gDNA isolation or if your whole plant is already mutagenized. You should therefore consider the following aspects.
    1. Isolation of gDNA from several different leaves followed by GOI-amplification should result in similar sequencing histograms.
    2. The mutation should show a Mendelian inheritance pattern in the progeny generation.
    3. Loss of the Cas9 T-DNA cassette by backcrossing and/or segregation should not affect the mutation. The presence of the Cas9 T-DNA can be detected by PCR using primers FH61/FH201 and plant gDNA as template.

Recipes

  1. LB-medium
    10 g/L Bacto-tryptone (BD)
    5 g/L yeast extract (BD)
    5 g/L NaCl (Fisher Scientific)
    Optional: 15 g/L Bacto agar for plates (BD)
  2. YEP-medium
    10 g/L yeast extract (BD)
    10 g/L Bacto-peptone (BD)
    5 g/L NaCl
    Optional: 15 g/L Bacto agar for plates (BD)
  3. ½ MS-medium
    2.2 g/L Murashige & Skoog medium (Duchefa Biochemie)
    0.5 g/L MES (Carl Roth)
    Adjust pH to 5.7 with KOH (Carl Roth)
    Optional: 8 g/L plant agar for plates (Duchefa Biochemie)

Acknowledgments

This work was funded by the Cluster of Excellence on Plant Science (CEPLAS, EXC 1028) and the HHU Center for Synthetic Life Sciences (CSL). We thank Peter Hegemann and André Greiner (Humboldt-Universität zu Berlin) for providing us with Cas9 and sgRNA genes. We thank Holger Puchta, Felix Wolter and Dr. Nadine Rademacher for helpful discussions. We thank Franziska Kuhnert for critical reading of the manuscript. The protocol is adapted from Hahn et al. (2017).

References

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  6. Fauser, F., Schiml, S., and Puchta, H. (2014). Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana. Plant J 79(2): 348-359.
  7. Feng, Z., Mao, Y., Xu, N., Zhang, B., Wei, P., Yang, D. L., Wang, Z., Zhang, Z., Zheng, R., Yang, L., Zeng, L., Liu, X. and Zhu, J. K. (2014). Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis. Proc Natl Acad Sci U S A 111(12): 4632-4637.
  8. Hahn, F., Mantegazza, O., Greiner, A., Hegemann, P., Eisenhut, M. and Weber, A. P. (2017). An efficient visual screen for CRISPR/Cas9 activity in Arabidopsis thaliana. Front Plant Sci 8: 39.
  9. Heigwer, F., Kerr, G. and Boutros, M. (2014). E-CRISP: fast CRISPR target site identification. Nat Methods 11(2): 122-123.
  10. Hofgen, R. and Willmitzer, L. (1988). Storage of competent cells for Agrobacterium transformation. Nucleic Acids Res 16(20): 9877.
  11. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A. and Charpentier, E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096): 816-821.
  12. Lei, Y., Lu, L., Liu, H. Y., Li, S., Xing, F. and Chen, L. L. (2014). CRISPR-P: a web tool for synthetic single-guide RNA design of CRISPR-system in plants. Mol Plant 7(9): 1494-1496.
  13. Liang, G., Zhang, H., Lou, D. and Yu, D. (2016). Selection of highly efficient sgRNAs for CRISPR/Cas9-based plant genome editing. Sci Rep 6: 21451.
  14. Mao, Y., Zhang, H., Xu, N., Zhang, B., Gou, F. and Zhu, J. K. (2013). Application of the CRISPR-Cas system for efficient genome engineering in plants. Mol Plant 6(6): 2008-2011.
  15. Norris, S. R., Meyer, S. E. and Callis, J. (1993). The intron of Arabidopsis thaliana polyubiquitin genes is conserved in location and is a quantitative determinant of chimeric gene expression. Plant Mol Biol 21(5): 895-906.
  16. Pacher, M. and Puchta, H. (2016). From classical mutagenesis to nuclease-based breeding - directing natural DNA repair for a natural end-product. Plant J.
  17. Paul, J. W., 3rd and Qi, Y. (2016). CRISPR/Cas9 for plant genome editing: accomplishments, problems and prospects. Plant Cell Rep 35(7): 1417-1427.
  18. Qiu, P., Shandilya, H., D'Alessio, J. M., O'Connor, K., Durocher, J. and Gerard, G. F. (2004). Mutation detection using Surveyor nuclease. Biotechniques 36(4): 702-707.
  19. Salomon, S. and Puchta, H. (1998). Capture of genomic and T-DNA sequences during double-strand break repair in somatic plant cells. EMBO J 17(20): 6086-6095.
  20. Sambrook, J. and Russell, D. W. (2006). The inoue method for preparation and transformation of competent E. coli: "ultra-competent" cells. CSH Protoc 2006(1).
  21. Schiml, S., Fauser, F., and Puchta, H. (2014). The CRISPR/Cas system can be used as nuclease for in planta gene targeting and as paired nickases for directed mutagenesis in Arabidopsis resulting in heritable progeny. Plant J 80: 1139-1150.
  22. Schiml, S. and Puchta, H. (2016). Revolutionizing plant biology: multiple ways of genome engineering by CRISPR/Cas. Plant Methods 12: 8.
  23. Semenova, E., Jore, M. M., Datsenko, K. A., Semenova, A., Westra, E. R., Wanner, B., van der Oost, J., Brouns, S. J. and Severinov, K. (2011). Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence. Proc Natl Acad Sci U S A 108(25): 10098-10103.
  24. Weigel, D. and Glazebrook, J. (2009). Quick miniprep for plant DNA isolation. Cold Spring Harb Protoc 2009(3): pdb prot5179.
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简介

CRISPR / Cas9系统已成为植物及其以外基因编辑的强大工具。 我们已经开发了植物载体系统,用于拟南芥中多达两个不同靶位点的基因的目标Cas9依赖性诱变。 该方案描述了对于含有Cas9和sgRNA的基因的单个T-DNA载体的简单的1周克隆程序,以及植物中诱导突变的检测。 该方法可能适用于其他可转化植物物种。
【背景】CRISPR / Cas9系统(Cas9)提供了一种简单且广泛适用的方法来修改感兴趣的基因组区域,因此成为植物和其他生物体基因组编辑的首选工具(Schiml和Puchta,2016)。该系统依赖于可以通过短的人造单指导RNA分子(sgRNA)向基因组DNA序列引导的化脓性链球菌(Cas9)的细菌Cas9核酸酶(Jinek等人, ,2012),在那里它创建一个双链断裂(DSB)。然后通过植物细胞的固有DNA修复机制修复这些DSB。在这里,可以区分两个主要途径(Salomon和Puchta,1998)。 (i)与DSB位点高度同源的DNA分子可用作修复模板。可以利用这种同源性定向修复(HDR)方法在DSB的现场引入特定的序列(Schiml等人,2014; Baltes and Voytas,2015)。然而,由于这些序列的低融合率,植物中HDR介导的基因编辑仍然具有挑战性。 (ii)更容易和更有效的方法是使用植物的非同源末端连接(NHEJ)修复途径,其是大多数植物中的主要修复途径,例如拟南芥(Arabidopsis thaliana)(拟南芥拟南芥。由于NHEJ易于出错,几个碱基对(bp)的少量插入或缺失(bp)经常发生在DSB位点,导致移码突变和基因敲除(Pacher和Puchta,2016)。在这里,我们提供了在拟南芥模拟植物中靶向基因敲除的详细方案,包括用于含有Cas9和sgRNA的植物载体系统的简单的1周克隆方案,然后是拟南芥< em>突变的转化和检测。

关键字:CRISPR/Cas9, 基因组编辑, 拟南芥, 植物, 基因敲除

材料和试剂

  1. 1.5ml微量离心管(SARSTEDT,目录号:72.690.001)
  2. 200μlPCR管(Labomedic,目录号:2081644AA)
  3. 培养皿(SARSTEDT,目录号:82.1472)
  4. 2 ml微量离心管(SARSTEDT,目录号:72.691)
  5. 20μl移液器吸头(SARSTEDT,目录号:70.1116)
  6. 200μl移液器吸头(SARSTEDT,目录号:70.760.012)
  7. 1,000μl移液器吸头(SARSTEDT,目录号:70.762.010)
  8. 农杆菌(Agrobacterium tumefaciens)(根癌农杆菌)菌株GV3101 :: pMP90
  9. 拟南芥种子(Col-0)
  10. 向量(见图1)


    图1. pUB-Cas9(A)和pFH6(B)的载体图。 pFH6用于整合sgRNA支架上游的20bp靶序列并在拟南芥U6-26 RNA聚合酶III启动子的控制下。然后将整个sgRNA盒通过吉布森克隆转移到用于植物转化的二元T-DNA载体pUB-Cas9(含有在Ubiquitin10启动子控制下的Cas9 基因) 。使用SnapGene Viewer生成地图( http://www.snapgene.com/ products / snapgene_viewer / )。

    1. 含有密码子优化(UBIQUITIN10启动子)密码子优化的植物T-DNA Cas9载体pUB-Cas9(GenBank登录号KY080691)表达Cas9 基因,用于细菌选择的卡那霉素抗性盒和作为植物选择标记的潮霉素抗性盒(Hahn等人,[2017];可在Addgene获得,目录号:86556)
    2. 含有拟南芥U6-26启动子的sgRNA亚克隆载体pFH6(GenBank登录号KY080689),20bp原始序列的整合位点,sgRNA支架和氨苄青霉素抗性盒(Hahn等) [2017];可在Addgene获得,目录号:86555)
      注意:pFH6在U6-26启动子的3'末端和原始聚合酶整合位点之间包含另外的9bp片段(GTCCCTTCG)。在几个实验中,我们可以显示这不影响基因编辑活动。然而,我们还克隆了新版本的亚克隆载体,而没有附加片段(pFH6_new),其在初步实验中显示出高的切割活性,并且可以从我们获得。该版本仅在U6-26启动子的3'末端和原始聚合酶整合位点之间仅含有另外的鸟嘌呤(G),其允许任何20bp原始样品的整合而不限制G作为第一个碱基对(比较例如Fauser等[2014])。 pFH6_new的克隆策略类似于本协议中描述的克隆策略,唯一的区别是克隆您的20 bp原始序列序列的正向引物含有不同的重叠,并且在开始时缺少初始G的需要(ATTG-N20,比较程序部分)。
  11. 感受态的大肠杆菌(E.coli)细胞(例如,,Mach1 TM感受态细胞,Thermo Fisher Scientific,Invitrogen TM ,目录号:C862003)
  12. I-HF + CutSmart缓冲液(New England Biolabs,目录号:R3539S)
  13. 蒸馏H 2 O O
  14. 质粒微型制备试剂盒和琼脂糖凝胶提取试剂盒(例如,1碱性DNA纯化试剂盒中的GeneMATRIX 3,Roboklon,目录号:E3545)
  15. T4 DNA连接酶与10x连接缓冲液(New England Biolabs,目录号:M0202S)
  16. 氨苄青霉素(Amp)(Carl Roth,目录号:K029.2)
  17. 引物(见表1)

    表1.寡核苷酸列表


  18. 5x Green GoTaq反应缓冲液(Promega,目录号:M791A)
  19. dNTP(每种10mM)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:R0192)
  20. GoTaq G2聚合酶(Promega,目录号:M7841)或其他标准PCR聚合酶
  21. III-HF + CutSmart缓冲液(New England Biolabs,目录号:R3104S)
  22. Kpn I-HF + CutSmart缓冲液(New England Biolabs,目录号:R3142S)
  23. Phusion高保真聚合酶(New England Biolabs,目录号:M0530S)或其他校对聚合酶
  24. 吉布森装配克隆试剂盒(New England Biolabs,目录号:E5510S)
  25. 硫酸卡那霉素(Kan)(Carl Roth,目录号:T832.4)
  26. 利福平(Rif)(Molekula,目录号:32609202)
  27. 硫酸庆大霉素(Gent)(Carl Roth,目录号:0233.3)
  28. 潮霉素B(Hyg)(Carl Roth,目录号:CP12.2)
  29. T7内切核酸酶I(可选; New England Biolabs,目录号:M0302S)
  30. LB培养基(琼脂平板和液体),补充200μg/ ml氨苄青霉素(参见食谱)
  31. LB培养基(琼脂平板和液体),补充有30μg/ ml硫酸卡那霉素(参见食谱)
  32. 补充有150μg/ ml利福平,50μg/ ml硫酸庆大霉素,50μg/ ml卡那霉素的YEP培养基(琼脂平板和液体)(参见食谱)
  33. 补充有33.3μg/ ml潮霉素B(参见食谱)的1/2 MS培养基琼脂平板

设备

  1. 琼脂糖凝胶电泳设备(例如,VWR,Peqlab,型号:PerfectBlue 凝胶系统Mini M,目录号:700-0434)
  2. 细菌培养箱(28℃,37℃,例如,Memmert,型号:IN55)和振荡器(28℃,37℃;例如,Eppendorf,New Brunswick TM ,型号:Innova ® 44,目录号:M1282-0002)
  3. 加热块(例如,Eppendorf,型号:Thermomixer Compact,目录号:T1317-1EA)
  4. PCR循环仪(例如,Thermo Fisher Scientific,Applied Biosytems TM,型号:Veriti 96孔热循环仪,目录号:4375786) br />
  5. 10μl移液管(例如,,Pipetman Neo P10N,Gilson,目录号:F144562)
  6. 20微升移液管(例如,,P20N,Gilson,目录号:F144563)
  7. 200μl移液管(例如,,P200N,Gilson,目录号:F144565)
  8. 1,000μl移液管(例如,,P1000N,Gilson,目录号:F144566)
  9. 植物生长室(例如,CLF Plant Climatics,Percival Scientifici,型号:AR-66L)

软件

  1. SerialCloner
  2. Cas-OFFinder
  3. 4Peaks软件
  4. MUSCLE(可选)

程序

  1. 基因敲除的sgRNA序列的设计
    sgRNA的设计可能是Cas9介导的基因编辑最关键的一步。因此,应仔细选择Cas9目标站点。 2014年的许多在线工具(Heigwer等人,2014; Lei等人,2014; Xie等人,2014)帮助找到合适的然而,我们仍然在标准分子生物学软件(例如,SerialCloner, http://serialbasics.free.fr/Serial_Cloner.html )。选择目标网站时,应考虑以下几个方面:
    1. 在两条链上搜索您感兴趣的基因( GOI )以获取GN 19 NGG序列,以找到推定的目标位点。 NGG是化脓性链球菌Cas9原始相邻动机(PAM),其必须存在于目标基因座中以有效地结合Cas9蛋白(Jinek等人, 2012)。 GN 19 代表20bp长的原始序列基序,定义要在其中创建突变的靶位点。这个标记sgRNA分子5'末端的这个20 bp序列应该以G开头,因为这是在U6-26启动子控制下sgRNA转录的前提条件, em>拟南芥。
      注意:如果没有合适的目标序列,您可以将G添加为21st bp,即使目标基因座中没有互补碱基。这些sgRNA可能仍然导致在靶位点的切割(例如,Hahn等,2017)。但是,如果可能,我们建议使用规范的20 bp原始序列。
    2. 选择您的GOI 外显子中的目标位点,该目标位置优选接近编码序列的5'末端,因为该区域的突变更可能破坏蛋白质的功能。如果您知道蛋白质的高度重要的结构域,例如核酸结合位点,则可能需要在这些区域中产生突变。
    3. 考虑选择几个不同的目标网站,因为并不是所有的sgRNA都能很好地工作(Liang等人,2016)。此外,您可能需要同时使用两个sgRNA(参见克隆策略)来创建大量的缺失,这可能会产生更大的影响。
    4. 搜索推定的非目标网站,以避免敲除共享20 bp目标网站或高度相似的网站的基因。为此,我们使用可在,2014) .net / cas-offinder /“target =”_blank“> http://www.rgenome.net/cas-offinder/ 。在这里,我们使用以下设置:
      PAM型:来自化脓性链球菌的SpCas9:5'-NRG-3'(R = A或G)
      目标基因组:拟南芥(TAIR 10)
      不匹配号码:3
      在参考基因组中搜索您的20 bp目标位点后,该程序将显示所有类似的站点,最多3个不匹配。如果您有多个不匹配的命中(一个总是您的 GOI ),则应选择其他目标站点。如果您有1-3个错配,您应该考虑到Cas9也可能会诱变这些位点,特别是如果这些错配位于20 bp序列的5'末端(Semenova等人,2011年) )。尽管对Cas9的亲和力将低于您的GOI ,但植物似乎一般较少受到目标定位的影响(Feng 等人,2014),我们建议在敲除植物中仔细测序这些目标位点,以确保这些基因不存在突变
    5. 考虑使用也含有市售限制酶的识别位点的20bp靶位点。限制酶的靶位点应该跨越Cas9切割位点(PAM前面3 bp)。这将允许您使用限制酶长度多态性检测来检测您的GOI 中的突变。

  2. 将20 bp原始序列亚克隆到pFH6中
    1. 概述如何将原始序列亚克隆到pFH6中,如图2所示
    2. 为您的候选靶序列N <20>订购正向和反向引物,重叠用于克隆,并将其稀释至10μM的最终浓度(f.c.)。
      正向引物:TTCG-GN
      反向引物:AAAC-N19(反向互补)C(参见图2)


      图2.原生质体序列在pFH6中的亚克隆。通过连接二聚引物可将20bp原始序列序列整合到sgRNA支架和U6-26启动子(U6p)之间进入 我消化的pFH6载体。

    3. 将10μl每个引物稀释液吸取到1.5 ml微量离心管中并混合
    4. 加热底漆混合至98℃,10分钟,然后55℃,10分钟,然后通过关闭加热块缓慢冷却至RT(例如,)。通过这样做,将产生包括您的原始序列(=退火引物)的双链DNA。
    5. 在37℃下将pFH6与bbs I-HF消化3小时。


    6. 在琼脂糖凝胶上运行限制性消化,并使用1碱基DNA纯化试剂盒中的GeneMATRIX 3从凝胶中提取载体骨架(3,612bp)。
    7. 在4℃下将经消化的pFH6的退火引物连接过夜

    8. 转换胜任的电机大肠杆菌细胞与连接反应,并将转化的细胞在补充有Amp。
      的LB琼脂平板上扩散 注意:通常,电镀100μl的转化混合物就足够了。转基因大肠杆菌细胞的方案是根据要求提供的。
    9. 验证八。大肠杆菌菌落通过菌落PCR,使用引物M13R和您的原标本引物TTCG-N <20>进行以下设置:


      PCR程序:


      注意:我们建议使用pFH6作为阴性对照。我们有时在阴性对照中观察到预期条带大小(281 bp)的片段,因为原始标记引物可能部分结合BbsI位点。然而,阳性菌落通常具有更强的PCR产物信号,因此可以区分于阴性克隆。
    10. 在补充有Amp的LB培养基中接种阳性菌落并生长过夜
    11. 使用1碱基DNA纯化试剂盒中的GeneMATRIX 3纯化质粒
    12. 使用引物M13R对质粒进行序列并验证原始序列插入序列(表1)
  3. 使用吉布森装配克隆最终植物T-DNA Cas9载体
    1. 概述如何克隆最终植物T-DNA Cas9载体在图3中给出。


      图3.产生具有用于Cas9诱导诱变的所有必需组分的植物T-DNA载体。包含20bp靶位点的sgRNA盒从pFH6扩增并整合到Kpn中> I / em> Hind III-消化的pUB-Cas9通过吉布森装配。所得载体包含在UBIQUITIN10启动子(UB10p)和潮霉素(UG10p)控制下的sgRNA盒以及Cas9基因, )抗性基因作为选择标记。 RB,右T-DNA边界; PolyT,多胸苷转录终止子; NLS,核定位信号; NosTm,胭脂碱合酶终止子; CaMV35S,花椰菜花叶病毒2x35S启动子; 35STm,35S终止子; LB,左T-DNA边界。

    2. 消化载体pUB-Cas9与Hind III-HF和Kpn I-HF在37℃下培养3小时。


      注意:pUB-Cas9质粒的高效消化对于进一步克隆步骤至关重要。如果您在随后的克隆步骤中遇到问题,请尝试用新鲜的酶和更长的孵育时间消化pUB-Cas9。
    3. 在琼脂糖凝胶上排除限制性消化反应,并使用Gene-Basic DNA纯化试剂盒中的GeneMATRIX 3从凝胶中提取载体骨架(14,097bp)。
    4. 使用引物FH41和FH42(表1)和Phusion高保真聚合酶扩增具有靶序列的pFH6的sgRNA盒(预期大小:771bp)和以下设置:


      PCR程序:


    5. 在琼脂糖凝胶上运行PCR反应,并使用Gene-Basic DNA纯化试剂盒中的GeneMATRIX 3从凝胶中提取sgRNA盒(771bp)。
    6. 组装pUB-Cas9骨架和sgRNA盒,根据制造商的说明书,孵育时间为1小时,使用Gibson Assembly Master Mix生成最终载体pUB-Cas9- @ GOI。
      注意:我们提出了最终结构的命名,其中目标基因由@ -sign标记。因此,针对拟南芥GL1基因设计的载体称为pUB-Cas9- @ GL1(Hahn等,2017)。
    7. 转变主管大肠杆菌细胞与吉布森反应混合物,并将转化的细胞在补充有Kan的LB琼脂平板上铺展。
      注意:新英格兰Biolabs在吉布森装配克隆试剂盒中提供高度感受态的大肠杆菌细胞。然而,我们也使用我们实验室生产的感受态大肠杆菌细胞。由于这些通常的能力较差,我们建议在LB琼脂平板上电镀所有转化的细胞。在Sambrook和Russell(2006)中可以找到用于产生感兴趣的MachI大肠杆菌的方案。
    8. 使用引物M13F和FH179(表1)通过菌落PCR验证8个阳性菌落,并使用以下设置:


      PCR程序:


      注意:预期产品大小为779bp,pUB-Cas9载体可用作阴性对照。
    9. 将阳性菌落接种到补充有Kan的LB培养基中,并使之生长过夜。
    10. 使用1碱基DNA纯化试剂盒中的GeneMATRIX 3纯化质粒
    11. 使用引物FH179和M13F(表1)对质粒进行序列测序以验证原始序列插入 注意:载体系统还允许将两个不同的sgRNA克隆到pUB-Cas9载体骨架中,以在靶基因组中诱导大的缺失或一次破坏两个基因。因此,如所述,两个原始序列必须分别克隆到pFH6中。然后,使用引物FH41和FH254,必须从其亚克隆pFH6构建体扩增第一个sgRNA盒(表1)。使用上述PCR条件,第二个sgRNA盒必须用FH42和FH255扩增(表1)。使用Gibson Assembly Master Mix,两个盒可以在一个步骤中整合到KpnI-HF / HindIII-HF消化的pUB-Cas9中。由于两个片段整合到消化的载体中,与一个片段的整合相比,菌落的量通常较低。然而,正确组装的载体的比例是可比的。菌落PCR可以用相同的引物进行,但使用较长的延长时间(1.5分钟),因为PCR反应扩增了两个sgRNA盒(预期产物大小:1,512bp)。应按上述方式进行排序。

      所有克隆步骤总结在表2中:

      表2.用于克隆靶向基因敲除的植物T-DNA Cas9载体的时间表


  4. 使用pUB-Cas9- @ GOI进行拟南芥转化
    1. 转变主管根瘤土壤杆菌细胞与1μg的pUB-Cas9- @ GOI接种,并将转化的细胞扩散到补充有Rif / Gent / Kan的YEP琼脂平板上。
      注意:在其他地方描述了用于产生根癌农杆菌细胞和转化根癌农杆菌细胞的方案(Hofgen和Willmitzer,1988)。
    2. 在30℃生长两天后,使用引物FH61和FH201通过菌落PCR验证质粒在至少三个菌落中的存在(表1)。为了验证菌落,用移液管吸头取一个菌落的一半,并将细胞悬浮在含有100μlddH 2 O的1.5ml微量离心管中。加热至98℃10分钟,并使用2μl作为PCR的DNA模板。将另一半的菌落在补充有Rif / Gent / Kan的新鲜YEP琼脂平板上。在30°C生长细胞一天,然后在4°C储存。对于菌落PCR,请使用以下设置:


      PCR程序:


      注意:预计产品大小为1,003 bp。
    3. 使用农杆菌介导的T-DNA转移与花浸渍方法(Clough and Bent,1998)一起转化拟南芥植物。
      注意:每个建筑物,我们通常用五个拟南芥植物改造四盆。我们建议在转化前删除所有现有的长角色,以避免以后对转化事件的过度筛选。
    4. 在补充有Hyg的1/2强度MS琼脂平板上选择T1代植物以获得成功的T-DNA插入事件。通过使用前面描述的参数的引物FH61 / FH201,通过分离叶基因组DNA(gDNA)和随后的PCR来验证T-DNA的整合。
      注意:通过异丙醇沉淀的植物gDNA提取的详细方案可以在别处找到(Weigel和Glazebrook,2009)。

  5. 检测Cas9诱导突变
    我们的载体系统中的Cas9蛋白质在拟南芥的UBIQUITIN10启动子的控制下转录,其允许Cas9在植物的几乎所有组织中表达(Norris et al。等人,1993)。由于Cas9系统的编辑机制在每个植物细胞中独立工作,所以T1代植物通常是在不同细胞中具有不同基因型的嵌合体。只有突变发生在种系细胞中,才能出现具有统一基因型的后代植物。这通常是进一步分析的前提条件。因此(由于T1代可用植物数量有限),检测突变的筛选方法应适用于T2或后代。我们建议筛选三到四个独立转化系的后代,因为由于T-DNA盒的整合位点,突变效率可能在不同的线之间变化。为了选择有希望的T1线进一步筛选,我们还搜索了T1代的突变事件。我们建议以这种方式筛选植物进行突变:
    1. 使用Phusion聚合酶扩增来自叶gDNA的基因特异性引物在您的GOI (上游和下游±300-500bp)的Cas9切割位点周围的基因组区域。


      PCR程序:


    2. 在琼脂糖凝胶上运行PCR产物,并使用1碱基DNA纯化试剂盒中的GeneMATRIX 3从凝胶中提取您的 GOI 。
    3. Sanger测序的序列PCR产物。当Cas9活跃且至少在叶细胞的一部分发生突变时,测序直方图将显示WT序列和突变序列的混合物(图4)。


      图4.来自嵌合叶gDNA的扩增靶基因的示例性测序结果直方图在Cas9切割位点(PAM动机NGG前面的3bp)处出现双峰,指示突变事件< em> GOI 在叶细胞的一部分。

      Cas9系统的效率差异很大,其诱变效应依赖于植物种类,Cas9和sgRNA表达水平以及目标基因座序列(Bortesi and Fischer,2015; Paul and Qi,2016)等各个方面, 。因此,很难预测有多少植物必须筛选以鉴定遗传突变。我们建议每行开始筛选30株植物。可以如前所述通过扩增和您的GOI 的Sanger测序来进行T2代突变的筛选。由于这可能变得昂贵,几种方法可以检测基因组水平上的突变而不进行测序
    4. 如果您希望由于您的基因敲除而导致特定的表型,则筛选突变的最简单的方法就是首先显示视觉屏幕。这些表型可能是植物在选择培养基上的解剖学或存活的变化。然后如上所述在基因组水平上分析显示表型的植物。
    5. 如果您同时在您的GOI 中定位两个网站,则同时使用两个sgRNA切割可导致大量缺失,这可能会在您的GOI扩增PCR过程中被检测到。 >(图5A)。理论上也可以使用一种sgRNA进行较大的缺失,但是我们通常会发现在几bp范围内的小变体,通常使用琼脂糖凝胶电泳不能通过PCR产物大小差异来检测。
      注意:如果期望只有少数细胞含有预期的突变(例如,在嵌合植物中),则可以通过用限制性酶切割未突变的gDNA来提高突变体等位基因的PCR扩增效率,该限制酶切割两个sgRNA靶标您的GOI的PCR扩增前的位点。


      图5.使用正向(FW)和反向(RV)引物(绿色箭头)扩增GOI后,允许检测诱导突变的方法的总结。 :一种。两种sgRNA的使用可以在GOI 中诱导更大的缺失,其可以通过WT和突变的GOI( )之间的PCR扩增子大小差异来检测, 。 B.如果在PAM位点前面3 bp的突变区域存在限制性位点(红色字母)(粗体,下划线),则突变(蓝色X)导致切割抗性PCR扩增子。 C.T7内切核酸酶测定允许异源双链体的切割,这是由于一条链上的突变而由不完美的DNA配对产生的DNA扩增子的一条链(红色星号)。

    6. 限制性片段长度多态性(RFLP)测定法是检测您的GOI 中的bp交换或小插入物的优雅方法。该方法的优点是可以检测到杂合,双重和纯合突变(图5B)。 RFLP测定依赖于Cas9诱导的突变导致的限制酶靶位点的破坏。因此,限制酶靶位点应跨越PAM动机前面3 bp的区域。为了检测突变,如上所述用基因特异性引物扩增GOI 。然后根据制造商的说明书,用选择的限制酶消化PCR扩增子。通过琼脂糖凝胶电泳分析限制性片段的大小。对于RFLP测定,如前所述放大您的GOI 。在琼脂糖凝胶上分离PCR产物,并使用Gene-Basic DNA纯化试剂盒中的GeneMATRIX 3从凝胶中提取PCR产物。根据制造商的说明书,用适当的限制性酶消化PCR产物。
      注意:
      1. WT gDNA应用作阴性对照。
      2. 如果您的GOI中有纯合子或杂合突变,您只需获取未消化的PCR扩增子或未消化和消化的PCR条带的混合物。
      3. 如果您的PCR产物含有多于一个限制性内切酶切位点(Cas9不是靶向的),那么PCR酶产物可以提供消化本身的内部控制功能。
      4. 如果您已经通过测序检测到T1植物中的突变,则可以使用测序PCR产物作为该测定的阳性对照。
      5. 如果您只想要少量的突变体等位基因(例如嵌合植物),则可以通过在您的GOI PCR扩增前用限制酶消化gDNA来丰富这些扩增子。在这种情况下,您的引物退火部位之间不应存在除sgRNA靶位点之外的其他切割位点。
    7. T7内切核酸酶测定和Surveyor测定允许检测目标位点中的小的indel和bp交换(图5C)。首先,使用校对DNA聚合酶PCR扩增 GOI 。然后将PCR扩增子变性并重新退火。在该步骤中,含有突变的ssDNA链将退火不含突变的DNA链,导致非完全匹配的dsDNA链。然后这些异源双链体被T7内切核酸酶I或测量员核酸酶识别和切割。通过琼脂糖凝胶电泳分析限制性片段模式。这些方法的优点是它们不需要您的gRNA靶位点内的限制性位点。有关此方法的详细信息,请参阅Endonuclease供应商的详细协议(例如, https://www.neb.com/protocols/2014/08/11/determining-genome-targeting - 效率 - 使用-17-内切核酸酶-II)和其他研究组的实验程序(Mao等人,2013; Xie和Yang,2013; Qiu等人。,2004) 注意:这些测定不能检测到纯合突变,因为没有异源双链体发生。

数据分析

  1. 大规模数据分析没有具体要求来产生Cas9介导的敲除突变体。 Cas9系统的诱变效应根据植物种类,Cas9和sgRNA表达水平以及目标基因座序列(Bortesi和Fischer,2015; Paul和Qi,2016)而有很大差异。此外,由于每个细胞中独立突变事件的可能性,Cas9系统产生嵌合植物。因此,突变效率的统计分析是复杂的,一个实验的结果可能不能转移到另一个实验中,特别是如果使用不同的sgRNA。
  2. 从转化植物获得的测序数据可以使用具有默认设置的对准工具(如MUSCLE)与来自WT植物的序列进行比较(Edgar,2004)。可以使用4Peaks软件分析排序直方图。

笔记

  1. 所有关于gDNA水平的分析(测序,RFLP测定,T7测定)均依赖于单叶的gDNA。如果您的敲除突变体不显示可见的表型,并且Cas9盒仍然整合到植物基因组中,则很难分辨您是否仅在您用于gDNA分离的叶中存在嵌合突变,或者如果您的整个植物已被诱变。因此,您应该考虑以下几个方面。
    1. 从几个不同叶子中分离gDNA,然后进行扩增,应该产生类似的测序直方图。
    2. 突变应在后代中显示孟德尔遗传模式。
    3. 通过回交和/或分离的Cas9 T-DNA盒的损失不应影响突变。使用引物FH61 / FH201和植物gDNA作为模板,可以通过PCR检测Cas9 T-DNA的存在。

食谱

  1. LB-medium
    10 g / L细菌胰蛋白胨(BD)
    5 g / L酵母提取物(BD)
    5g / L NaCl(Fisher Scientific)
    可选:15g / L板式(B)的Bacto琼脂
  2. YEP-medium
    10g / L酵母提取物(BD)
    10 g / L细菌蛋白胨(BD)
    5克/升NaCl
    可选:15g / L板式(B)的Bacto琼脂
  3. ½MS-medium
    2.2 g / L Murashige&amp; Skoog培养基(Duchefa Biochemie)
    0.5 g / L MES(Carl Roth)
    用KOH(Carl Roth)将pH调节至5.7 可选:8克/升植物琼脂(Duchefa Biochemie)

致谢

这项工作由植物科学卓越丛书(CEPLAS,EXC 1028)和HHU合成生命科学中心(CSL)资助。我们感谢Peter Hegemann和AndréGreiner(Humboldt-Universitätzu Berlin)为我们提供了Cas9和sgRNA基因。我们感谢Holger Puchta,Felix Wolter和Nadine Rademacher博士进行了有益的讨论。感谢Franziska Kuhnert对手稿的批判性阅读。该协议改编自Hahn等人(2017)。

参考

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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Hahn, F., Eisenhut, M., Mantegazza, O. and Weber, A. P. (2017). Generation of Targeted Knockout Mutants in Arabidopsis thaliana Using CRISPR/Cas9. Bio-protocol 7(13): e2384. DOI: 10.21769/BioProtoc.2384.
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