Manipulation of mouse genome with minimal off-target by microinjection of one-cell embryos with paired sgRNAs anf nickase基因敲除小鼠系统
While CRISPR/Cas9 technique has been widely used in genome editing regarding multiple organisms, the off-target effect can’t be neglected. Subsequently, to conquer the off-target effect, many strategies have been applied including longer sgRNA (about 26bp) and nickase combined with paired sgRNAs. Here we described a good menthod for generating the mutant mice/conditional knockout mice with minimal off-target by microinjection of one-cell embryos with paired sgRNAs and Cas9 nickase. Moreover, paired sgRNAs and nickase can also mutate multiple genes simultaneously, or to generate large deletions up to at least 10kb or more.Comparison of Cas9 and nickase (Cas9D10A)Figure 1. Cas9 nickase strategy. Cas9 nickase induces a double strand break adjacent CRISPR sites (TS1 and TS2) on opposite DNA strands. Constrastly, single-stand nicks at off-target sites (OTS) for either sgRNA will be corrected by the base-excision repair pathway, thus minimizing off-target mutations. P, PAM site.Plasmids used in the protocolT7-Nickase(Cas9-D10A):T7-sgRNA:ReagentsPlasmids: T7-Nickase(Cas9-D10A) and T7-sgRNAmMESSAGE mMACHINE®T7 Ultra kit (Ambion, AM1345)MEGAshortscript TM Kit (Ambion, AM1354)RNeasy Mini Kit (QIAGEN, 74104)MEGAclear TM Kit (Ambion, AM1908)RNAsecureTM Reagent (Ambion, AM7005)QIAprep Spin Miniprep Kit (QIAGEN, 27104)MiniElute PCR Purification Kit (QIAGEN, 28004)BsaI (NEB, R0535S)AgeI (NEB, R0552S)DraI (TAKARA, D1037A)T4 DNA Ripid ligation Kit (NEB,M2200S)PMSG (Sansheng, China, 50IU/ml in normal saline, Aliquot and store at -80℃)HCG (Sansheng, China, 50IU/ml in normal saline, Aliquot and store at -80℃)EmbryoMax® Injection Biffer (Millipore, MR-095-10F)Proteinase K(Merck,1245680100, 20 mg/ml in water, Aliquot and store at -20℃)Lysis buffer (10 μM Tris-HCl, 0.4 M NaCl, 2 μM EDTA, 1% SDS)Phenol (Tris-saturated), Chloroform and alcoholPCR clearing Kit (Axygen, AP-PCR-50)T7EN1 (NEB, M0302L)PrimerSTAR HS DNA Polymerase (TAKARA, DR010A)pMD19T-vector kit (TAKARA, 3271)EquipmentCentrifuge (RT and 4℃)VortexOne Drop OD-1000+ SpectrophotometerThermocyclerThermomixerThermo-controlled water bath(37℃,42℃ and 58℃)ProcedureConstruction of sgRNA expression vectors- Design of paired sgRNA oligos.
Select paired sgRNAs in a tail-to-tail orientation and separated by 10-30 bp, which have the sequence 5’-CCN(52-72)GG. All possible paired sites for mouse and human exons are available on website (http://www.sanger.ac.uk/htgt/wge/). For each sgRNA, the 5’-GGN(19)GG motif is preferred, however, 5’-GN(20)GG or 5’-N(21)GG are also satisfactory. BLAT or BLAST the sgRNA target sites in UCSC or ENSEMBL genome browsers to find those with few or no highly related sites in the genome.Order oligos as below:For 5’-GGN(19)GG motift -2℃/s; 85-25℃ at -0.1℃/s; hold at 4℃.2.Preparation of T7-sgRNA plasmid.
2 μg T7-sgRNA plasmid. 1 μl CutSmart Buffer 1 μl BsaI3. Add H2O up to 50 μl and incubAnnealing oligos prior to cloning.
4.5μl Top Oligo (100 μM) 4.5μl Bottom Oligo (100 μM)1μl NEB buffer 2Annealing oligos using a thermocycler with the following program:95℃,5 min; 95-85℃ aate at 37℃ for 2 h with occasional shake. Purify the digeston product using MinElute PCR Purification Kit.4.Ligation of annealed oligos with BsaI-digested T7-sgRNA
4 μl annealed oligos 2 μl (25 ng/μl) digested T7-sgRNA 10×NEB ligation buffer 1 μl ddH2O 2 μl NEB T4 DNA ligase 1 μl Up to 10 μl Incubate at 22℃ for 30 min5.Transformation and plate on Kan+ plate (50 μg/ml).
6.Confirm correct Insertion of sgRNA oligos by sequencing using M13-47 primer.
7.Mini-prep T7-sgRNA plasmid using QIAprep Spin Miniprep Kit.
Transcription of sgRNAs in vitro- Ensure that reagents, tubes and tips are RNase-free and that the work is done in a ribonuclease-free enviroment.
- Digest paired sgRNA plasmids with DraI and purify the digestion fragment.
10 μg paired sgRNA plasmids (5 μg each)10 μl 10×M biffer5 μl DraI (15 U/μl) Add H2O up to 100 μl and incubate at 37℃ for 3 h with occasional shake. Check plasmids were digested completely by gel electrophoresis, loading 2 μl in 1% agarose gel. Two bands (1621 and 1152 bp) will be observed. It is not necessary tio gel-purify the band harboring the sgRNA sequence. Add 4 μl RNAsecure and incubate at 60℃ for 10 min in a thermomixer.Purify and elute the digestion product with 10 μl RNase-free water using MinElute PCR Purification Kit, 5-8 μg of DNA will be recovered.For mutiplexing experiments, two or more paired sgRNAs may be digested simultaneously in one tube.Alternatively, the transcription template containing the T7 promoter sgRNA sequence may be prepared by PCR amplification from a bacterial colony using the following primers and PCR program:sgRNA-For: 5’-TCTCGCGCGTTTCGGTGATGACGGsgRNA-Rev: 5’-AAAAAAAGCACCGACTCGGTGCCACTTTTTCProgram:94℃,5 min; ((98 ℃ ,10s; 72-62 ℃, -1℃/cycle, 15s; 72 ℃, 30s) 10 cycles, (98℃, 10s; 62 ℃, 15s; 72 ℃, 30s) 25 cycles); 72 ℃, 5 min; hold at 4℃.Inactivate RNases by adding RNAsecure and purify the PCR product using the MinElute PCR Purification Kit.- In vitro transcription of sgRNAs using MEGAshortscriptTM Kit.
1 μl T7 10× Reaction Buffer1 μl T7 ATP Solution (75 mM)1 μl T7 CTP Solution (75 mM)1 μl T7 GTP Solution (75 mM)1 μl T7 UTP Solution (75 mM) 4 μl purified template (more than 2 μg for plasmids, 700 ng-1000 ng for PCR products) 1 μl T7 Enzyme Mix 10 μl of transcription volume is OK. Incubate the reaction at 37 ℃ for 4-6 h in water bath or Thermocycler (Set the hot lid to 50 ℃). Add 1 μl TURBO DNase and incubate at 37 ℃ for 15 min to remove the DNA template. - Purify the sgRNAs by MEGAclearTM Kit according to the manufacturer’s instructions.
RNA elution option 2 in the manual is preferred. Precipitate with 5 M Ammonium Acetate and ethanol. Resuspend the pellet using the 30 μl RNase free water. 20-50 μg RNA will be obtained depending on the quality of DNA template.5.Assess sgRNA yield using the One Drop OD-1000+ Spectrophotometer (or equivalent) and sgRNA quality by gel electrophoresis. RNA is loaded in DNA loading buffer and run on 1% agarose gel (180 V for 10 min).6.Aliquot and store at -80 ℃. The sgRNAs are stable for one year without freeze-thaw cycles.Transcription of Nickase (Cas9-D10A) in vitro- Ensure that reagents, tubes and tips are RNase-free and that the work is done in a ribonuclease-free enviroment.
- Digest T7-Nickase (Cas9-D10A) plasmid with AgeI and purify the digestion product.
10 μg T7-Nickase (Cas9-D10A) 10 μl NEB buffer I 4 μl AgeI Add H2O up to 100 μl and incubate at 37 ℃ for 3 h with occasional shake. Add 4 μl RNAsecure and incubate at 60 ℃ for 10 min in a thermomixer.Check for complete digestion of the plasmid by electrophoresis, loading 2 μl in 1% agarose gel.Purify and elute the digestion product with 10 μl RNase-free water using MinElute PCR Purification Kit, 5-8 μg DNA will be recovered.- In vitro transcribe Cas9-D10A using mMESSAGE mMACHINE® T7 Ultra Kit according to the manufacturer’s instruction.
- Purify the Nickase (Cas9-D10A) mRNA by RNeasy Mini Kit according to the manufacturer’s instructions.
- Assess sgRNA yield using the One Drop OD-1000+ Spectrophotometer (or equivalent) and sgRNA quality by gel electrophoresis. RNA is loaded in DNA loading buffer and run on a 1% agarose gel (180V for 10 min). A yield of 30-60 μg mRNA is expected.
Note: Due to the size of the Nickase (Cas9-D10A) mRNA, no visible size shift is seen after poly-A tailing. The mRNA quality is good if a smear is not observed.- Aliquot and store at -80 ℃. Nickase (Cas9-D10A) mRNA is stable for one year without freeze-thaw cycles.
Collection of zygotes- Superovulate 4-week-old female C57BL/6J (about 12-14g) mice by intraperitoneal injection with PMSG (5 IU/100 μl) at 14:00 of day 1 and with HCG (5 IU/100 μl) at 13:00 of day 3.
- Cross superovulated females with males (C57BL/6J or CBA).
- Identify plugged females at 9:00 of day4. Collect one-cell embryos as decribed in Reyon, D. et al, 2012.
Preparation of microinjection mixture- Thaw aliquot of the Cas9-D10A mRNA and sgRNAs on ice. Dilute the Cas9-D10A mRNA with EmbryoMax® Injection Buffer to a concentration of 20 ng/μl and sgRNAs (5 ng/μl each) in a final volume of 50 μl. Pipette the mixture up and down several times
- Centrifuge at 4 ℃ for 1 min at top sped, and carefully transfer 45 μl supernatant to a new tube. Always keep the tube on ice.
Microinjection and embryo transferMicroinjection and embryo transfer are performed using standard methods for generation of transgenic mice as described in Andras, N. et al., 2003, Cold Spring Harb Protoc.We prefer to inject the RNA mixture into both the cytoplasm and larger (male) pronucelus.Genotyping founders- Tail tips from founders (5-day-old) are collected and digested overnight at 55 ℃ with lysis buffer containing 100 μg/ml Proteinase K. Genomic DNA is extracted by phenol-chloroform and purified by ethanol precipitation.
- Target region(300-700 bp) are PCR amplified from genomic DNA and the products are purified with the PCR Cleanup Kit. Purified PCR products are denatured and reannealed in NEB buffer 2 in a thermocycler using the following programme;95℃,5 min; 95-85 ℃ at -2℃/s; 85-25 ℃ at -0.1℃/s; hold at 4℃.
- Hybridized PCR products are digested with 0.5 μl T7EN1 at 37℃ for 30 min and separated by 2% agarose gel. Mutant founders will yield lower molecular weight cleavage bands.
- Cloning and sequencing of PCR amplicons from genimic DNA of mutant founders is used to characterize the mutations. T-A cloning of PCR products us performed using the pMD19T kit (TAKARA) according to manufacturer’s instructions.
Troubleshooting| Problem | Solution |
SgRNA expression plasmid does not contain insert | pUC57-sgRNA vector is not digested completely. Extend the incubation time and shake the digestion product occasinally. Colony PCR can be used to identify the positive colonies using 5’-TTGTACTGAGAGTGCACCATATG-3’ and the bottom strand sgRNA oligo |
Low yield of sgRNAs | - Use the recommended kits to improve the quality of plasmids and template
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- Increase the amount of template or use the PCR product as template.
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Electrophoresis of sgRNAs shows more than one band | - sgRNAs can form dimers. Always keep sgRNAs on ice. A low amount of dimer will not affect the function of sgRNA.
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- DNA template is incompletely digested. Circular template can produce longer transcripts. Extend the incubation time and shake the digestion product occasionally.
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- DNA template contamination. Add more TURBO DNase and extend incubation time.
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| Cas9-D10A mRNA produces a smear on an agrose gel | - Use RNAsecure to inactivate RNase contamination
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- Use the recommended kits to improve the quality of the DNA template.(QIAGEN Mini-prep and PCR clean-up kits are recommended)
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Time Taken4 days for the construction of sgRNA expression vectors.1 day for the in vitro transcription and preparation of sgRNAs.1 day for the in vitro transcription and preparation of Cas9-D10A mRNA.4 days for the superovulationb of females, collection of 1-cell embryos and microinjections1 week for the genotyping of founder animals. 
