CRISPR/Cas9 system-based gene knockout technology in animals
CRISPR/Cas9 system-based gene knockout technology in animals
CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated system) is present in most bacteria and almost all archaea, and is an immune defense mechanism that bacteria have developed over the course of evolution to help bacteria resist the invasion of foreign viruses and DNA. Among them, Cas9 (CRISPR-associated protein 9) has the potential to be used for genetic manipulation.
Principle
The principle of the CRISPR/Cas9 system is that Cas9 is an endonuclease with two endonuclease activity centers, RuvC and HNH. It has been shown that Cas9 has a strong ability to cleave double-stranded DNA in bacteria and test tubes, but this cleavage requires two types of small RNA intermediates-crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA). tracrRNA 5' and crRNA 3' conserved sequences form a hybrid molecule through base complementary pairing. The sequence at the 5' end of tracrRNA and the conserved sequence at the 3' end of crRNA form a hybrid molecule through base complementary pairing, and this hybrid molecule combines with Cas9 through its special spatial structure to form a protein-RNA complex, which binds to the target DNA through the 20 bases at the 5' end of crRNA, and the Cas9 in the complex cuts the double-stranded DNA through two endonuclease active centers, where the HNH active center cuts one strand complementary to crRNA and the other strand complementary to crRNA. The HNH active center cuts the strand that is complementary to the crRNA and the RuvC active center cuts the non-complementary strand (Figure 5-2-22b). The cleavage activity of protein-RNA complexes on DNA is also dependent on the presence of a PAM (protospacer adjacentmotif) on the target DNA, which is generally referred to as the NGG sequence, and mutations in either of the two GG bases can lead to a reduction or even loss of cleavage activity of protein-RNA complexes.
Operation method
CRISPR/Cas9 system-based gene knockout technology in animals
Principle
Cas9 is an endonuclease with two endonuclease activity centers, RuvC and HNH, and has a strong cleavage ability for double-stranded DNA, which requires two types of small RNA intermediates - crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA). tracrRNA 5' end sequence and crRNA 3' end conserved sequence form a hybrid molecule through base complementary pairing, and this hybrid molecule binds to Cas9 through its special spatial structure to form a protein-RNA complex. The sequence at the 5' end of tracrRNA and the conserved sequence at the 3' end of crRNA form a hybrid molecule through base complementary pairing, and this hybrid molecule binds to Cas9 through its special spatial structure to form a protein-RNA complex, which binds to the target DNA through the 20 bases specific to the 5' end of crRNA, and Cas9 in the complex cuts the double-stranded DNA through two endonuclease activity centers, of which HNH activity center is the HNH activity center, which cuts the double-stranded DNA. Cas9 in the complex cuts the double-stranded DNA through its two endonuclease active centers, the HNH active center cuts the strand that is complementary to crRNA and the RuvC active center cuts the non-complementary strand.
Materials and Instruments
Equipment: disposable sterile gloves, surgical forceps, syringes, PCR instrument, water bath, centrifuge, sequencing platform, incubator Move I. Selection of Cas9 target sites 1. Cas9 target site contains 20 bases, of which the 5' end should be GG: the 3 bases immediately adjacent to the 3' end of the target site constitute the PAM region, and the sequence is required to be NGG (N is an arbitrary base), and the target site can be selected in the justified or antisense strand. You can refer to the following website for Cas9 target prediction: http://zifit.partner.org/zifit/csquare9nuclease.aspx. 2. PCR amplification of target sequences. Select sequences for PCR amplification of the target and nearby sequences from the mouse genome ready for targeting. Design primers around the target so that they are greater than 100 bp from both sides of the target, and the PCR amplification product should preferably be no more than 500 bp and a single band. 3. Confirm the target sequence by sequencing. The PCR product is sent directly to sequencing (no TA cloning required). If the measured sequence differs from the predicted sequence of the target, the gRNA sequence should in principle be designed based on the measured sequence; however, if the measured sequence no longer conforms to the principles of target selection (e.g., the PAM region is not an NGG or the 5' end is not a GG), the target should be reselected; and if the sequencing results show that the target sequence is heterozygous, it is advisable to reselect the experimental material. 4. Selection of experimental materials. Adopt the above PCR and sequencing strategies to screen out a sufficient number of mice with correct and pure targets, and then use these mice for all subsequent experiments on the target.
Materials: mice, gonadotropins, anesthetics, restriction endonuclease, DNA polymerase, ligase, etc., culture medium
1. p-T7-gRNA is a gRNA cloning and in vitro transcription vector. The vector backbone is from pMD18-T simple (Amp resistant), and the gRNA sequences are inserted in the direction of RV-M->T7->M12-47. 2.
2. pT7-gRNA was digested with BbsI, and the cuttings were recovered to obtain the gRNA cloning backbone pT7-gRNA_BbsI, and the resulting sticky ends were as follows: (a 4 bp overhang was left in each 5' of the backbone, and the small fragments in the middle were discarded): 5'- TAATACGACTCACTACT. TAATACGACTCACTATAGGaGTCTTCTAGAAGACgttttagag-3', 3'-ATTATGCTGAGTGATATC CTCAGAAGATCTTCTGcaaaat cte-5'.
3. Order two oligos according to the target site (both 25 nt, s sequence is isotropic to the target sequence, As is inverse). Take PKHD1 gene for example: PKHD1 target oligo s: 5'-ataGGAAGATTGAGTGCACTACCgt-3', PKHDI target oligo As: 5'-taaaacGGGTAGTGCACTCAATCTTC-3'. The uppercase bases in the upper row are the target sequence, and the lowercase bases are fixed sequences necessary for the formation of sticky ends and cannot be changed; the lower row of uppercase bases need to be changed according to the target sequence (note that the uppercase bases in the target oligo As are the complementary sequences of the target, only 19 of them are needed, and the last G does not need to be synthesized).
4. Dissolve the oligo to 10 μmol/L with ddH2O and anneal to obtain a small fragment with the following sticky ends: PKHD1 target_an: 5'-ataGGAAGATTGAGTGCACTACCgt-3', 3'-CTTCTAACTCACGTGTGGcaaaat-5'.
5. The annealed fragment was ligated with the above gRNA cloning backbone pT7-gRNA BbsI, transformed, and a single clone was picked, and then identified by bacteriophage PCR (annealing at 58 ℃, extension for 30 seconds, 30 cycles) using RV-M and targetoligo As as primers. The target band was about 130 bp. Positive clones were selected for sequencing, and the clone with correct sequence was selected for glycerol preservation and plasmid extraction.
Preparation of Cas9 mRNA and gRNA1. Preparation of Cas9 mRNA
(1) Preparation of in vitro transcription template of Cas9 mRNA: linearize pSP6-2sNLS-spCas9 vector (Amp resistance) by XbaI single enzyme cleavage (37 ℃, more than 4 hours), take a small amount of electrophoresis to confirm that the linearization is complete, and then recover the linearized product directly.
(2) In vitro transcription of Cas9 mRNA.
(3) Add polyA sequence and recover Cas9 mRNA for microinjection (adding polyA sequence can enhance the stability and translation efficiency of mRNA).
2. Preparation of gRNA
(1) Preparation of gRNA template for PCR in vitro transcription: Using T7-er fwd and tracr rev primer pairs (mRNA size of about 2,000 bp) as templates for the constructed gRNA in vitro transcription vectors, PCR was performed using a high-fidelity enzyme to obtain the gRNA template for PCR in vitro (anneal at 58 ℃ for 30 seconds, 40 cycles, 40 μl of the system). One μl of the PCR product was electrophoresed to confirm a single band (125 bp), and the PCR product was recovered directly for use in subsequent steps. t7-cr fwd sequence: 5'-GAAATTAATACACGACTCACTATA-3' Tracr rev sequence: 5'-AAAAAAAAGCACCGACTCGGGTGCCAC-3'.
(2) Transcription of gRNA in vitro
(3) Recovery of gRNA (Ambion mirVana miRNA Isolation Kit): 1) Dilute the gRNA transcription system to 300 μl with RNase-free water and add 330 μl of anhydrous ethanol. 2) Add the solution to a recovery column and centrifuge at 10,000 g for 15 seconds. 3) Add 700 μl of miRNA Wash Solution I, and then centrifuge at 10,000 g for 15 seconds. Add 700 μl of miRNA Wash Solution I and centrifuge for 5-10 seconds. 4) Add 500 μl of wash solution II, centrifuge at 10 000 g for 5-10 seconds and repeat. 5) Discard the liquid in the collection tube and centrifuge at 10 000 g for 1 minute to remove the residual liquid. 6) Add appropriate amount of RNase-free water or Elution Solution preheated at 95 ℃ and spin at maximum speed. 7) Add the RNase-free water or Elution Solution preheated at 95 ℃. 6) Add an appropriate amount of preheated RNase-free water or Elution Solution at 95 ℃, centrifuge at maximum speed for 20~30 seconds, and collect the obtained gRNA.
3. Microinjection and identification of the first constructed mouse
(1) Cas9 mRNA and gRNA were mixed and microinjected into the cytoplasm of fertilized mouse eggs, the process is basically the same as TALEN injection.
(2) The genome of the mouse was extracted with high quality, and the DNA fragments containing the target site were amplified by high-fidelity enzyme and sequenced, and the sequencing results were compared with those of wild-type mice.
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