WO2019227640A1 - 利用碱基编辑修复fbn1t7498c突变的试剂和方法 - Google Patents

利用碱基编辑修复fbn1t7498c突变的试剂和方法 Download PDF

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WO2019227640A1
WO2019227640A1 PCT/CN2018/096968 CN2018096968W WO2019227640A1 WO 2019227640 A1 WO2019227640 A1 WO 2019227640A1 CN 2018096968 W CN2018096968 W CN 2018096968W WO 2019227640 A1 WO2019227640 A1 WO 2019227640A1
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fbn1
sgrna
repair
mutation
site
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黄行许
李广磊
李佳楠
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上海科技大学
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Priority to EP18889963.7A priority patent/EP3816296A4/en
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Definitions

  • the invention relates to the field of gene repair, and more specifically, to a method for repairing a FBN1 T7498C mutation related to Marfan syndrome using base editing.
  • CRISPR / Cas9 Gene editing technology, especially CRISPR / Cas9, has been widely used in genetic manipulation and can be used to precisely repair pathogenic mutations ( Komor et al., 2017). At the same time, the technology of gene editing on human embryos has also shown great advantages, suggesting its clinical value in the treatment of human genetic diseases (Kang et al., 2016). However, limited by ethical and therapeutic efficiencies and the presence of off-target, there is still huge room for improvement in the application of gene editing in human embryos (Ruzo and Brivanlou, 2017).
  • CRISPR / Cas9-mediated gene editing uses sgRNA (single-guided RNA) to guide Cas9 protein to locate and cut double-stranded DNA through complementary target sequences, resulting in double-strand breaks (DSB).
  • sgRNA single-guided RNA
  • Non-homologous end joining (NHEJ) repair occurred, resulting in frameshift mutation (knockout); in the presence of a template, repair by homologous recombination (homology) -directed repair (HDR), to achieve knockin, due to low HDR efficiency (integration rarely occurs), and non-homologous end joining mechanisms are prone to random insertion and deletion (indel), making it possible near the breakpoint New bases are introduced randomly, resulting in inaccurate gene editing (Hsu et al., 2014).
  • CRISPR / Cas9-mediated gene editing always has some off-target effects [Gorski et al., 2017].
  • the recently developed base editing system base editor, is based on the nicking enzyme nCas9 and adds rat cytosine deaminase APOBEC1.
  • the base editing system can achieve the target without cutting the double-stranded DNA C at the site is converted to uracil ( Komor et al., 2016). Later, through DNA replication or repair, uracil is converted to thymine (T), and then C to T conversion is achieved. Similarly, it can also convert a single base G to A. Because DSB is not cut, the indel formed is less than 1%, and the gene editing is more accurate.
  • the base editing system has been successfully applied to in vivo base editing, and CT mutations have been achieved in mice. We also used BE3 to achieve efficient editing of target sites in human discarded embryos.
  • Marfan syndrome is an autosomal dominant genetic disease that accounts for 0.2 ⁇ of the world's population. It mainly causes abnormal development of human connective tissue. At present, sudden death of many well-known athletes is related to Marfan syndrome (Arbustini et al., 2005). Although some patients can be treated by surgery, there is still a risk of disease for future generations, and genetically treating the mutations that cause the disease will be the fundamental method. It has been shown earlier that mutations in the FBN1 gene are the main cause of Marfan syndrome. Therefore, whether we can find effective and safe treatment is the direction we are pursuing. The present invention is to find a more efficient and safe method for repairing mutations related to Marfan syndrome, and to achieve the method of repairing the mutation at the human embryonic stage, thereby reducing the incidence of the disease and the huge social burden.
  • Arbustini E., Grasso, M., Ansaldi, S., Malattia, C., Pilotto, A., Porcu, E., Disabella, E., Marziliano, N., Pisani, A., Lanzarini, L., et al. (2005). Identification of sixty-two novel and twelve known FBN1 mutations in eighty-one unrelated probands with Marfan symndrome and other fibrillinopathies. Human, 26,494.
  • the object of the present invention is to provide a kit and method for efficiently repairing the FBN1 T7498C mutation.
  • the present invention provides a kit for efficiently repairing FBN1 T7498C mutation, which is characterized in that it includes a base editor and a repair re-sgRNA for the FBN1 T7498C site.
  • the base editing system is one of BE3, YE1-BE3, YE2-BE3, or YEE-BE3.
  • the sequence of the repaired re-sgRNA for the FBN1 T7498C site is SEQ ID NO.3.
  • the present invention further provides a method of making a combination of mutation and mutation repair, characterized by comprising editing according FBN1 T7498C mutation sites are engineered and mt-sgRNA corresponding mutant ssODN, repair for re-sgRNA FBN1 T7498C site and bases At least one of the systems.
  • the invention also provides a method for repairing mutations by base editing, which comprises: in a mutant cell containing FBN1 T7498C , using a repair re-sgRNA directed to the FBN1 T7498C site to guide the base editing system to the mutation site Base edit repair was performed and transfected cells were collected.
  • the mutant cell containing FBN1 T7498C is a HEK293T cell or an embryonic cell.
  • the method for constructing the mutant cell containing FBN1 T7498C is: designing a mutant mt-sgRNA and a corresponding mutant ssODN according to the FBN1 T7498C site; constructing an expression vector for mt-sgRNA, and cassing the Cas9 protein and the transcribed mt in vitro -sgRNA constitutes RNP-binding ssODN and transfects HEK293T cells, and single cells are identified by flow cytometry to identify mutant cell lines containing FBN1 T7498C .
  • the repaired re-sgRNA for the FBN1 T7498C site is obtained by designing a repaired re-sgRNA based on the FBN1 T7498C site and constructing an U6- and / or T7-driven expression vector.
  • the method for base editing repair of Marfan's disease mutation further comprises: Sanger sequencing to detect repair efficiency; injection of mRNA and re-sgRNA of the base editing system into human embryo cells containing mutations of FBN1 T7498C to detect embryos Repair efficiency, and high-throughput sequencing on-target and off-target efficiency.
  • the sequence of the mt-sgRNA is SEQ ID NO. 1
  • the sequence of ssODN is SEQ ID NO. 2
  • the sequence of the re-sgRNA is SEQ ID NO. 3.
  • the method of obtaining an embryo is to inject sperm containing a mutation of the FBN1 T7498C site into normal oocytes by ICSI, or to inject normal sperm to an egg containing a mutation site of FBN1 T7498C by ICSI, or to contain FBN1 Sperm with mutation at T7498C site was injected into the egg containing FBN1 T7498C mutation site to obtain heterozygous or homozygous mutant embryos containing this site.
  • the invention also provides a method for base-editing and repairing Marfan's disease mutation, which comprises: designing a mutant mt-sgRNA and a corresponding mutant ssODN according to the FBN1 T7498C site; constructing a T7-driven expression vector for mt-sgRNA, and injecting Cas9 in vitro
  • the protein and the transcribed mt-sgRNA constitute RNP and ssODN to electrotransform 293T cells.
  • Flow cytometry sorted single cells identified homozygous mutant cell lines containing FBN1 T7498C .
  • the re-sgRNA was designed and repaired according to the FBN1 T7498C site, and U6 was constructed separately.
  • the invention uses the base editing technology to prove the efficiency and safety of the method at two different levels of cells and embryos.
  • the mutation of FBN1 is the main cause of Marfan syndrome. Its incidence is about 0.2 ⁇ . Completely repairing this mutation genetically will be the most effective measure to treat the disease.
  • the base editing system provides a way to precisely change DNA, changing C to T.
  • the invention uses a new base editing tool, base editor, to repair mutant human embryos.
  • base editor a new base editing tool
  • the safety and effectiveness of the method will be verified at the cell and embryo levels.
  • the inventors will use CRISPR / Cas9 and ssODN-based homologous recombination methods to make cell lines containing the FBN1 T7498C mutation, and then use a base editor in conjunction with appropriate sgRNA to repair mutations at this site.
  • the transcribed mRNA and sgRNA were injected into human embryos containing the FBN1 T7498C mutation. Embryos were collected three days later, and deep sequencing was used to detect repair efficiency and off-target conditions.
  • the present invention utilizes base editing technology to correct the mutation of FBN1 T7498C through precise CT single base mutation, thereby providing an efficient and safe method for treating Marfan syndrome caused by such mutations.
  • Figure 1 shows the identification of mutation sites in samples from patients with Marfan syndrome.
  • A a sample from blood
  • B a sample from semen.
  • FIG. 2 is a method for making mutant cell lines in 293T cells by using Cas9 / sgRNA in combination with ssODN.
  • A Schematic drawing of mutations and repair mutations in cells.
  • B Mutant sgRNA and ssODN are designed for the FBN1 gene to make corresponding mutations.
  • C T7EN1 digestion and identification of transfection efficiency after transfection of cells.
  • D 22 single-cell clones were selected by flow cytometry, and the mutation type of the cells was confirmed by Sanger sequencing.
  • E Sanger sequencing peak map of homozygous mutations.
  • Figure 3 shows off-target detection and sorting of mutant sgRNA.
  • A Potential off-target sites of mutant sgRNA were analyzed by software. For homozygous mutant cell lines, corresponding off-target sites were detected, and T7EN1 was used to detect off-target sites.
  • B Sanger sequencing to detect mutations.
  • Figure 4 shows the use of base editing to repair mutant cell lines.
  • A Design the corresponding repair sgRNA at the mutation site and repair it using base editing.
  • B Mutation was detected by sequencing after cell transfection.
  • C TA clones were performed on panel B to identify repair types.
  • D Sanger sequencing peak map after complete repair.
  • Figure 6 shows off-target detection of repaired sgRNA.
  • A Off-target detection using T7EN1 digestion.
  • B Sanger sequencing was used to confirm off-target conditions.
  • Figure 7 shows repair of human mutant embryos.
  • A Schematic representation of human embryo repair using base editing.
  • B Embryo state after mutation-containing embryos are injected with related RNA.
  • C Genotypes of representative embryos injected with repaired sgRNA.
  • D Genotypes of representative embryos injected with random sgRNA.
  • E High-throughput sequencing for genotyping of repaired and control embryos.
  • Figure 8 shows the genotypes of the repaired and control embryos used.
  • A Repairing the genotype of the embryo.
  • B Genotype of control embryos.
  • FIG. 9 shows the use of high-throughput sequencing to detect off-target repaired sgRNA in repaired embryos.
  • BE3 build different versions of BE3, namely YE1-BE3, YE2-BE3, YEE-BE3, and BE3.
  • the original version BE3 was purchased from Addgene (73021).
  • BE3 differ only in the coding frame of rAPOBEC1. They were synthesized by bio-organisms (http://www.sangon.com/). NotI and SmaI sites were added to the two ends of the synthesized fragment. The synthesized fragment was cloned into a commonly used pmd19t vector (TAKARA: 6013).
  • BE3 and the above three synthetic vectors were digested with NotI (NEB: R0189L) and SmaI (NEB: R0141L).
  • the system is as follows: Buffer (NEB: R0189L) 6uL; plasmid 2ug; NotI 1 ⁇ L; SmaI 1 ⁇ L; ddH 2 O To 60 ⁇ L. After mixing, the enzyme was digested overnight at 37 ° C.
  • the digested product was recovered on a 1% agarose gel (Axygen: AP-GX-250G). Among them, BE3 recovered the large fragment backbone vector, and the synthetic vector recovered the APOBEC1 small fragment vector. Recovery was performed according to the instructions of the kit (Axygen: AP-PCR-250G). The recovered fragments were tested for concentration by Nanodrop 2000.
  • the recovered backbone vector and APOBEC1 fragment were ligated, and the connection system was as follows: T4 ligation buffer (NEB: M0202L) 1 ⁇ L, backbone vector 20 ng, APOBEC1 fragment 50 ng, T4 ligase (NEB: M0202L) 0.5 ⁇ L, ddH 2 O supplemented to 10 ⁇ L Connect at 16 ° C overnight.
  • the transformation steps are as follows: 20 ⁇ L of competent cells (TransGen: CD201) are thawed on ice; 2 ⁇ L of the ligation product is mixed with the competent cells and left on ice for 20 minutes; heat shocked at 42 ° C. for 60 seconds; Resuscitate LB medium (MDBio: L001-1kg) and shake for 30 minutes; take 70 ⁇ L ampicillin-coated plates (50 ⁇ g / ml, 37 ° C incubator, and incubate for 14 hours.
  • Monoclonal bacteria were selected, expanded in 4 ml liquid LB medium, and plasmids were extracted after 14 hours (Axygene: AP-MN-P-250G).
  • the steps are as follows: Centrifuge the bacterial solution for 10 minutes at 4000 rpm and discard the supernatant medium; add 350 ⁇ L of buffer S1, blow off the bacteria and transfer to a 2 ml centrifuge tube; add 250 ⁇ L of buffer S2, and turn it upside down 8 times Add 250 ⁇ L of buffer S3 and mix upside down 6 times to produce a precipitate; Centrifuge at 12,000 rpm for 10 minutes and take the supernatant through the column; Centrifuge for 1 minute, discard the waste solution, add 500 ⁇ L of W1, centrifuge for one minute, and discard Waste liquid; add 750 ⁇ L of W2, centrifuge, and discard the supernatant; add 500 ⁇ L of W2, centrifuge, discard the supernatant; idle for 1 minute; add 50 ⁇ L of elu
  • any of the above-mentioned BE3 may be used, preferably (1) or (4).
  • a mutant FBN1 T7498C mutant cell line was made on the cell line using Cas9 / sgRNA in combination with ssODN, and the mutant line was repaired using a base editing system ( Figure 2).
  • a mutant mt-sgRNA (SEQ ID NO.1) was designed to synthesize oligos.
  • the upstream sequence was: 5'-taggCGCCAATGGTGTTAACACAT-3 '(SEQ ID NO. (14)), and the downstream sequence was: 5'- aaac ATGTGTTAACACCATTGGCG-3 '(SEQ ID NO.
  • the upstream and downstream sequences passed the program (95 ° C, 5min; 95 ° C-85 ° C at-2 ° C / s; 85 ° C-25 ° C at-0.1 ° C / s; hold at 4 ° C) annealing, and ligated to the PUC57-T7sgRNA vector (addgene: 51132) linearized by BsaI (NEB: R0539L).
  • the linearization system is as follows: PUC57-T7sgRNA 2 ⁇ g; buffer (NEB: R0539L) 6 ⁇ L; BsaI 2 ⁇ L; ddH 2 O to 60 ⁇ L. Digest at 37 ° C overnight.
  • the homologous template ssODN (SEQ ID NO. 2) used was synthesized by Shengong Biological Company (http://www.sangon.com/) by PAGE purification. At the same time, near the mutation site, according to the characteristics of base editing, design and repair re-sgRNA (SEQ ID NO.3) and synthesize oligos.
  • the upstream sequence is 5'-accgCTACGTGTTAACACCATTGG-3 '(SEQ ID NO.16), and the downstream The sequence is 5'-aaacCCAATGGTGTTAACACGTAG-3 '(SEQ ID NO. 17).
  • the upstream and downstream sequences were annealed by a program (95 ° C, 5min; 95 ° C-85 ° C at-2 ° C / s; 85 ° C-25 ° C at-0.1 ° C / s; hold at 4 ° C), and connected to the BsaI (NEB: R0539L) ) On a linearized PGL3-U6sgRNA vector.
  • the ligated vector was transformed, picked, identified, and the primer was identified as the U6 vector as the upstream sequence: 5'-TTTCCCATGATTCCTTCATA-3 '(SEQ ID NO. 20), the downstream sequence It is the downstream sequence of the corresponding oligo.
  • the T7 vector is the upstream sequence: 5'-CGCCAGGGTTTTCCCAGTCACGAC-3 '(SEQ ID NO.21), and the downstream sequence is the corresponding oligo downstream sequence.
  • a positive clone of Shake bacteria was extracted from the plasmid (Axygene: AP-MN -P-250G) to determine the concentration for future use.
  • mutant plasmids were named mt-T7-sgRNA (SEQ ID NO.11), re-U6-sgRNA (SEQ ID NO.12), and re-T7-sgRNA (SEQ ID NO .13).
  • the sgRNA-containing fragment was amplified using the primers: F: 5'-TCTCGCGCGTTTCGGTGATGACGG-3 ', (SEQ ID NO.22) R: 5'-AAAAAAAGCACCGACTCGGTGCCACTTTTTC-3' (SEQ ID NO .twenty three).
  • the amplification system is as follows: 2Xbuffer (Novizan: P505) 25 ⁇ L; dNTP 1 ⁇ L; F (10 pmol / ⁇ L) 2 ⁇ L; R (10 pmol / ⁇ L) 2 ⁇ L; template 1 ng; DNA polymerase (Novizan: P505) 0.5 ⁇ L; ddH 2 O was made up to 50 ⁇ L.
  • the amplified PCR product was purified by the following steps: 4 ⁇ L RNAsecure (Life: AM7005) was added per 100 ⁇ L volume; 60 ° C for 15 minutes; three times the volume of PCR-A (Axygen: AP-PCR-250G) was added to the column, and centrifuged Centrifuge at 12000 rpm for 1 minute; add 500 ⁇ L of W2 and centrifuge for 1 minute; idle for 1 minute; add 20 ⁇ L of RNAase-free water to elute.
  • 4 ⁇ L RNAsecure (Life: AM7005) was added per 100 ⁇ L volume; 60 ° C for 15 minutes; three times the volume of PCR-A (Axygen: AP-PCR-250G) was added to the column, and centrifuged Centrifuge at 12000 rpm for 1 minute; add 500 ⁇ L of W2 and centrifuge for 1 minute; idle for 1 minute; add 20 ⁇ L of RNAase-free water to elute.
  • reaction system is: reaction buffer 1 ⁇ L; enzyme mix 1 ⁇ L; A 1 ⁇ L; T 1 ⁇ L; G 1 ⁇ L; C 1 ⁇ L; template 800ng; H 2 O complement To 10 ⁇ L. After the above system was mixed, the reaction was performed at 37 ° C for 5 hours. Add 1 ⁇ L DNase and react at 37 ° C for 15 minutes.
  • the steps are as follows: 90 ⁇ L Elution solution was added to the reaction volume in the previous step to transplant 1.5 ml EP tubes; 350 ⁇ L Binding solution was added to mix; 250 ⁇ L absolute ethanol was added to mix; ; Centrifuge at 10,000 rpm for 30 seconds and discard the waste solution; add 500 ⁇ L Washing solution and centrifuge at 10,000 rpm for 30 seconds and discard the waste solution; idling for 1 minute; change the collection tube and add 100 ⁇ L Elution solution to elute; add 10 ⁇ L acetic acid Mix with ammonium (Ambion, Life Technologies, AM1908); add 275 ⁇ L of absolute ethanol and mix; place at -20 ° C for 30 minutes, and prepare 70% ethanol at -20 ° C; centrifuge at 13,000 rpm for 15 minutes at 4 ° C. Discard the supernatant and add 500 ⁇ L of 70% ethanol; centrifuge
  • HEK293T cells purchased from ATCC
  • the present invention performs eukaryotic cell culture and transfection: HEK293T cells are inoculated and cultured in DMEM high-sugar culture medium supplemented with 10% FBS (HyClone, SH30022.01B) It contains penicillin (100 U / ml) and streptomycin (100 ⁇ g / ml).
  • the off-target sites were identified by designing corresponding primers.
  • the primer sequences are shown in Table 3.
  • the amplified PCR product was identified by T7EN1 and sequencing, and no off-target was found (Figure 3).
  • the mutant cell line is cultured and transfected: HEK293T cells are inoculated and cultured in DMEM high glucose medium (HyClone, SH30022.01B) supplemented with 10% FBS, which contains penicillin (100U / ml) and streptomycin ( 100 ⁇ g / ml).
  • DMEM high glucose medium HyClone, SH30022.01B
  • FBS penicillin
  • streptomycin 100 ⁇ g / ml
  • the upstream sequence is 5'-taggCTACGTGTTAACACCATTGG-3 '(SEQ ID NO.18) and the downstream sequence. 5'-aaacCCAATGGTGTTAACACGTAG-3 '(SEQ ID NO. 19).
  • the upstream and downstream sequences were annealed by a program (95 ° C, 5min; 95 ° C-85 ° C at-2 ° C / s; 85 ° C-25 ° C at-0.1 ° C / s; hold at 4 ° C), and connected to the BsaI (NEB: R0539L) ) On a linearized PUC57-T7sgRNA vector.
  • the linearization system and procedures are as above.
  • the ligation system is as follows: T4 ligation buffer (NEB: M0202L) 1 ⁇ L, linearized carrier 20 ng, annealed oligo fragment (10 ⁇ M) 5 ⁇ L, T4 ligase (NEB: M0202L) 0.5 ⁇ L, ddH 2 O supplemented to 10 ⁇ L. 16 ° C ligation overnight.
  • the ligated vector was transformed, picked, identified, and the primer was identified as the upstream sequence: 5'-CGCCAGGGTTTTCCCAGTCACGAC-3 '(SEQ ID NO. 21), and the downstream sequence was the corresponding oligo downstream sequence.
  • the positive clone Shake bacteria extraction plasmid (Axygene: AP-MN-P-250G) was measured for concentration before use.
  • the sgRNA-containing fragment was amplified using the primers: F: 5'-TCTCGCGCGTTTCGGTGATGACGG-3 '(SEQ ID NO.22), R: 5'-AAAAAAAGCACCGACTCGGTGCCACTTTTTC-3' (SEQ ID NO.23).
  • the amplification system is as follows: 2Xbuffer (Novizan: P505) 25 ⁇ L; dNTP 1 ⁇ L; F (10 pmol / ⁇ L) 2 ⁇ L; R (10 pmol / ⁇ L) 2 ⁇ L; template 1 ⁇ g; DNA polymerase (Novizan: P505) 0.5 ⁇ L; ddH 2 O was made up to 50 ⁇ L.
  • the amplified PCR product was purified by the following steps: 4 ⁇ L RNAsecure (Life: AM7005) was added per 100 ⁇ L volume; 60 ° C for 15 minutes; three times the volume of PCR-A (Axygen: AP-PCR-250G) was added to the column, and centrifuged Centrifuge at 12000 rpm for 1 minute; add 500 ⁇ L of W2 and centrifuge for 1 minute; idle for 1 minute; add 20 ⁇ L of RNAase-free water to elute.
  • 4 ⁇ L RNAsecure (Life: AM7005) was added per 100 ⁇ L volume; 60 ° C for 15 minutes; three times the volume of PCR-A (Axygen: AP-PCR-250G) was added to the column, and centrifuged Centrifuge at 12000 rpm for 1 minute; add 500 ⁇ L of W2 and centrifuge for 1 minute; idle for 1 minute; add 20 ⁇ L of RNAase-free water to elute.
  • reaction system is: reaction buffer 1 ⁇ L; enzyme mix 1 ⁇ L; A 1 ⁇ L; T 1 ⁇ L; G 1 ⁇ L; C 1 ⁇ L; template 800ng; H 2 O complement To 10 ⁇ L. After the above system was mixed, the reaction was performed at 37 ° C for 5 hours. Add 1 ⁇ LDNase and react at 37 ° C for 15 minutes.
  • the steps are as follows: 90 ⁇ L Elution solution was added to the reaction volume in the previous step to transplant the 1.5 ml EP tube; 350 ⁇ L Binding solution was added to mix; 250 ⁇ L absolute ethanol was added to the column; Centrifuge at 10,000 rpm for 30 seconds, discard the waste solution; add 500 ⁇ L Washing solution, centrifuge at 10,000 rpm for 30 seconds, discard the waste solution; idling for 1 minute; change the collection tube, add 100 ⁇ LElution solution to elute; add 10 ⁇ L ammonium acetate ( Ambion, Life Technologies, AM1908); add 275 ⁇ L of absolute ethanol and mix; place at -20 ° C for 30 minutes, while preparing 70% ethanol and place at -20 ° C; centrifuge at 13,000 rpm for 15 minutes at 4 ° C. Discard the supernatant and add 500 ⁇ L of 70% ethanol; centrifuge for
  • the system was as follows: BE3 / YE1-BE3 / YE2-BE3 / YEE-BE3 10 ⁇ g; buffer I (NEB: R0539L) 10 ⁇ L; BbsI 4 ⁇ L (NEB: R0539L); H 2 O was made up to 100 ⁇ L. After mixing, digest at 37 ° C overnight.
  • RNAsecure Life: AM7005
  • QIAGEN 28004
  • the specific system is as follows: 20 ⁇ L reaction product; 36 ⁇ L H 2 O; 20 ⁇ L 5xE-PAP buffer; 10 ⁇ L 25mM MnCl 2 ; 10 ⁇ L ATP solution; 4 ⁇ L PEP. After the reaction system was mixed, the reaction was carried out at 37 ° C for 30 minutes.
  • Recycle This was performed using a recovery kit (QIAGEN: 74104). The steps are as follows: 350 ⁇ L of buffer RLT was added to the reaction product in the previous step; 250 ⁇ L of absolute ethanol was added, passed through the column, and centrifuged; 500 ⁇ L of RPE was added, centrifuged, and 500 ⁇ L of RPE was added, and centrifuged; After measuring the concentration, store at -80 ° C.
  • the volume of the base-editing mRNA is about 0.2 ⁇ L by micromanipulation.
  • BE3 is used, and the sgRNA cytoplasm used for repair is injected into the fertilized egg.
  • BE3 and sgRNA The concentrations were 100 ng / ⁇ L and 50 ng / ⁇ L, respectively.
  • the treated embryo cells were further cultured in a three-gas incubator for three days ( Figure 7).
  • PE150 high-throughput sequencing was used to detect the target fragment. Three control embryos and all 7 repair embryos were selected. It was found that 3 embryos in the control group were heterozygous genotypes. The 7 repaired embryos were all normal genotypes, which proved that the base editing system can efficiently achieve target site repair.

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Abstract

一种利用碱基编辑修复FBN1 T7498C突变的试剂和方法。所述的高效修复FBN1 T7498C突变的试剂盒，其特征在于，包括碱基编辑系统以及针对FBN1 T7498C位点的修复re-sgRNA。利用碱基编辑技术，通过精准的CT单碱基突变，从而可以修复FBN1 T7498C的突变，从而为治疗该类突变引起的马凡氏综合征提供了高效安全的方法。

Description

利用碱基编辑修复FBN1 T7498C突变的试剂和方法 技术领域
本发明涉及基因修复领域，更具体来说，利用碱基编辑修复马凡氏综合征相关的FBN1 T7498C突变的方法。
背景技术
目前为止医学上已经鉴定出了接近一万种遗传病，对家庭和社会造成了巨大的负担，然而只有大约6％的遗传病目前可以治疗(Austin and Dawkins,2017)。诊断和治疗遗传病已经是医学上的重要研究内容。高通量测序技术的发展已经使得遗传病的诊断变得较为容易。虽然附植前遗传学诊断(PGD)可以阻断部分遗传病的发生，然而对一些诸如纯合突变等遗传病，尚需要开发更有效的遗传学治疗手段(Dunbar et al.,2018)。
基因编辑技术，尤其是CRISPR/Cas9，已经广泛应用于基因操作，并且可以应用于精准修复致病突变(Komor et al.,2017)。同时目前该技术在人胚胎上的基因编辑也展示出了巨大的优势，提示其在人遗传病治疗中的临床价值(Kang et al.,2016)。然而限于伦理和治疗的效率及脱靶的存在，基因编辑在人胚胎中的应用还有巨大的提升空间(Ruzo and Brivanlou,2017)。CRISPR/Cas9介导的基因编辑是在sgRNA(single guided RNA)通过靶序列互补引导Cas9蛋白定位剪切双链DNA，造成双链DNA断裂(double-strand breaks,DSB)，在没有模板的条件下，发生非同源末端连接(non-homologous end joining,NHEJ)修复，造成移码突变(frameshift mutation)，导致基因敲除(knockout)；在有模板的条件下，通过同源重组进行修复(homology-directed repair,HDR)，实现基因敲入(knockin)，由于HDR效率低(整合很少发生)，而且非同源性末端接合机制容易产生随机插入和删除(indel)，使得在断裂点附近可能随机引入新的碱基，从而导致不精确的基因编辑(Hsu et al.,2014)。此外，CRISPR/Cas9介导的基因编辑总有一些脱靶效应[Gorski et al.,2017]。
最近开发出来的碱基编辑系统，base editor，是在切口酶nCas9的基础上加上了大鼠的胞嘧啶脱氨酶APOBEC1，碱基编辑系统可以在不切割DNA双链的 情况下，实现靶位点上C转化为尿嘧啶(Komor et al.,2016)。之后，通过DNA复制或修复，尿嘧啶被转化成胸腺嘧啶(T)，进而实现C到T的转换，类似地，其也能将单碱基G转化成A。由于不切割DNA造成DSB，形成的indel低于1％，实现的基因编辑更精确。碱基编辑系统已经被成功应用于体内碱基编辑，实现了小鼠的CT突变。我们利用BE3在人的废弃胚胎中也实现了靶位点的高效编辑。
马凡氏综合征是一种常染色体显性遗传病，占世界人口的0.2‰，其主要造成人结缔组织的发育异常，目前已有多名知名运动员的猝死和马凡氏综合征有关(Arbustini et al.,2005)。虽然部分患者可以通过手术治疗，但对于后代仍然具有患病的风险，从遗传上治疗引起该病的突变将是根本的方法。前期已有表明FBN1基因的突变是造成马凡氏综合征的主因。因此能否找到高效同时安全性的治疗方法是我们追求的方向。本发明就是寻找一种更加高效和安全的修复马凡氏综合征相关的突变，达到在人胚胎阶段修复该突变的方法，降低此病的发病比例和巨大的社会负担。
参考文献
Arbustini,E.,Grasso,M.,Ansaldi,S.,Malattia,C.,Pilotto,A.,Porcu,E.,Disabella,E.,Marziliano,N.,Pisani,A.,Lanzarini,L.,et al.(2005).Identification of sixty-two novel and twelve known FBN1 mutations in eighty-one unrelated probands with Marfan syndrome and other fibrillinopathies.Human mutation 26,494.
Austin,C.P.,and Dawkins,H.J.S.(2017).Medical research:Next decade's goals for rare diseases.Nature 548,158.
Dunbar,C.E.,High,K.A.,Joung,J.K.,Kohn,D.B.,Ozawa,K.,and Sadelain,M.(2018).Gene therapy comes of age.Science 359.
Hsu,P.D.,Lander,E.S.,and Zhang,F.(2014).Development and applications of CRISPR-Cas9 for genome engineering.Cell 157,1262-1278.
Kang,X.,He,W.,Huang,Y.,Yu,Q.,Chen,Y.,Gao,X.,Sun,X.,and Fan,Y.(2016).Introducing precise genetic modifications into human 3PN embryos by CRISPR/Cas-mediated genome editing.Journal of assisted reproduction and genetics 33,581-588.
Komor,A.C.,Badran,A.H.,and Liu,D.R.(2017).CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes.Cell 168,20-36.
Komor,A.C.,Kim,Y.B.,Packer,M.S.,Zuris,J.A.,and Liu,D.R.(2016).Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.Nature 533,420-424.
Ruzo,A.,and Brivanlou,A.H.(2017).At Last:Gene Editing in Human Embryos to Understand Human Development.Cell Stem Cell 21,564-565.
发明内容
本发明的目的是提供一种高效修复FBN1 T7498C突变的试剂盒和方法。
为了达到上述目的，本发明提供了一种高效修复FBN1 T7498C突变的试剂盒，其特征在于，包括碱基编辑系统(base editor)以及针对FBN1 T7498C位点的修复re-sgRNA。
优选地，所述的碱基编辑系统为BE3，YE1-BE3，YE2-BE3或者YEE-BE3中的一种。
优选地，所述的针对FBN1 T7498C位点的修复re-sgRNA的序列为SEQ ID NO.3。
本发明还提供了一种制作突变和修复突变的组合，其特征在于，包括根据FBN1 T7498C位点设计突变mt-sgRNA和相应的突变ssODN、针对FBN1 T7498C位点的修复re-sgRNA以及碱基编辑系统中的至少一种。
本发明还提供了一种碱基编辑修复突变的方法，其特征在于，包括：在含有FBN1 T7498C的突变细胞中，利用针对FBN1 T7498C位点的修复re-sgRNA引导碱基编辑系统到突变位点进行碱基编辑修复，收集转染后的细胞。
优选地，所述的含有FBN1 T7498C的突变细胞为HEK293T细胞或胚胎细胞。
优选地，所述的含有FBN1 T7498C的突变细胞的构建方法为：根据FBN1 T7498C位点设计突变mt-sgRNA和相应的突变ssODN；构建mt-sgRNA的表达载体，体外将Cas9蛋白和转录出来的mt-sgRNA组成RNP结合ssODN的方式并电转HEK293T细胞，流式分选单细胞鉴定出含有FBN1 T7498C的突变细胞株。
优选地，所述的针对FBN1 T7498C位点的修复re-sgRNA通过根据FBN1 T7498C位点设计修复re-sgRNA，并构建U6启动和/或T7启动的表达载体得到。
优选地，所述的碱基编辑修复马凡氏疾病突变方法还包括：Sanger测序检测修复效率；在含有FBN1 T7498C的突变的人胚胎细胞中注射碱基编辑系统的mRNA和re-sgRNA，检测胚胎中的修复效率，并高通量测序on-target和off-target的效率。
优选地，所述的mt-sgRNA的序列为SEQ ID NO.1，ssODN的序列为SEQ ID NO.2，re-sgRNA的序列为SEQ ID NO.3。
优选地，获得胚胎的方法是通过ICSI将含有FBN1 T7498C位点突变的精子注射到正常的卵母细胞中，或通过ICSI将正常精子注射到含有FBN1 T7498C突变位点的卵子中，或者将含有FBN1 T7498C位点突变的精子注射到含有FBN1 T7498C突变 位点的卵子中获得含有此位点的杂合或纯合的突变胚胎。
本发明还提供了一种碱基编辑修复马凡氏疾病突变方法，包括：根据FBN1 T7498C位点设计突变mt-sgRNA和相应的突变ssODN；构建mt-sgRNA的T7启动的表达载体，体外将Cas9蛋白和转录出来的mt-sgRNA组成RNP结合ssODN的方式电转293T细胞，流式分选单细胞鉴定出含有FBN1 T7498C的纯合突变细胞株；根据FBN1 T7498C位点设计修复re-sgRNA，并分别构建U6启动和T7启动的表达载体；在制作的纯合突变细胞株中，利用U6启动的re-sgRNA引导base editor到突变位点，3天之后收集转染后的细胞，Sanger测序检测修复效率；在含有此位点突变的人胚胎细胞中注射BE3的mRNA和re-sgRNA，检测胚胎中的修复效率，并高通量测序on-target和off-target的效率。
本发明利用碱基编辑技术在细胞和胚胎两个不同层面进行方法的高效性和安全性证明。
FBN1的突变是造成马凡氏综合征的主要成因，它的发病率在0.2‰左右，从遗传上彻底修复此突变，将是治疗该疾病最有效的措施。碱基编辑系统提供了一种精确改变DNA，即将C变为T的方法。
本发明利用新型碱基编辑工具base editor修复突变的人胚胎。首先将在细胞和胚胎两个水平验证方法的安全性和有效性。在细胞方面，发明人将利用基于CRISPR/Cas9和ssODN的同源重组方法制作含有FBN1 T7498C突变的细胞株，其后利用base editor结合合适的sgRNA修复此位点的突变。验证了系统的安全性和有效性之后，将转录出来的mRNA和sgRNA注射到含有FBN1 T7498C突变的人胚胎中，三天之后收集胚胎，利用深度测序的方式检测修复效率和脱靶情况。
本发明利用碱基编辑技术，通过精准的CT单碱基突变，从而可以修复FBN1 T7498C的突变，从而为治疗该类突变引起的马凡氏综合征提供了高效安全的方法。
附图说明
图1为对患有马凡氏综合征病人的样本进行突变位点的确认。(A)来自血液的样本，(B)来自精液的样本。
图2为利用Cas9/sgRNA结合ssODN的方式在293T细胞中制作突变细胞株。(A)在细胞中制作突变和修复突变的模式图。(B)对FBN1基因设计突变型的 sgRNA和ssODN来制作相应突变。(C)转染细胞后对转染效率进行T7EN1酶切鉴定。(D)通过流式分选出22个单细胞克隆，sanger测序确认细胞的突变类型。(E)纯合突变的sanger测序峰图。
图3为对突变的sgRNA进行脱靶检测分选。(A)通过软件分析出突变sgRNA的潜在脱靶位点，对纯合突变细胞株，检测相应的脱靶位点，T7EN1检测脱靶。(B)Sanger测序检测突变。
图4为对突变的细胞株利用碱基编辑进行修复。(A)在突变位点设计相应的修复sgRNA，利用碱基编辑将其修复。(B)细胞转染后的测序检测突变情况。(C)对图B进行TA克隆鉴定修复类型。(D)完全修复后的sanger测序峰图。
图5利用YE1-BE3，YEE-BE3和BE3对突变细胞株修复。
图6为对修复sgRNA进行脱靶检测。(A)利用T7EN1酶切检测脱靶。(B)利用sanger测序确认脱靶情况。
图7为对人突变胚胎进行修复。(A)利用碱基编辑对人胚胎修复示意图。(B)含有突变的胚胎注射相关RNA后的胚胎状态。(C)注射修复sgRNA的代表性胚胎的基因型。(D)注射随机sgRNA的代表性胚胎的基因型。(E)高通量测序对修复胚胎和对照胚胎进行基因型分析。
图8为所使用的修复胚胎和对照胚胎检测的基因型。(A)修复胚胎的基因型。(B)对照胚胎的基因型。
图9为在修复胚胎中利用高通量测序检测修复sgRNA的脱靶。
具体实施方式
下面将结合实施例对本发明的实施方案进行清楚、完整的描述，显然，所描述的实施例仅用于说明本发明的一部分实施例，而不应视为限制本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，视为可以通过市售购买获得的常规产品。
以上所述仅为本发明较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
首先，构建不同版本的BE3，即YE1-BE3、YE2-BE3、YEE-BE3和BE3。原始版本BE3购自Addgene(73021)。
1、不同类型APOBEC1的合成。
上述四种BE3只是在rAPOBEC1的编码框上有所差异，YE1-rAPOBEC1(SEQ ID NO.4)、YE2-rAPOBEC1(SEQ ID NO.5)、YEE-rAPOBEC1(SEQ ID NO.6)，其序列分别由生工生物(http://www.sangon.com/)合成。所合成片段的两端分别加有NotI和SmaI的酶切位点。合成的片段克隆到常用的pmd19t载体(TAKARA：6013)上。
2、载体的酶切与纯化
BE3和上述三种合成的载体经过NotI(NEB：R0189L)、SmaI(NEB：R0141L)酶切，体系如下：Buffer(NEB：R0189L)6uL；质粒2ug；NotI 1μL；SmaI 1μL；ddH 2O补齐到60μL。混样后于37℃过夜酶切。
酶切产物经过1％的琼脂糖凝胶，进行回收(Axygen：AP-GX-250G)。其中BE3回收大片段的骨架载体，合成载体回收APOBEC1的小片段载体。回收按照试剂盒的使用说明进行(Axygen：AP-PCR-250G)。回收后的片段经过Nanodrop 2000检测浓度。
3、载体的连接、转化与质粒提取。
回收的骨架载体和APOBEC1片段经过连接，连接体系如下：T4连接buffer(NEB：M0202L)1μL，骨架载体20ng，APOBEC1片段50ng，T4连接酶(NEB：M0202L)0.5μL，ddH 2O补齐到10μL，16℃连接过夜。转化步骤如下：取20μL感受态细胞(TransGen：CD201)在冰上解冻；2μL的连接产物与感受态细胞混合，冰上放置20分钟；42℃热激60秒；冰上放置2分钟，加入400μL复苏LB培养基(MDBio：L001-1kg)，摇床30分钟；取70μL涂氨苄平板(50μg/ml，37℃培养箱，培养14个小时。
挑选单克隆菌，在4ml液体LB培养基中扩大培养，14小时后提取质粒(Axygene：AP-MN-P-250G)。步骤如下：菌液经过4000转/分钟离心10分钟，倒掉上清培养基；加入350μL的buffer S1，将菌体吹散，转移到2ml离心管中；加入250μL的buffer S2，上下颠倒8次；加入250μL的buffer S3，颠倒混匀6次，产生沉淀；12000转/分钟离心10分钟，取上清过柱；离心1分钟，倒掉废 液，加入500μL的W1，离心一分钟，倒掉废液；加入750μL的W2，离心，倒掉上清；加入500μL的W2，离心，倒掉上清；空转1分钟；加入50μL的洗脱液，静置2分钟，离心。获得质粒经过浓度检测，取10μL送测序，阳性质粒保存在-20℃。
最终构建成如下四种BE3：
(1)YE1-BE3，SEQ ID NO.7；
(2)YE2-BE3，SEQ ID NO.8；
(3)YEE-BE3，SEQ ID NO.9；
(4)BE3，SEQ ID NO.10。
在下面进行突变位点修复时，可以采用上述任一种BE3，优选为(1)或(4)。
实施例1
本实施例中，在细胞株上利用Cas9/sgRNA结合ssODN制作突变FBN1 T7498C突变细胞株，并利用碱基编辑系统对突变株进行修复(图2)。
1.1质粒构建
在突变位点附近，设计突变mt-sgRNA(SEQ ID NO.1)，合成oligos，上游序列为：5’-taggCGCCAATGGTGTTAACACAT-3’(SEQ ID NO.(14))，下游序列为：5’-aaac ATGTGTTAACACCATTGGCG-3’(SEQ ID NO.(15))，上下游序列通过程序(95℃，5min；95℃-85℃ at-2℃/s；85℃-25℃ at-0.1℃/s；hold at 4℃)退火，连接到经过BsaI(NEB：R0539L)线性化的PUC57-T7sgRNA载体(addgene：51132)上。线性化体系如下所示：PUC57-T7sgRNA 2μg；buffer(NEB：R0539L)6μL；BsaI 2μL；ddH 2O补齐到60μL。37℃酶切过夜。所使用的同源模板ssODN(SEQ ID NO.2)，利用PAGE纯化的方式由生工生物公司(http://www.sangon.com/)合成。同时在突变位点附近，依据碱基编辑作用的特点，设计修复re-sgRNA(SEQ ID NO.3)，合成oligos，上游序列为5’-accgCTACGTGTTAACACCATTGG-3’(SEQ ID NO.16)，下游序列为5’-aaacCCAATGGTGTTAACACGTAG-3’(SEQ ID NO.17)。上下游序列通过程序(95℃，5min；95℃-85℃ at-2℃/s；85℃-25℃ at-0.1℃/s；hold at 4℃)退火，连接到经过BsaI(NEB：R0539L)线性化的PGL3-U6sgRNA载体上。同时 合成上游引物：5’-taggCTACGTGTTAACACCATTGG-3”(SEQ ID NO.18)和下游引物：5’-aaacCCAATGGTGTTAACACGTAG-3’(SEQ ID NO.19)，经过退火连接到线性化的PUC57-T7sgRNA载体上。退火程序、线性化体系与程序如上。连接体系如下：T4连接buffer(NEB：M0202L)1μL，线性化载体20ng，退火的oligo片段(10μM)5μL，T4连接酶(NEB：M0202L)0.5μL，ddH 2O补齐到10μL.16℃连接过夜。连接的载体通过转化，挑菌，鉴定，鉴定引物为U6载体为上游序列：5’-TTTCCCATGATTCCTTCATA-3’(SEQ ID NO.20)，下游序列为相应oligo的下游序列。T7载体为上游序列：5’-CGCCAGGGTTTTCCCAGTCACGAC-3’(SEQ ID NO.21)，下游序列为相应oligo的下游序列。对阳性克隆摇菌提取质粒(Axygene：AP-MN-P-250G)测定浓度备用。获得的突变质粒命名为mt-T7-sgRNA(SEQ ID NO.11)、re-U6-sgRNA(SEQ ID NO.12)和re-T7-sgRNA(SEQ ID NO.13)。
1.2 sgRNA的体外转录
以构建的PUC57-T7sgRNA为模板，扩增含有sgRNA的片段，所用引物为：F:5’-TCTCGCGCGTTTCGGTGATGACGG-3’,(SEQ ID NO.22)R:5’-AAAAAAAGCACCGACTCGGTGCCACTTTTTC-3’(SEQ ID NO.23)。扩增体系如下：2Xbuffer(诺唯赞：P505)25μL；dNTP 1μL；F(10pmol/μL)2μL；R(10pmol/μL)2μL；模板1ng；DNA聚合酶(诺唯赞：P505)0.5μL；ddH 2O补齐到50μL。扩增出来的PCR产物经过下述步骤纯化：每100μL体积加4μL RNAsecure(Life：AM7005)；60℃15分钟；加入三倍体积的PCR-A(Axygen：AP-PCR-250G)过柱，离心，12000转/分钟离心1分钟；加入500μL W2，离心1分钟；空转1分钟；加入20μL无RNAase水洗脱。
利用体外转录试剂盒(Ambion,Life Technologies,AM1354)转录，步骤如下：反应体系为：reaction buffer 1μL；enzyme mix 1μL；A 1μL；T 1μL；G 1μL；C 1μL；模板800ng；H 2O补齐到10μL。上述体系混匀后37℃反应5个小时。加入1μL DNase，37℃反应15分钟。利用回收试剂盒(Ambion,Life Technologies,AM1908)回收转录的sgRNA，步骤如下：上步反应体积加入90μL Elution solution移植1.5mlEP管；加入350μL Binding solution混匀；加入250μL无水乙醇混匀；上柱；10000转/分钟离心30秒，倒掉废液；加入500μL Washing solution，10000 转/分钟离心30秒，倒掉废液；空转1分钟；换收集管，加入100μL Elution solution洗脱；加入10μL醋酸铵(Ambion,Life Technologies,AM1908)混匀；加入275μL无水乙醇混匀；-20℃放置30分钟，同时准备70％乙醇放置-20℃；4℃环境下13000转/分钟离心15分钟。弃上清，加入500μL 70％乙醇；离心5分钟，吸走废液，晾干5分钟；加入20μL的水溶解；取1μL测浓度。
1.3细胞的培养与电转
(1)以HEK293T细胞(购自ATCC)为例，本发明进行真核生物细胞的培养与转染：HEK293T细胞接种培养于添加10％FBS的DMEM高糖培养液中(HyClone,SH30022.01B),其中含penicillin(100U/ml)和streptomycin(100μg/ml)。
(2)转染前两个小时换成无抗生素的培养基，利用LONZA转染试剂(SF KIT)按照说明书转染，细胞通过计数得1X10 6个。将Cas9(Sigma：ESPCAS9PRO-50UG)，sgRNA和ssODN按照3μg，1.5μg和3μg的质量混合。电转程序采用DS150，电转后的细胞在6cm的平皿中培养两天。
(3)细胞通过流式分选仪，分选单细胞培养，等两周以后，通过裂解鉴定基因型，裂解液的成分为50mM KCl,1.5mM MgCl 2,10mM Tris pH 8.0,0.5％Nonidet P-40,0.5％Tween 20,100μg/ml protease K。挑选纯合突变的细胞株扩大培养。
(4)对突变的细胞株，通过http://crispr.mit.edu/,https://crispr.cos.uni-heidelberg.de/鉴定出7个相关的脱靶位点，如表1所示。
(5)通过设计相应的引物对脱靶位点进行鉴定，引物序列如表3所示。扩增的PCR产物经过T7EN1和测序鉴定，并没有发现脱靶(图3)。
1.4突变细胞株的修复(图4，图5)
(1)本发明对突变细胞株进行培养与转染：HEK293T细胞接种培养于添加10％FBS的DMEM高糖培养液中(HyClone,SH30022.01B),其中含penicillin(100U/ml)和streptomycin(100μg/ml)。
(2)在转染前分至6孔板中，待密度达到70％-80％时进行转染。
(3)转染以脂质体转染为例。按照Lipofectamine TM 2000 Transfection Reagent(Invitrogen,11668-019)的操作手册，将2μg BE3或YE1-BE3或YEE-BE3质 粒与1μg re-sgRNApGL3-U6-sgRNA质粒混匀,共转染至每孔细胞中，6-8小时后换液，72小时后收取细胞。
(4)对收集到的细胞首先进行PCR产物测序，发现存在修复的峰值(图3)。进一步得，对PCR产物进行TA克隆，在挑选出来的克隆里面，修复的克隆效率有50％。
(5)我们对修复的re-sgRNA进行脱靶分析，同样，我们找到了8个潜在的脱靶位点，如表2和图3所示，同样，我们对潜在脱靶位点进行了分析和检测(表3，图6)，同样，没有发现脱靶现象。
表1 mt-sgRNA潜在的脱靶位点
Figure PCTCN2018096968-appb-000001
表2 re-sgRNA潜在的脱靶位点
Figure PCTCN2018096968-appb-000002
表3 本项目所使用的引物序列
Figure PCTCN2018096968-appb-000003
Figure PCTCN2018096968-appb-000004
Figure PCTCN2018096968-appb-000005
实施例2
本实施例中，在人胚胎中利用碱基编辑系统对突变进行修复(图7)。
2.1质粒构建
在突变位点附近，依据碱基编辑作用的特点，设计修复re-sgRNA(SEQ ID NO.3)，合成oligos，上游序列为5’-taggCTACGTGTTAACACCATTGG-3’(SEQ ID NO.18)，下游序列为5’-aaacCCAATGGTGTTAACACGTAG-3’(SEQ ID NO.19)。上下游序列通过程序(95℃，5min；95℃-85℃ at-2℃/s；85℃-25℃ at-0.1℃/s；hold at 4℃)退火，连接到经过BsaI(NEB：R0539L)线性化的PUC57-T7sgRNA载体上。线性化体系与程序如上。连接体系如下：T4连接buffer(NEB：M0202L)1μL，线性化载体20ng，退火的oligo片段(10μM)5μL，T4连接酶(NEB：M0202L)0.5μL，ddH 2O补齐到10μL.16℃连接过夜。连接的载体通过转化，挑菌，鉴定，鉴定引物为上游序列：5’-CGCCAGGGTTTTCCCAGTCACGAC-3’(SEQ ID NO.21)，下游序列为相应oligo的下游序列。对阳性克隆摇菌提取质粒(Axygene：AP-MN-P-250G)测定浓度备用。
2.2 sgRNA的体外转录
以构建的PUC57-T7sgRNA为模板，扩增含有sgRNA的片段，所用引物为：F:5’-TCTCGCGCGTTTCGGTGATGACGG-3’(SEQ ID NO.22)，,R: 5’-AAAAAAAGCACCGACTCGGTGCCACTTTTTC-3’(SEQ ID NO.23)，。扩增体系如下：2Xbuffer(诺唯赞：P505)25μL；dNTP 1μL；F(10pmol/μL)2μL；R(10pmol/μL)2μL；模板1μg；DNA聚合酶(诺唯赞：P505)0.5μL；ddH 2O补齐到50μL。扩增出来的PCR产物经过下述步骤纯化：每100μL体积加4μL RNAsecure(Life：AM7005)；60℃15分钟；加入三倍体积的PCR-A(Axygen：AP-PCR-250G)过柱，离心，12000转/分钟离心1分钟；加入500μL W2，离心1分钟；空转1分钟；加入20μL无RNAase水洗脱。
利用体外转录试剂盒(Ambion,Life Technologies,AM1354)转录，步骤如下：反应体系为：reaction buffer 1μL；enzyme mix 1μL；A 1μL；T 1μL；G 1μL；C 1μL；模板800ng；H 2O补齐到10μL。上述体系混匀后37℃反应5个小时。加入1μLDNase，37℃反应15分钟。利用回收试剂盒(Ambion,Life Technologies,AM1908)回收转录的sgRNA，步骤如下：上步反应体积加入90μL Elution solution移植1.5mlEP管；加入350μLBinding solution混匀；加入250μL无水乙醇混匀；上柱；10000转/分钟离心30秒，倒掉废液；加入500μL Washing solution，10000转/分钟离心30秒，倒掉废液；空转1分钟；换收集管，加入100μLElution solution洗脱；加入10μL醋酸铵(Ambion,Life Technologies,AM1908)混匀；加入275μL无水乙醇混匀；-20℃放置30分钟，同时准备70％乙醇放置-20℃；4℃环境下13000转/分钟离心15分钟。弃上清，加入500μL的70％乙醇；离心5分钟，吸走废液，晾干5分钟；加入20μL的水溶解；取1μL测浓度。
2.3 BE3的体外转录
BE3酶切回收。本步骤是将质粒BE3进行线性化。体系如下：BE3/YE1-BE3/YE2-BE3/YEE-BE3 10μg；buffer I(NEB：R0539L)10μL；BbsI 4μL(NEB：R0539L)；H 2O补齐到100μL。混匀之后，37℃酶切过夜。
线性化质粒的回收。酶切产物中加入4μL RNAsecure(Life：AM7005),60℃反应10分钟；利用回收试剂盒(QIAGEN：28004)进行操作其余步骤，加入5倍体积buffer PB，过柱；加入750μL buffer PE离心；空转1分钟；用10μL水洗脱，测定浓度。
体外转录。按照试剂盒(Invitrogen：AM1345)的要求依次加入体系：1入g线性化载体；10μL2XNTP/ARCA；补齐到20μL水；2μL T7ezyme mix；2μL 10xreaction buffer。混合之后37℃反应2小时。加入1μL DNasea反应15分钟。
加尾。转录产物进行加尾处理保证转录mRNA的稳定性。具体体系如下：20μL反应产物；36μL H 2O；20μL 5xE-PAP buffer；10μL 25mM MnCl 2；10μL ATP solution；4μL PEP。反应体系混匀后37℃反应30分钟。
回收。利用回收试剂盒进行(QIAGEN：74104)。步骤如下：上步反应产物加入350μL buffer RLT；加入250μL无水乙醇，过柱，离心；加入500μL RPE，离心，加入500μL RPE，离心；空转；加入30μL水洗脱。测定浓度后-80℃保存。
2.4突变胚胎的获得
所有的胚胎操作均在广州医科大学附属第三医院的生殖医学中心进行，本实验已经通过了医院的伦理委员会的审核，捐献卵子和精子的患者已经签署了知情同意书。本实验获得了带有FBN1 T7498C的杂合突变患者，并通过血液和精液对其基因型进行了鉴定(图1)。所使用的卵子均是生殖障碍患者不成熟卵，经过体外培养成熟。通过ICSI将突变精子与卵子结合，获得了突变的受精卵。
2.5胚胎的修复操作
当观察受精卵是2PN阶段时，通过显微操作，将体积大概为0.2μL的碱基编辑mRNA，本实施中使用的是BE3，和修复所用的sgRNA胞浆注射到受精卵中，BE3和sgRNA的浓度分别是100ng/μL和50ng/μL。处理过的胚胎细胞在三气培养箱中继续培养三天(图7)。
2.6胚胎的扩增与鉴定
对收集到的胚胎进行单细胞扩增，所用试剂为Vazyme,N601-01。扩增后的基因组经过100倍稀释，用来扩增目的片段，以检测突变的效率(图8)。扩增目的片段引物见表3。
2.7深度测序确定编辑效率
为了更进一步确认编辑的效率，采用PE150高通量测序的方式，对目的片段进行检测，选择了三个对照胚胎和所有的7个修复胚胎，发现对照组有3个胚胎是杂合子基因型，而7个修复胚胎都是正常基因型，证明了碱基编辑系统可以高效的实现靶位点的修复。
2.8对胚胎脱靶位点的检测
为确保碱基编辑系统的安全性。我们将三个对照胚胎和所有的修复胚胎进行了脱靶检测，结果显示(图9)，在修复胚胎中并没有发现明显的脱靶现象。
Figure PCTCN2018096968-appb-000006
Figure PCTCN2018096968-appb-000007
Figure PCTCN2018096968-appb-000008
Figure PCTCN2018096968-appb-000009
Figure PCTCN2018096968-appb-000010
Figure PCTCN2018096968-appb-000011
Figure PCTCN2018096968-appb-000012
Figure PCTCN2018096968-appb-000013
Figure PCTCN2018096968-appb-000014
Figure PCTCN2018096968-appb-000015
Figure PCTCN2018096968-appb-000016
Figure PCTCN2018096968-appb-000017
Figure PCTCN2018096968-appb-000018
Figure PCTCN2018096968-appb-000019
Figure PCTCN2018096968-appb-000020
Figure PCTCN2018096968-appb-000021
Figure PCTCN2018096968-appb-000022
Figure PCTCN2018096968-appb-000023
Figure PCTCN2018096968-appb-000024
Figure PCTCN2018096968-appb-000025
Figure PCTCN2018096968-appb-000026
Figure PCTCN2018096968-appb-000027
Figure PCTCN2018096968-appb-000028
Figure PCTCN2018096968-appb-000029
Figure PCTCN2018096968-appb-000030
Figure PCTCN2018096968-appb-000031
Figure PCTCN2018096968-appb-000032
Figure PCTCN2018096968-appb-000033
Figure PCTCN2018096968-appb-000034

Claims (9)

  1. 一种高效修复FBN1 T7498C突变的试剂盒，其特征在于，包括碱基编辑系统以及针对FBN1 T7498C位点的修复re-sgRNA。
  2. 如权利要求1所述的高效修复FBN1 T7498C突变的试剂盒，其特征在于，所述的碱基编辑系统为BE3，YE1-BE3，YE2-BE3或者YEE-BE3中的一种。
  3. 如权利要求1所述的高效修复FBN1 T7498C突变的试剂盒，其特征在于，所述的针对FBN1 T7498C位点的修复re-sgRNA的序列为SEQ ID NO.3。
  4. 一种制作突变和修复突变的组合，其特征在于，包括根据FBN1 T7498C位点设计突变mt-sgRNA和相应的突变ssODN、针对FBN1 T7498C位点的修复re-sgRNA以及碱基编辑系统中的至少一种。
  5. 一种碱基编辑修复突变的方法，其特征在于，包括：在含有FBN1 T7498C的突变细胞中，利用针对FBN1 T7498C位点的修复re-sgRNA引导碱基编辑系统到突变位点进行碱基编辑修复，收集转染后的细胞。
  6. 如权利要求5所述的碱基编辑修复突变的方法，其特征在于，所述的含有FBN1 T7498C的突变细胞为HEK293T细胞。
  7. 如权利要求5所述的碱基编辑修复突变的方法，其特征在于，所述的含有FBN1 T7498C的突变细胞的构建方法为：根据FBN1 T7498C位点设计突变mt-sgRNA和相应的突变ssODN；构建mt-sgRNA的表达载体，体外将Cas9蛋白和转录出来的mt-sgRNA组成RNP结合ssODN的方式并电转HEK293T细胞，流式分选单细胞鉴定出含有FBN1 T7498C的突变细胞株。
  8. 如权利要求5所述的碱基编辑修复突变的方法，其特征在于，所述的针对FBN1 T7498C位点的修复re-sgRNA通过根据FBN1 T7498C位点设计修复re-sgRNA，并构建U6启动和/或T7启动的表达载体得到。
  9. 如权利要求7所述的碱基编辑修复突变的方法，其特征在于，所述的mt-sgRNA的序列为SEQ ID NO.1，ssODN的序列为SEQ ID NO.2，re-sgRNA的序列为SEQ ID NO.3。
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