WO2022247629A1 - 非同源双链寡聚核苷酸片段在基因敲除系统中的应用 - Google Patents

非同源双链寡聚核苷酸片段在基因敲除系统中的应用 Download PDF

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WO2022247629A1
WO2022247629A1 PCT/CN2022/092012 CN2022092012W WO2022247629A1 WO 2022247629 A1 WO2022247629 A1 WO 2022247629A1 CN 2022092012 W CN2022092012 W CN 2022092012W WO 2022247629 A1 WO2022247629 A1 WO 2022247629A1
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stranded oligonucleotide
sequence
oligonucleotide fragment
homologous double
gene knockout
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谢红娴
兰凯
黄龙
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珠海舒桐医疗科技有限公司
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    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
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Definitions

  • the invention belongs to the technical field of gene editing, and in particular relates to the application of non-homologous double-stranded oligonucleotide fragments in a gene knockout system.
  • the knockout efficiency of the existing gene knockout system (CRISPR/Cas nuclease system) on the target gene is not high, which limits the development and application of the CRISPR/Cas nuclease system; at the same time, in order to detect off-target effects, it is necessary to assume the off-target site in advance Similar to the on-target site, but the number and location of off-target breakpoints are difficult to predict because many off-target mutations occur in places that are very different from the on-target site. How to improve the target gene knockout efficiency of the gene knockout system and monitor off-target effects at the same time has become the focus of research. For example, using CRISPR/Cas9 to knock out the HPV18 E7 gene, the editing efficiency needs to be further improved, and the off-target situation is unknown, which limits its further clinical application.
  • patent CN111718418A discloses a fusion protein that enhances gene editing
  • patent CN112601812A discloses a reagent for promoting homology-directed DNA repair used in hematopoietic stem cell and/or progenitor cell gene therapy
  • patent CN107532162A discloses the use of editing oligonucleotide Modification of the genome sequence in the cell
  • Patent CN109295060A discloses a paired sgRNA for gene editing and its application. Multiple sgRNAs are designed according to the specific editing requirements of the target gene or target genomic site, and two specific spacings are selected corresponding to the specific position of the PAM.
  • the combined sgRNA acts as a paired sgRNA, and the paired Cas9-sgRNA can improve gene editing efficiency.
  • Replacing cas9 proteins, such as sacas9 has the limitations of insufficient cleavage efficiency and stricter PAM sequence requirements.
  • the modification of sgRNA only achieves better editing efficiency for specific target genes, and there is a problem of low universality.
  • the purpose of the present invention is to provide the application of non-homologous double-stranded oligonucleotide fragments in the gene knockout system, which can increase the cutting breakpoint of the gene knockout system to the target gene And it can be integrated into the cutting breakpoint, so as to enhance the knockout effect of the target gene and accurately determine the off-target site.
  • the first aspect of the present invention provides the application of a non-homologous double-stranded oligonucleotide fragment in a gene knockout system, and the non-homologous double-stranded oligonucleotide fragment can improve the cleavage of the target gene by the gene knockout system Efficiency and ability to integrate at cutting breakpoints.
  • non-homologous double-stranded oligonucleotide fragments refer to double-stranded oligonucleotide fragments that have no homologous sequence with the whole human genome, preferably, the length of the non-homologous double-stranded oligonucleotide fragments Less than or equal to 50bp.
  • the non-homologous double-stranded oligonucleotide fragment can stimulate non-homologous end-joining repair in cells and introduce more insertions or deletions.
  • cells are co-transfected with the CRISPR system for knocking out target genes and non-homologous double-stranded oligonucleotide fragments.
  • the non-homologous double-stranded oligonucleotide fragment can also be used as a marker for monitoring the off-target effect of the gene knockout system.
  • GUIDE-seq method was used to analyze the integration of non-homologous double-stranded oligonucleotide fragments Position determination of off-target sites.
  • the inventors found that the cutting effect of the CRISPR system constructed with sgRNA is still not ideal only by screening out sgRNA with high target activity.
  • the inventors combined non-homologous oligonucleotide fragments with a gene knockout system, explored and verified non-homologous single-stranded oligonucleotide fragments (non-homologous ssODN) and non-homologous ssODN respectively.
  • non-homologous dsODN homologous double-stranded oligonucleotide fragments on the target gene in the gene knockout system was found to be better than that of non-homologous ssODN, which can significantly enhance the gene knockout of the CRISPR/Cas9 system remove the effect. It can be seen that even though both are non-homologous oligonucleotide fragments, there are significant differences between dsODN and ssODN in terms of target gene knockout efficiency.
  • CRISPR/Cas9-induced cellular double-strand DNA breaks are mainly repaired through two pathways: non-homologous end-joining repair (NHEJ) and homologous recombination-mediated repair (HDR).
  • NHEJ non-homologous end-joining repair
  • HDR homologous recombination-mediated repair
  • NHEJ includes: accurate non-homologous end-joining repair (accurate NHEJ) and inaccurate non-homologous end-joining repair (inaccurate NHEJ).
  • DSBs are mainly repaired by the NHEJ pathway and less by the HDR pathway.
  • the inventors used the experimental method of amplicon sequencing to detect the insertion or deletion (Indel) of the target gene locus, which represents inaccurate non-homologous end joining repair, and found that co-transfection of non-homologous dsODN can significantly improve the target locus. Indel rate. According to the results of amplicon sequencing, the inventors found that, unlike ssODN (which tends to be repaired by the HDR pathway), non-homologous dsODN is more inclined to stimulate inaccurate non-homologous end-joining repair pathways, which is expected to be applied to gene knockout systems, Improve the efficiency of target gene knockout.
  • non-homologous double-stranded oligonucleotide fragments are inserted into the cutting breakpoints, and they can also be used as markers to accurately locate off-target sites, and monitor off-target effects while significantly enhancing the knockout efficiency of target genes.
  • the CRISPR system is preferably a CRISPR/Cas9 system.
  • the research on the CRISPR/Cas9 system is relatively comprehensive, but its target gene knockout efficiency is low and there are still serious off-target effects. Knockout effects and pinpoint off-target sites.
  • sequence of the non-homologous double-stranded oligonucleotide fragment is preferably as shown in SEQ ID NO.6, or a sequence with at least 80% and above homology, preferably at least 85% and above homology Sex, more preferably at least 90% and above homology, more preferably at least 95% and above homology, still more preferably at least 98% and above homology; and reverse to the sequence Complementary paired sequences.
  • the 5' end of the non-homologous double-stranded oligonucleotide fragment is modified by phosphorylation, two of the three nucleotides at the 5' end are connected by glucosinolate modification, and the 3' A glucosinolate modification is carried out between two consecutive nucleotides among the three nucleotides.
  • Phosphorylation modification at the 5' end is conducive to the integration of non-homologous double-stranded oligonucleotide fragments with the cutting end; glucosinolate modification is conducive to improving the stability of non-homologous double-stranded oligonucleotide fragments when they exist in cells not easily degraded.
  • the sequence of the non-homologous double-stranded oligonucleotide fragment is: 5'-P-G*T*TTAATTGAGTTGTCATATGTTAATAACGGT*A*T-3' and 5'-P-A*T*ACCGTTATTAACATATGACAACTCAATTAA *A*C-3', wherein, "P” indicates 5' phosphorylation, and "*” indicates a glucosinolate-modified linkage between two nucleotides.
  • the second aspect of the present invention provides a gene knockout system, which includes a CRISPR system and a non-homologous double-stranded oligonucleotide fragment.
  • the CRISPR system is a CRISPR/Cas9 system.
  • the CRISPR system for knocking out the target gene and the non-homologous double-stranded oligonucleotide fragments are co-transfected into the cells.
  • the sequence of the non-homologous dsODN is shown in SEQ ID NO.6, or a sequence having at least 80% or more homology, preferably at least 85% or more homology, more preferably having At least 90% and above homology, more preferably at least 95% and above homology, still more preferably at least 98% and above homology; and a sequence that is reverse complementary to said sequence.
  • the 5' end of the non-homologous double-stranded oligonucleotide fragment is modified by phosphorylation, two of the three nucleotides at the 5' end are connected by glucosinolate modification, and the 3' A glucosinolate modification is carried out between two consecutive nucleotides among the three nucleotides.
  • Phosphorylation modification at the 5' end is conducive to the integration of non-homologous double-stranded oligonucleotide fragments and cleavage endpoints; glucosinolate modification is conducive to improving the stability of non-homologous double-stranded oligonucleotide fragments inserted into the cleavage endpoints.
  • the gene knockout system is aimed at the knockout of human papillomavirus type 18 E7 gene.
  • the sequence of the sgRNA of the CRISPR system is shown in SEQ ID NO.1, or a sequence having at least 80% and above homology, preferably at least 85% and above homology , more preferably having a homology of at least 90% and above, more preferably having a homology of at least 95% and above, and still more preferably having a homology of at least 98% and above;
  • sequence of the sgRNA is as shown in SEQ ID NO.2, or a sequence having at least 80% and above homology, preferably at least 85% and above homology, more preferably at least 90% and above
  • the above homology is more preferably at least 95% and above, and still more preferably at least 98% and above.
  • the sgRNA and Cas9 cloning expression vector PX330 capable of combining with the human papillomavirus type 18 E7 gene were obtained to obtain a plasmid targeting knockout of the human papillomavirus type 18 E7 gene;
  • Cells were co-transfected with a plasmid for targeted knockout of the human papillomavirus E7 gene and non-cognate dsODN.
  • the expression plasmid and non-homologous dsODN were co-transfected into the cells to specifically induce frameshift mutations in the HPV oncogenic elements of the corresponding HPV subtype positive cells , lose carcinogenic properties, and even lead to apoptosis directly due to excessive DSBs. It effectively enhances the efficiency of the CRISPR/Cas9 system to specifically knock out the high-risk HPV E7 gene, so as to achieve the purpose of reducing viral load, clearing virus and diseased cells, and reversing canceration, which has important clinical application value.
  • the system is used for targeted knockout of the human papillomavirus type 18 E7 gene, and the sequence of the non-homologous dsODN is shown in SEQ ID NO.6, or has at least 80% or more homology with it
  • the sequence preferably has a homology of at least 85% and above, more preferably has a homology of at least 90% and above, still more preferably has a homology of at least 95% and above, and still more preferably has at least 98% and above Homology of the above; and a sequence that is a reverse complementary pair to said sequence.
  • the present invention has the following advantages compared with the prior art:
  • non-homologous double-stranded oligonucleotide fragments can stimulate non-homologous end-joining repair in cells, introducing more insertions/deletions, and non-homologous double-stranded oligonucleotide fragments can be inserted
  • the GUIDE-seq method can be used as a marker to accurately locate the off-target site, which helps to enhance the knockout efficiency of the target gene and monitor the off-target effect at the same time.
  • Fig. 1 is the sequence diagram of the non-homologous double-stranded oligonucleotide fragment (dsODN) used in the embodiment;
  • Figure 2 is a schematic diagram of the effect of CRISPR/Cas9 co-transfection of non-homologous dsODN knockout of high-risk HPV E7 gene;
  • FIG. 3 shows the application of CRISPR/Cas9 targeting HPV18 E7 to co-transfect non-homologous dsODN in the embodiment, and ODN integration was detected at the breakpoint of the target gene.
  • blade represents the breakpoint PCR result of the untreated group
  • - represents the breakpoint PCR result of the HPV18 E7-targeted CRISPR/Cas9 plasmid group transfected only
  • ss represents the co-transfected HPV18 E7-targeting CRISPR/Cas9 Plasmid and non-homologous single-stranded oligonucleotide fragment (ssODN) group breakpoint PCR results
  • ds represents the co-transfected CRISPR/Cas9 plasmid targeting HPV18 E7 and non-homologous double-stranded oligonucleotide fragment ( dsODN) group breakpoint PCR results;
  • Fig. 4 is the Sanger sequencing result of the PCR product Sanger sequencing of the non-homologous dsODN group breakpoint using the CRISPR/Cas9 targeting HPV18 E7 in the embodiment;
  • Fig. 5 is the expression level of HPV18 E7 mRNA after applying the CRISPR/Cas9 plasmid targeting HPV18 E7 and non-homologous dsODN to cotransfect HeLa cells in the embodiment.
  • “blank” represents the expression level of HPV18 E7 mRNA in the untreated group
  • "-” represents the expression level of HPV18 E7 mRNA in the group transfected only with CRISPR/Cas9 plasmids targeting HPV18 E7
  • ss represents the co-transfection of CRISPR targeting HPV18 E7 /Cas9 plasmid and non-homologous ssODN group HPV18 E7 mRNA expression level
  • “ds” represents the HPV18 E7 mRNA expression level of co-transfected CRISPR/Cas9 plasmid targeting HPV18E7 and non-homologous dsODN group
  • ds represents the HPV18 E7 m
  • Fig. 6 is the HPV18-E7 gene knockout efficiency (Indel%) after applying the CRISPR/Cas9 plasmid targeting HPV18 E7 and non-homologous dsODN co-transfecting HeLa cells in the embodiment.
  • "-" in the figure represents only the CRISPR/Cas9 plasmid group targeting HPV18 E7 transfection
  • "ss” represents the co-transfection group of CRISPR/Cas9 plasmid targeting HPV18 E7 and non-homologous ssODN group
  • ds represents the co-transfection targeting CRISPR/Cas9 plasmid and non-homologous dsODN set of HPV18 E7.
  • NON-ODN% represents the proportion of insertion or deletion (Indel) but no ODN insertion at the breakpoint
  • ODN% represents the proportion of ODN insertion at the breakpoint;
  • Figure 7 is a schematic diagram of the GUIDE-Seq library construction process
  • Fig. 8 shows the off-target situation monitored by co-transfected dsODN as a marker in the embodiment.
  • 20bp target site refers to the DNA sequence of the targeted target gene
  • PAM is the protospacer sequence adjacent motif.
  • ⁇ indicates that the bases of the monitored sites are consistent with the target DNA sequence, all " ⁇ ” indicate that the site is on the target site, and any inconsistency indicates that the site is an off-target site.
  • the primers used in the examples were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., and the unmarked primers were commonly used primers in this field; qRT-PCR reagents were Premix Ex Taq TM was purchased from Bao Biological Engineering (Dalian) Co., Ltd. (Code No. RR420A).
  • Embodiment 1 non-homologous ODN sequence (SEQ ID NO.6) and method of obtaining.
  • sequence of ssODN donor is:
  • Oligo 1 5′-P-G*T*TTAATTGAGTTGTCATATGTTAATAACGGT*A*T-3′
  • Oligo 2 5′-P-A*T*ACCGTTATTAACATATGACAACTCAATTAA*A*C-3′
  • Embodiment 2 construction of the CRISPR/Cas9 plasmid expression vector targeting HPV18 E7
  • the uppercase letters are the crRNA sequence, that is, the sequence that binds to the target DNA
  • the lowercase letters are the scaffold, that is, the secondary structure region where the sgRNA plays a role.
  • CRISPR/Cas9-E7 expression vector construction method refers to the construction method in the citation to complete the construction (Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819-823, doi: 10.1126/science.1231143 (2013).):
  • Example 3 Verification that dsODN can be integrated into DSB after co-transfection of CRISPR/Cas9-E7 plasmid and non-homologous dsODN
  • the CRISPR/Cas9-E7 system targeting HPV18 E7 expressed after the CRISPR/Cas9-E7 plasmid is transfected into cells can quickly recognize the HPV18 E7 sequence and play a cutting role. After cleavage, cells are rapidly repaired mainly through the NHEJ pathway that is not restricted by the cell cycle, thereby introducing small fragment insertions or deletions (indels), resulting in frameshift mutations, and ultimately loss of E7 function and inhibition of HPV18 E7 oncoprotein expression.
  • indels small fragment insertions or deletions
  • dsODN can be integrated into the breakpoint, on the other hand, it can stimulate the cellular NHEJ pathway and introduce more indels, thereby improving the knockout efficiency of the target gene.
  • the specific operation method is:
  • HPV18-positive cervical cancer cell line HeLa was cultured in DMEM complete medium containing 10% serum in a 37°C, 5% CO 2 incubator. When the cell confluence reached 90%, it was digested with 0.25% trypsin, then digested with DMEM complete medium, seeded into a 6-well plate, and cultured for 24 hours.
  • the transfection can be carried out. Transfect each well with 2 ⁇ g of CRISPR/Cas9-E7 plasmid and 2 ⁇ L of non-homologous dsODN, and use Roche’s X-tremeGENE HP DNA Transfection Reagent transfection reagent to perform transfection according to the instructions.
  • the transfected cells continued to be cultured in a 37°C, 5% CO 2 incubator.
  • digest with 0.25% trypsin routinely, stop the digestion with DMEM complete medium, collect the cells into a centrifuge tube, centrifuge at 300g for 5 minutes, discard the medium, wash once with PBS, centrifuge again at 300g for 5 minutes, discard Remove PBS, obtain cell slag, use cell genome extraction kit (Quanshijin Biotechnology Co., Ltd., article number: EE101-01) to extract cell genome DNA, and measure DNA concentration.
  • cell genome extraction kit Quanshijin Biotechnology Co., Ltd., article number: EE101-01
  • Primers were designed according to the HPV18 E7 gene sequence and non-homologous dsODN, one end of the primer was on the dsODN, and the other end was on the target gene.
  • the corresponding primer sequences in this example are:
  • ODN-F TTGAGTTGTCATATGTTAATAACGGT (SEQ ID NO. 7).
  • HPV18-E7-R GTTGCTTACTGCTGGGATGC (SEQ ID NO. 8).
  • Example 4 qRT-PCR clarifies the impact of cotransfection of CRISPR/Cas9-E7 plasmid and dsODN on HPV 18 E7 mRNA transcription level
  • Two-phase separation Add 0.2ml of chloroform to every 1ml of TRIZOL reagent lysed sample, and tightly cap the tube. Shake the tube vigorously by hand for 15 seconds, and incubate at 15 to 30°C for 2 to 3 minutes. Centrifuge at 12000 rpm for 15 minutes at 4°C. After centrifugation, the mixed liquid will be divided into the lower red phenol chloroform phase, the middle layer and the upper layer of the colorless aqueous phase. The RNA was all partitioned into the aqueous phase. The volume of the upper layer of the aqueous phase is about 60% of the TRIZOL reagent added during homogenization.
  • RNA precipitation Transfer the upper layer of the aqueous phase to a clean RNase-free centrifuge tube. Add an equal volume of isopropanol and mix to precipitate the RNA. After mixing, incubate at 15 to 30°C for 10 minutes, then centrifuge at 12000rpm at 4°C for 10 minutes. At this point the RNA pellet that was not visible prior to centrifugation will form a gelatinous pellet on the bottom and sides of the tube.
  • RNA washing Remove the supernatant, add at least 1 ml of 75% ethanol (75% ethanol prepared with DEPCH 2 O) to each 1 ml of the sample lysed by TRIZOL reagent, and wash the RNA precipitate. After mixing, centrifuge at 7000 rpm for 5 minutes at 4°C.
  • RNA drying Carefully suck off most of the ethanol solution, and dry the RNA pellet in air at room temperature for 5-10 minutes.
  • Dissolving RNA precipitation When dissolving RNA, first add 40 ⁇ l of RNase-free water and pipette several times with a gun to dissolve it completely, and store the obtained RNA solution at -80°C until use.
  • reaction solution preparation is carried out on ice
  • the Applied Biosystems 7500 Fast Real-Time PCR System was used for the Real Time PCR reaction, and the reaction procedure used the two-step PCR amplification standard procedure:
  • FIG. 5 shows that the HPV18 E7 mRNA transcript level in the HeLa cell group co-transfected with CRISPR/Cas9-E7 plasmid and dsODN was significantly reduced, indicating that the effect of targeted knockout of HPV18 E7 gene is better than that of the same amount of CRISPR/Cas9-E7 plasmid transfected Cell group, cell group transfected with equal amount of CRISPR/Cas9-E7 plasmid and co-transfected with non-homologous ssODN.
  • Example 5 Amplicon sequencing further evaluates the editing efficiency of co-transfected CRISPR/Cas9-E7 plasmids and non-homologous dsODN on target genes
  • Example 3 Using the genomic DNA extracted in Example 3 as a template, primers were designed according to the target and a PCR reaction was performed.
  • the reagent used in this experiment is KAPA HiFi HotStart ReadyMix (product number KK2602) from KAPA Biosystems.
  • Primers are designed according to the target, and the two ends of the primer are located on both sides of the target.
  • the corresponding primer sequences in this example are:
  • E7-NGS-F ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGCATGGACCTAAGGCAACA (SEQ ID NO. 9).
  • E7-NGS-R gtgactggagttcagacgtgtgctcttccgatctgctcaattctggcttcacact (SEQ ID NO. 10).
  • Reaction conditions 98°C for 3min; 25cycles of(98°C for 20s, 65°C for 15s, 72°C for 15s), 72°C for 1min, 4°C for ⁇
  • Reaction conditions 98°C for 3min; 11cycles of(98°C for 20s, 65°C for 15s, 72°C for 15s), 72°C for 1min, 4°C for ⁇
  • the Indel% of the HeLa cell group co-transfected with CRISPR/Cas9-E7 plasmid and non-homologous dsODN can reach 40%.
  • the cell group with the same amount of CRISPR/Cas9-E7 plasmid and non-homologous ssODN increased by about 25% respectively, and the ODN insertion at the representative breakpoint accounted for about 66% of the total indel; when the sgRNA was E7-sgRNA2 , the Indel% of the HeLa cell group co-transfected with CRISPR/Cas9-E7 plasmid and non-homologous dsODN can reach about 65%.
  • the cell group of E7 plasmid and non-homologous ssODN increased by about 20% respectively, wherein "ODN%" means that the ODN insertion at the breakpoint accounted for about 17% of the total indel.
  • Cotransfection of non-homologous dsODN with CRISPR/Cas9-E7 plasmid can significantly enhance the efficiency of targeted knockout of human papillomavirus E7 gene.
  • the Y-shaped adapter is made by annealing the Miseq universal oligonucleotide (MiSeq Common Adapter) to each sample barcode adapter primer (A##adapter): wherein, the sequence of the Miseq universal oligonucleotide adapter is P- GATCGGAAGAGC*C*A (“P” means phosphorylation, “*” means glucosinolate modification), the sequence of the sample barcode adapter primer is:
  • NWNNWNN is the molecular index label
  • * indicates glucosinolate modification
  • the annealing reaction system for preparing the Y-joint is:
  • the DNA concentration was measured by Qubit.
  • the A260/280 must be between 1.8-2.0 to be qualified.
  • the concentration is greater than 20ng/ ⁇ L and the total amount is greater than 1 ⁇ g.
  • the DNA of each sample was fragmented to an average length of 500bp.
  • reaction system is:
  • reaction system is:
  • PEG/NaCl Solution was taken out in advance and returned to room temperature.
  • reaction system In a 200 ⁇ L reaction tube, add the following (per reaction), the reaction system is:
  • the primer sequence of P5-1 is: aatgatacggcgaccaccgagatcta (SEQ ID NO.11), synthesized by Suzhou Jinweizhi Company.
  • GSP1Primer mixed by GSP1 (+) and GSP1 (-), the sequence of GSP1 (+) primer is: ggatctcgacgctctccctgtttaattgagttgtcatatgttaataac (SEQ ID NO.12); the sequence of GSP1 (-) primer is: ggatctcgacgctctcctataccgttattaacatatgaca (SEQ ID NO.13 ).
  • PEG/NaCl Solution was taken out in advance and returned to room temperature.
  • the primer sequence of P5-2 is: aatgatacggcgaccaccgagatctacac (SEQ ID NO.14), synthesized by Suzhou Jinweizhi Company.
  • GSP2Primer mixed by GSP2 (+) and GSP2 (-), the sequence of GSP2 (+) primer is: cctctctatgggcagtcggtgatacatatgacaactcaattaaac (SEQ ID NO.15); the sequence of GSP2 (-) primer is: cctctctatgggcagtcggtgatttgagttgtcatatgttaataacggta (SEQ ID NO. .16).
  • the sequence of the P7-# primer is: caagcagaagacggcatacgagat(nnnnnnnn)gtgactggagtcctctctatgggcagtcggtga, wherein "nnnnnn” is an 8bp barcode sequence for distinguishing samples.
  • reaction system In a 200 ⁇ L reaction tube, add the following (per reaction), the reaction system is:
  • PEG/NaCl Solution was taken out in advance and returned to room temperature.
  • the samples after the second round of PCR purification were first quantified using qubit, and the purity of the library DNA was checked according to A260/280, and it was qualified between 1.8 and 2.0. For subsequent high-throughput sequencing on the machine.
  • qPCR library quantification Dilute each sub-library to 4nM and then mix equal volumes into a pool.
  • phix library (4nM): pooling library 2 ⁇ L: 6 ⁇ L, total 8 ⁇ L volume.
  • Library denaturation Take 5 ⁇ L from the 8 ⁇ L in the previous step and mix with 5 ⁇ L 0.2M NaOH for denaturation for 5 min (the library concentration at this time is 2 nM).
  • the library in the previous step needs to be diluted to a final concentration of 1.2pM before loading to obtain better data quality.
  • the dilution method is as follows: take 10 ⁇ L of the denatured library obtained in the above step and mix with 990 ⁇ L of HT1 buffer by shaking (the library concentration at this time is 20 pM). Then take 90 ⁇ L of the upper mixture and 1410 ⁇ L of HT1 buffer for shaking and mixing to obtain the upper library with a final concentration of 1.2 pM.
  • test reagent square plate
  • Dilution of upper machine primers the upper machine primers were diluted to a concentration of 0.3 ⁇ M.
  • sequence reads In the sequence obtained by sequencing, the sequence with the same first 6 bases and the sequence of the molecular index tag with the same 8 bases are normalized and integrated together, and they are identified as coming from the same PCR Samples before processing.
  • the integrated sequence was aligned to the human genome reference sequence (GrCh38) using the BWA-MEM software program.
  • the starting matching positions of the reads with an alignment quality ⁇ 50 are retained and displayed, and a 10bp sliding alignment window is used to group the alignment regions.
  • Analyze the reads containing the integrated dsODN sequence and then use the molecular index and SAMtools computer analysis tools based on the bin-consensus mutation calling algorithm to call these positions with dsODN sequences as SNPs and indels, and then compare them to the reference genome
  • the non-target sequence of the dsODN sequence can be determined to determine the specific reference coordinates of the integration of the dsODN sequence on the cell genome, so as to determine whether it is on-target or off-target.
  • the co-electroporated dsODN donor monitored the on-target and off-target status of the CRISPR-Cas9 system at the HPV18 E7 target.
  • Figure 8 shows that when single E7-sgRNA1 is used to target and cut the HPV18 E7 gene, 36505 reads can be detected at the target site, and 1 off-target site can be detected.
  • single E7-sgRNA2 was used to target and cut the HPV18 E7 gene, 75596 reads could be detected at the target site, and 2 off-target sites could be detected.

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Abstract

一种非同源双链寡聚核苷酸片段在基因敲除系统中的应用。基于现有基因敲除系统的靶基因敲除效率不高，且脱靶位点难以准确定位，该方法首次将非同源双链寡聚核苷酸片段与基因敲除系统相结合用于基因敲除，非同源双链寡聚核苷酸片段能够激发细胞的非同源末端连接修复，引入更多插入或缺失，同时非同源双链寡聚核苷酸片段可插入切割断点处，可作为标记物监测脱靶位点，有助于增强靶基因的敲除效率的同时准确监测脱靶效应。

Description

非同源双链寡聚核苷酸片段在基因敲除系统中的应用 技术领域
本发明属于基因编辑技术领域，具体涉及非同源双链寡聚核苷酸片段在基因敲除系统中的应用。
背景技术
随着对基因功能研究的深入，临床把抗病毒治疗方向转向个体化基因编辑领域。然而，现有基因敲除系统(CRISPR/Cas核酸酶系统)对靶基因的敲除效率不高，限制CRISPR/Cas核酸酶系统的发展和应用；同时为了检测脱靶效应，需事先假定脱靶位点与靶标位点相似，但是由于许多脱靶突变发生在与目标位点差异很大的地方，因此脱靶断点的数量和位置是很难预测的。如何提高基因敲除系统的靶基因敲除效率并同时监测脱靶效应成为研究的重点。例如利用CRISPR/Cas9靶向敲除HPV18 E7基因，存在编辑效率有待进一步提高，并且脱靶情况不明等问题，限制了其在临床上的进一步转化应用。
现有技术为了获得更高的编辑效率，通常采用寻找新的核酸酶、或采用筛选靶活性高的sgRNA、或添加外源增效蛋白、或敲入外源基因片段的同源供体、或对sgRNA进行改造等。例如专利CN111718418A公布了一种增强基因编辑的融合蛋白；专利CN112601812A公开了在造血干细胞和/或祖细胞基因疗法中使用的促进同源定向DNA修复的试剂；专利CN107532162A公开了利用编辑寡核苷酸修饰细胞内的基因组序列；专利CN109295060A公开了一种用于基因编辑的配对sgRNA及应用，根据靶标基因或靶标基因组位点的特定编辑需求设计多条sgRNA，选择两条特定间距、对应PAM特定位置组合的sgRNA作为配对sgRNA，配对Cas9-sgRNA可以提高基因编辑效率。更换cas9蛋白，例如sacas9，存在切割效率不足、PAM序列要求更严格的局限性。通过同时添加外源性的小分子化合物提高编辑效率，存在化合物不便于同时递送到细胞内的问题。而对sgRNA的改造仅针对特定的靶基因获得较好的编辑效率，存在普适性不高的问题。
发明内容
本发明的目的是提供非同源双链寡聚核苷酸片段在基因敲除系统中的应用，非同源双链寡聚核苷酸片段能够增加基因敲除系统对靶基因的切割断点并能够整合到切割断点处，从而增强靶基因的敲除效果并能够准确判定脱靶位点。
为达到上述目的，本发明采用的技术方案是：
本发明第一方面提供非同源双链寡聚核苷酸片段在基因敲除系统中的应用，所述非同源双链寡聚核苷酸片段能够提高基因敲除系统对靶基因的切割效率并能够整合到切割断点处。
本发明中非同源双链寡聚核苷酸片段是指与人类全基因组无同源序列的双链寡聚核苷酸片段，优选地，非同源双链寡聚核苷酸片段的长度小于等于50bp。
具体地，所述非同源双链寡聚核苷酸片段能够激发细胞的非同源末端连接修复，引入更多插入或缺失。
具体地，通过将用于敲除靶基因的CRISPR系统和非同源双链寡聚核苷酸片段共转染细胞。
优选地，将所述非同源双链寡聚核苷酸片段还可作为标记物用于监测基因敲除系统的脱靶效应。
具体地，将用于敲除靶基因的CRISPR系统和非同源双链寡聚核苷酸片段共转染细胞后，采用GUIDE-seq方法分析非同源双链寡聚核苷酸片段的整合位置判定脱靶位点。
基于现有基因敲除系统的敲除效果不好且脱靶位点难以准确定位的问题，发明人发现仅通过筛选出靶活性高的sgRNA，以此sgRNA构建的CRISPR系统的切割效果依旧不够理想。为解决此类问题，发明人将非同源寡聚核苷酸片段与基因敲除系统相结合，分别探讨并验证了非同源单链寡聚核苷酸片段(非同源ssODN)和非同源双链寡聚核苷酸片段(非同源dsODN)在基因敲除系统对靶基因的编辑效率，发现非同源dsODN优于非同源ssODN，能够显著增强CRISPR/Cas9系统的基因敲除效果。可见即使同为非同源寡聚核苷酸片段，dsODN和ssODN在靶基因敲除效率方面也存在显著差异。CRISPR/Cas9诱导的细胞双链DNA断裂(DSB)主要通过两种途径修复：非同源末端连接修复(NHEJ)和同源重组介导的修复(HDR)。其中，NHEJ又包括：准确的非同源末端连接修复(accurate NHEJ)和不准确的非同源末端连接修复(inaccurate NHEJ)。在真核细胞中，DSB主要通过NHEJ途径修复，而较少通过HDR途径修复。发明人通过扩增子测序的实验方法，检测靶基因位点的插入或缺失(Indel)，其代表不准确的非同源末端连接修复，发现共转染非同源dsODN可显著提高靶位点Indel率。依据扩增子测序结果，发明人发现，与ssODN(倾向于通过HDR途径修复)不同，非同源dsODN更倾向于激发不准确的非同源末端连接修复途径，有望应用于基因敲除系统，提高靶基因敲除效率。同时将非同源双链寡聚核苷酸片段插入切割断点处，还可以其作为标记物准确定位脱靶位点，在显著增强靶基因的敲除效率的同时监测脱靶效应，可谓一举两得。
以上应用中，所述CRISPR系统优选为CRISPR/Cas9系统。目前CRISPR/Cas9系统的研究比较全面，但是其靶基因敲除效率较低且依然存在严重的脱靶效应，将非同源双链寡聚核苷酸片段与之结合能够显著提高CRISPR/Cas9系统的敲除效果并准确定位脱靶位点。
所述非同源双链寡聚核苷酸片段的序列优选如SEQ ID NO.6所示，或与其具有至少80％及以上的同源性的序列，优选具有至少85％及以上的同源性，进一步优选具有至少90％及以上的同源性，更进一步优选具有至少95％及以上的同源性，再进一步优选具有至少98％及以上的同源性；以及与所述序列反向互补配对的序列。
进一步优选地，所述非同源双链寡聚核苷酸片段的5′端进行磷酸化修饰、5′端的三个核苷酸中相连两个核苷酸之间进行硫甙修饰、3′的三个核苷酸中相连两个核苷酸之间进行硫甙修饰。
5′端进行磷酸化修饰有利于非同源双链寡聚核苷酸片段与切割端点整合；硫甙修饰有利于提高非同源双链寡聚核苷酸片段在细胞内存在时的稳定性而不易被降解。
根据一些具体且优选的实施方式，所述非同源双链寡聚核苷酸片段的序列为：5′-P-G*T*TTAATTGAGTTGTCATATGTTAATAACGGT*A*T-3′和5′-P-A*T*ACCGTTATTAACATATGACAACTCAATTAA*A*C-3′，其中，“P”表示5′磷酸化，“*”表示2个核苷酸之间的硫甙修饰连接。
本发明第二方面提供一种基因敲除系统，其包括CRISPR系统和非同源双链寡聚核苷酸片段。
优选地，所述CRISPR系统为CRISPR/Cas9系统。
优选地，用于敲除靶基因的CRISPR系统和非同源双链寡聚核苷酸片段共转染细胞。
优选地，所述非同源dsODN的序列如SEQ ID NO.6所示，或与其具有至少80％及以上的同源性的序列，优选具有至少85％及以上的同源性，进一步优选具有至少90％及以上的同源性，更进一步优选具有至少95％及以上的同源性，再进一步优选具有至少98％及以上的同源性；以及与所述序列反向互补配对的序列。
进一步优选地，所述非同源双链寡聚核苷酸片段的5′端进行磷酸化修饰、5′端的三个核苷酸中相连两个核苷酸之间进行硫甙修饰、3′的三个核苷酸中相连两个核苷酸之间进行硫甙修饰。5′端进行磷酸化修饰有利于非同源双链寡聚核苷酸片段与切割端点整合；硫甙修饰有利于提高非同源双链寡聚核苷酸片段插入切割端点后的稳定性。
优选地，所述基因敲除系统针对人乳头瘤病毒18型E7基因的敲除。
根据一些具体实施方式，所述的CRISPR系统的sgRNA的序列如SEQ ID NO.1所示，或与其具有至少80％及以上的同源性的序列，优选具有至少85％及以上的同源性，进一步优选具有至少90％及以上的同源性，更进一步优选具有至少95％及以上的同源性，再进一步优选具有至少98％及以上的同源性；
或者所述的sgRNA的序列如SEQ ID NO.2所示，或与其具有至少80％及以上的同源性的序列，优选具有至少85％及以上的同源性，进一步优选具有至少90％及以上的同源性，更进一步优选具有至少95％及以上的同源性，再进一步优选具有至少98％及以上的同源性。
具体地，将能够与人乳头瘤病毒18型E7基因相结合的sgRNA、Cas9克隆表达载体PX330得到一种靶向敲除人乳头瘤病毒18型E7基因的质粒；
将用于靶向敲除人乳头瘤病毒E7基因的质粒和非同源dsODN共转染细胞。
利用构建的靶向高危型HPV E7基因的CRISPR/Cas9表达质粒，通过将表达质粒和非同源dsODN共同转染到细胞中，特异性的诱导相应HPV亚型阳性细胞的HPV致癌元件移码突变，失去致癌特性，甚至直接由于DSB过多导致细胞凋亡。有效增强了CRISPR/Cas9系统特异性敲除高危型HPV E7基因的效率，从而达到降低病毒负荷、清除病毒及病变细胞、逆转癌变的目的，具有重要的临床应用价值。
优选地，所述系统用于靶向敲除人乳头瘤病毒18型E7基因，所述非同源dsODN的序列如SEQ ID NO.6所示，或与其具有至少80％及以上的同源性的序列，优选具有至少85％及以上的同源性，进一步优选具有至少90％及以上的同源性，更进一步优选具有至少95％及以上的同源性，再进一步优选具有至少98％及以上的同源性；以及与所述序列反向互补配对的序列。
由于上述技术方案运用，本发明与现有技术相比具有下列优点：
基于现有基因敲除系统的靶基因敲除效率不高，且脱靶情况不明朗，脱靶位点难以准确定位等问题，首次将非同源双链寡聚核苷酸片段与基因敲除系统相结合用于基因敲除，非同源双链寡聚核苷酸片段能够激发细胞的非同源末端连接修复，引入更多插入/缺失，同时非同源双链寡聚核苷酸片段可插入切割断点处，可作为标记物采用GUIDE-seq方法准确定位脱靶位点，有助于增强靶基因的敲除效率并同时监测脱靶效应。
附图说明
图1为实施例中使用的非同源双链寡聚核苷酸片段(dsODN)的序列示意图；
图2为CRISPR/Cas9共转非同源dsODN敲除高危型HPV E7基因作用示意图；
图3为实施例中应用靶向HPV18 E7的CRISPR/Cas9共转非同源dsODN，靶基因断点处检测到ODN整合。图中“blank”代表未处理组断点PCR结果，“-”代表仅转染靶向HPV18 E7的CRISPR/Cas9质粒组断点PCR结果，“ss”代表共转靶向HPV18 E7的CRISPR/Cas9质粒和非同源单链寡聚核苷酸片段(ssODN)组断点PCR结果，“ds”代表共转靶向HPV18 E7的CRISPR/Cas9质粒和非同源双链寡聚核苷酸片段(dsODN)组断点PCR结果；
图4为实施例中应用靶向HPV18 E7的CRISPR/Cas9共转非同源dsODN组断点PCR产物Sanger测序结果；
图5为实施例中应用靶向HPV18 E7的CRISPR/Cas9质粒和非同源dsODN共转HeLa细胞后HPV18 E7 mRNA表达水平。图中“blank”代表未处理组HPV18 E7 mRNA表达水平，“-”代表仅转染靶向HPV18 E7的CRISPR/Cas9质粒组HPV18 E7 mRNA表达水平，“ss”代表共转靶向HPV18 E7的CRISPR/Cas9质粒和非同源ssODN组HPV18 E7 mRNA表达水平，“ds”代表共转靶向HPV18E7的CRISPR/Cas9质粒和非同源dsODN组HPV18 E7 mRNA表达水平；
图6为实施例中应用靶向HPV18 E7的CRISPR/Cas9质粒和非同源dsODN共转HeLa细胞后HPV18-E7基因敲除效率(Indel％)。图中“-”代表仅转染靶向HPV18 E7的CRISPR/Cas9质粒组，“ss”代表共转靶向HPV18 E7的CRISPR/Cas9质粒和非同源ssODN组，“ds”代表共转靶向HPV18 E7的CRISPR/Cas9质粒和非同源dsODN组。NON-ODN％代表断点处有插入或缺失(Indel)但无ODN插入的比例，ODN％代表断点处有ODN插入的比例；
图7为GUIDE-Seq建库流程示意图；
图8为实施例中共转染的dsODN作为标记物监测的脱靶情况。“20bp target site”指的是靶向的目标基因的DNA序列，PAM为前间区序列临近基序。“·”代表监测到的位点的碱基和目标DNA序列匹配一致，全部为“·”表示这个位点是在靶位点，有不一致的表明这个位点是脱靶位点。
具体实施方式
为更好地说明本发明的目的、技术方案和优点，下面将结合具体实施例对本发明作进一步说明。
实施例中使用的引物由苏州金唯智生物科技有限公司合成，未注明的引物为本领域常用引物；qRT-PCR试剂采用
Figure PCTCN2022092012-appb-000001
Premix Ex Taq TM，购自宝生物工程(大连)有限公司(货号为Code No.RR420A)。
实施例1、非同源ODN序列(SEQ ID NO.6)以及获得方式。
(1)ssODN donor的获得：
ssODN donor的序列为：
5′-P-G*T*TTAATTGAGTTGTCATATGTTAATAACGGT*A*T-3′
其中“P”表示5′磷酸化，“*”表示2个核苷酸之间的硫甙修饰。序列合成和修饰均由苏州金唯智生物公司完成。合成的干粉加入无菌的去离子水，稀释成50μM的浓度用于后续转染。
(2)dsODN donor的获得：
首先合成2条反向互补配对的ssODN，序列如下：
oligo 1：5′-P-G*T*TTAATTGAGTTGTCATATGTTAATAACGGT*A*T-3′
oligo 2：5′-P-A*T*ACCGTTATTAACATATGACAACTCAATTAA*A*C-3′
同样的，其中“P”表示5′磷酸化，“*”表示2个核苷酸之间的硫甙修饰连接。2条互补的ssODN序列合成和修饰均由苏州金唯智生物公司完成。合成的干粉加入无菌的去离子水，稀释成125μM的浓度用于后续的退火步骤。
2条单链ssODN退火形成双链dsODN donor
首先，配置10x的Oligoduplex Annealing Buffer(STE)作为退火所需要的缓冲液：
Figure PCTCN2022092012-appb-000002
然后，在200μL无菌反应管中加入如下反应体系：
Figure PCTCN2022092012-appb-000003
Figure PCTCN2022092012-appb-000004
最后，配置好的退火反应体系在PCR仪中完成，反应程序为：95℃，5min→每30s降1℃(共70个循环)→4℃hold。
实施例2、靶向HPV18 E7的CRISPR/Cas9质粒表达载体的构建
(1)从NCBI网站中查询获得HPV18 E7癌基因的全长序列信息，针对HPV18的E7设计sgRNA，并初步筛选出效果较好的以下sgRNA：
E7-sgRNA1
CGAGCAATTAAGCGACTCAGgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc(SEQ ID NO.1)。
E7-sgRNA2
TCGTGACATAGAAGGTCAACgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc(SEQ ID NO.2)。
E7-sgRNA3
AGAGCCCCAAAATGAAATTCgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc(SEQ ID NO.3)。
E7-sgRNA4
CATTGTGTGACGTTGTGGTTgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc(SEQ ID NO.4)。
E7-sgRNA5
ACGTTGTGGTTCGGCTCGTCgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc(SEQ ID NO.5)。
其中，大写字母为crRNA序列，即与靶DNA结合的序列，小写字母部分为scaffold，即sgRNA发挥作用的二级结构区域。经进一步实验验证，E7-sgRNA3、E7-sgRNA4和E7-sgRNA5的效果不佳，确定用于靶向HPV18 E7的CRISPR/Cas9系统的sgRNA为E7-sgRNA1(SEQ ID NO.1)或E7-sgRNA2(SEQ ID NO.2)。
(2)CRISPR/Cas9-E7的表达载体构建方法参考引文中的构建方法完成构建(Cong,L.et al.Multiplex genome engineering using CRISPR/Cas systems.Science 339,819-823,doi:10.1126/science.1231143(2013).)：
将E7-sgRNA1或E7-sgRNA2和Cas9序列克隆到表达载体PX330上，构建针对HPV18 E7的真核表达载体CRISPR/Cas9-E7质粒，构建完成后，通过常规测序比对确定构建载体序列正确无突变，挑选出完全正确的克隆进行扩增并提取质粒。
实施例3、CRISPR/Cas9-E7质粒及非同源dsODN共转后dsODN能整合至DSB处的验证
CRISPR/Cas9-E7质粒转染到细胞后表达出来的靶向HPV18 E7的CRISPR/Cas9-E7系统，可以迅速识别HPV18 E7序列，发挥切割作用。切割后细胞主要通过不受细胞周期限制的NHEJ途径快速修复，从而引入小片段的插入或缺失(indel)，导致移码突变，最终使得E7功能丧失，HPV18 E7癌蛋白的表达受到抑制。共转dsODN后，一方面dsODN能够整合至断点处，另一方面能够激发细胞NHEJ途径，引入更多indel，从而提高靶基因的敲除效率。
具体操作方法是：
(1)细胞培养
HPV18阳性宫颈癌细胞系HeLa用含有10％血清的DMEM完全培养基在37℃、5％CO 2培养箱里培养。待细胞融合度达到90％时用0.25％的胰酶消化后，用DMEM完全培养基终止消化，接种到6孔板中，继续培养24小时。
(2)CRISPR/Cas9-E7质粒和非同源dsODN共同转染
24小时后，确认细胞贴壁良好，细胞融合度达到80％，即可进行转染。每孔转染2μg CRISPR/Cas9-E7质粒和2μL非同源dsODN，使用Roche公司的X-tremeGENE HP DNA Transfection Reagent转染试剂按照说明书要求进行转染，与未处理的细胞组、转染等量CRISPR/Cas9-E7质粒的细胞组、转染等量CRISPR/Cas9-E7质粒和非同源ssODN的细胞组对照。转染后的细胞继续在37℃、5％CO 2培养箱中培养。
(3)基因组DNA提取
转染48小时后，常规0.25％的胰酶消化，用DMEM完全培养基终止消化，收集细胞到离心管中，300g离心5分钟，弃除培养基，PBS洗涤一次，再次300g离心5分钟，弃除PBS，获得细胞渣，使用细胞基因组提取试剂盒(全式金生物技术有限公司，货号：EE101-01)提取细胞基因组DNA，测量DNA浓度。
(4)引物的设计
根据HPV18 E7基因序列及非同源dsODN设计引物，引物一端在dsODN上，另一端位于靶基因上。本实施例中对应的引物序列为：
ODN-F:TTGAGTTGTCATATGTTAATAACGGT(SEQ ID NO.7)。
HPV18-E7-R:GTTGCTTACTGCTGGGATGC(SEQ ID NO.8)。
(5)PCR反应
以上述提取的基因组DNA为模版，用上述引物进行PCR反应。本实验使用的高保真DNA聚合酶为北京全式金生物科技有限公司的
Figure PCTCN2022092012-appb-000005
PCR SuperMix(货号：AS111-02)。
PCR反应体系：
Figure PCTCN2022092012-appb-000006
PCR反应条件：
Figure PCTCN2022092012-appb-000007
PCR反应完成后，取少量PCR产物进行琼脂糖凝胶电泳，根据电泳结果初步判定PCR产物的浓度以及条带大小是否正确等。
(6)目标条带Sanger测序
图3的琼脂糖凝胶电泳中的ds处有目标条带，说明共转CRISPR/Cas9-E7质粒和非同源dsODN的HeLa细胞组已将dsODN整合至DSB处，图4试验结果显示条带测序结果比对正确。图3的琼脂糖凝胶电泳中的未处理的细胞组、转染等量CRISPR/Cas9-E7质粒的细胞组、转染等量CRISPR/Cas9-E7质粒和ssODN的细胞组均无目标条带显示。
实施例4、qRT-PCR明确共转CRISPR/Cas9-E7质粒和dsODN对HPV 18 E7 mRNA转录水平的影响
(1)样品RNA的抽提
①取冻存已裂解的细胞，室温放置5分钟使其完全溶解。
②两相分离：每1ml的TRIZOL试剂裂解的样品中加入0.2ml的氯仿，盖紧管盖。手动剧烈振荡管体15秒后，15到30℃孵育2到3分钟。4℃下12000rpm离心15分钟。离心后混合液体将分为下层的红色酚氯仿相，中间层以及无色水相上层。RNA全部被分配于水相中。水相上层的体积大约是匀浆时加入的 TRIZOL试剂的60％。
③RNA沉淀：将水相上层转移到一干净无RNA酶的离心管中。加等体积异丙醇混合以沉淀其中的RNA，混匀后15到30℃孵育10分钟后，于4℃下12000rpm离心10分钟。此时离心前不可见的RNA沉淀将在管底部和侧壁上形成胶状沉淀块。
④RNA清洗：移去上清液，每1ml TRIZOL试剂裂解的样品中加入至少1ml的75％乙醇(75％乙醇用DEPCH 2O配制)，清洗RNA沉淀。混匀后，4℃下7000rpm离心5分钟。
⑤RNA干燥：小心吸去大部分乙醇溶液，使RNA沉淀在室温空气中干燥5-10分钟。
⑥溶解RNA沉淀：溶解RNA时，先加入无RNA酶的水40μl用枪反复吹打几次，使其完全溶解，获得的RNA溶液保存于-80℃待用。
(2)按下列组份配制PCR反应液(反应液配制均在冰上进行)
试剂 使用量 终浓度
SYBR Premix Ex Taq(Tli RNaseH Plus)(2×) 10μl 1×
PCR Forward Primer(10μM) 0.4μl 0.2μM ＊1
PCR Reverse Primer(10μM) 0.4μl 0.2μM ＊1
DNA模板(＜100ng) ＊2 2μl  
灭菌水 7.2μl  
Total 20μl  
应用Applied Biosystems 7500 Fast Real-Time PCR System进行Real Time PCR反应，反应程序采用两步法PCR扩增标准程序：
Stage 1：预变性
Reps：1
95℃ 30秒
Stage 2：PCR反应
Reps：40
95℃ 5秒
60℃ 30～34秒
各组HPV 18 E7 mRNA转录水平的测试结果见图5。图5显示，共转CRISPR/Cas9-E7质粒和dsODN的HeLa细胞组HPV18 E7 mRNA转录水平明显降低，说明其靶向敲除HPV18 E7基因的效果优于转染等量CRISPR/Cas9-E7质粒的细胞组、转染等量CRISPR/Cas9-E7质粒和共转非同源ssODN的细胞组。
实施例5、扩增子测序进一步评估共转CRISPR/Cas9-E7质粒和非同源dsODN对靶基因的编辑效率
以实施例3中提取的基因组DNA为模版，根据靶点设计引物并进行PCR反应。本实验使用试剂为KAPA Biosystems公司的KAPA HiFi HotStart ReadyMix(货号KK2602)。
(1)引物的设计
根据靶点设计引物，引物两端分别位于靶点两侧。本实施例中对应的引物序列为：
E7-NGS-F:ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGCATGGACCTAAGGCAACA(SEQ ID NO.9)。
E7-NGS-R:gtgactggagttcagacgtgtgctcttccgatctgctcaattctggcttcacact(SEQ ID NO.10)。
(2)第一轮PCR
反应体系：
Nuclease-free H2O 补齐至25ul
KAPA HiFi HotStart ReadyMix 12.5ul
E7-NGS-F(10μM) 0.75ul
E7-NGS-R(10μM) 0.75ul
Genome DNA 1ug
Total 25ul
反应条件：98℃ for 3min；25cycles of(98℃ for 20s,65℃ for 15s,72℃ for 15s),72℃ for 1min,4℃ for∞
(3)第二轮PCR
反应体系：
Nuclease-free H2O 补齐至25ul
KAPA HiFi HotStart ReadyMix 12.5ul
NEBNext i5 primer(10μM) 2.5ul
NEBNext i7 primer(10μM) 2.5ul
DNA from PCR1 2ul
Total 25ul
反应条件：98℃ for 3min；11cycles of(98℃ for 20s,65℃ for 15s,72℃ for 15s),72℃ for 1min,4℃ for∞
(4)第二轮PCR扩增产物切胶回收，定量，进行测序及数据分析。
图6实验结果显示，共转CRISPR/Cas9-E7质粒和非同源dsODN的HeLa细胞组Indel％明显高于转染等量CRISPR/Cas9-E7质粒的细胞组、转染等量CRISPR/Cas9-E7质粒和非同源ssODN的细胞组，其中一部分是由于ODN的整合插入。
当sgRNA为E7-sgRNA1时，共转CRISPR/Cas9-E7质粒和非同源dsODN的HeLa细胞组Indel％可达40％，相比转染等量CRISPR/Cas9-E7质粒的细胞组、转染等量CRISPR/Cas9-E7质粒和非同源ssODN的细胞组分别提高了25％左右，其中代表断点处有ODN插入占总插入缺失(Indel)的66％左右；当sgRNA为E7-sgRNA2时，共转CRISPR/Cas9-E7质粒和非同源dsODN的HeLa细胞组Indel％可达65％左右，相比转 染等量CRISPR/Cas9-E7质粒的细胞组、转染等量CRISPR/Cas9-E7质粒和非同源ssODN的细胞组分别提高了20％左右，其中“ODN％”代表断点处有ODN插入占总插入缺失(Indel)的17％左右。CRISPR/Cas9-E7质粒共转非同源dsODN可显著增强靶向敲除人乳头瘤病毒E7基因的效率。
实施例6、共转染的dsODN作为标记物监测的脱靶情况
1)GUIDE-Seq建库流程示意图见图7。
2)准备Y型接头：
Y型接头是通过将Miseq通用寡核苷酸(MiSeq Common Adapter)分别与每个样品条形码接头引物(A##adapter)进行退火而制成的：其中，Miseq通用寡核苷酸接头序列为P-GATCGGAAGAGC*C*A(“P”表示磷酸化，“*”表示硫甙修饰)，样品条形码接头引物序列为：
AATGATACGGCGACCACCGAGATCTACACTAGATCGCNNWNNWNNACACTCTTTCCCTACACGACGCTCTTCCGATC*T
其中，“NNWNNWNN”为分子index标签，“*”表示硫甙修饰。
准备Y型接头的退火反应体系为：
Figure PCTCN2022092012-appb-000008
3)准备DNA样品：
电转3天后收细胞，按照前述抽提DNA的方法抽提DNA，DNA采用Qubit测定浓度，A260/280需在1.8-2.0之间为合格，浓度大于20ng/μL、总量大于1μg为合格。
使用1xTE缓冲液(即10mm Tris-HCL，无EDTA)稀释到最终体积为120μL。
根据Covaris S2仪器的标准操作方案，将每个样品的DNA打断至平均长度为500bp。
4)打断的DNA样本纯化：
将纯化磁珠提前拿至室温，震荡混匀室温孵育30min后再使用。
将打断后的样品转入1.5mL专用纯化EP管中，加入120μL磁珠。
轻轻吸打混匀6次，室温静置孵育10min，将PCR管置于磁力架上3min使溶液澄清。
移除上清，PCR管继续放置在磁力架上，向PCR管内加入200μL新鲜配制的80％乙醇溶液，静置30s。
移除上清，再次向PCR管内加入200μL 80％乙醇溶液，静置30s后彻底移除上清。
室温静置5min，使残留乙醇彻底挥发。
加入20μL的1x TE缓冲液，轻轻吸打重悬磁珠后移开磁力架，室温静置2min。
将PCR管置于磁力架上2min使溶液澄清。
用移液器小心吸取15μL上清液(磁珠洗脱后，吸取上清液时不要吸取磁珠)，转移到新的PCR管中，标记好样本，用于下一步反应。
5)末端修复
在200μL PCR管中，添加以下(每个反应)，反应体系为：
Figure PCTCN2022092012-appb-000009
6)加A尾和连接
在上一步完成的反应管中，添加以下(每个反应)，反应体系为：
Figure PCTCN2022092012-appb-000010
7)加A尾后的产物纯化
将PEG/NaCl
Figure PCTCN2022092012-appb-000011
Solution预先拿出来恢复到室温。
每个样品中加入0.9x(也即是22.95μL)的PEG/NaCl
Figure PCTCN2022092012-appb-000012
Solution后，充分混匀，转移到1.5mL低吸附管中，室温孵育15min后按照常规纯化步骤进行纯化。
每个磁珠样品中加入12μL1xTE缓冲液进行洗脱后，转移到标注好的PCR小管。
8)第一轮PCR及纯化
在200μL的反应管中，添加以下(每个反应)，反应体系为：
Figure PCTCN2022092012-appb-000013
所对应的引物使用如下：
P5-1的引物序列为：aatgatacggcgaccaccgagatcta(SEQ ID NO.11)，由苏州金维智公司合成。
GSP1Primer：由GSP1(+)和GSP1(-)混合而成，GSP1(+)引物的序列为：ggatctcgacgctctccctgtttaattgagttgtcatatgttaataac(SEQ ID NO.12)；GSP1(-)引物的序列为：ggatctcgacgctctccctataccgttattaacatatgaca(SEQ ID NO.13)。
第一轮PCR的磁珠纯化：
将PEG/NaCl
Figure PCTCN2022092012-appb-000014
Solution预先拿出来恢复到室温。
每个样品中加入1.2x(也即是36μL)的PEG/NaCl
Figure PCTCN2022092012-appb-000015
Solution后，充分混匀，转移到1.5mL低吸附管中，室温静置孵育15min后按照常规纯化步骤进行纯化。
每个磁珠样品中加入15μL的1xTE缓冲液进行洗脱。
第二轮PCR及纯化
所对应的引物使用如下：
P5-2的引物序列为：aatgatacggcgaccaccgagatctacac(SEQ ID NO.14)，由苏州金维智公司合成。
GSP2Primer的序列：由GSP2(+)和GSP2(-)混合而成，GSP2(+)引物的序列为：cctctctatgggcagtcggtgatacatatgacaactcaattaaac(SEQ ID NO.15)；GSP2(-)引物的序列为：cctctctatgggcagtcggtgatttgagttgtcatatgttaataacggta(SEQ ID NO.16)。
P7-#引物的序列为：caagcagaagacggcatacgagat(nnnnnnnn)gtgactggagtcctctctatgggcagtcggtga，其中“nnnnnnnn”为8bp的barcode序列，用于区分样品。
在200μL的反应管中，添加以下(每个反应)，反应体系为：
Figure PCTCN2022092012-appb-000016
第二轮PCR的磁珠纯化步骤：
将PEG/NaCl
Figure PCTCN2022092012-appb-000017
Solution预先拿出来恢复到室温。
每个样品中加入0.7x(也即是21μL)的PEG/NaCl
Figure PCTCN2022092012-appb-000018
Solution后，充分混匀，转移到1.5mL低吸附管中，室温静置孵育15min后按照常规纯化步骤进行纯化。
每个磁珠样品中加入30μL的1xTE缓冲液进行洗脱。
第二轮PCR纯化后的样品，先使用qubit定量，根据A260/280查看文库DNA的纯度，1.8～2.0之间为合格。用于后续的高通量测序上机。
10)上机测序：
qPCR文库定量：将每个子文库稀释到4nM然后等体积混合成pool。
文库平衡：用25％文库比例的phix进行文库平衡：先将phix稀释至4nM(原浓度为10nM)，然后以体积比phix文库(4nM)：pool文库＝1：3进行混合得到25％phix的pooling文库。具体地，就是phix取0.8μL原液(即8nM)加入1.2μL水，phix总的就2μL。phix文库(4nM)：pooling文库＝2μL：6μL，总共8μL体积。
文库变性：从上一步的8μL中取5μL，与5μL 0.2M的NaOH混合变性5min(此时文库浓度为2nM)。
文库稀释：上一步的文库上机前需稀释成1.2pM终浓度，以得到更好的上机数据质量。稀释方法为：取10μL上步骤得到的变性文库与990μL的HT1缓冲液进行震荡混合(此时文库浓度为20pM)。然后取90μL上混合液与1410μL的HT1缓冲液进行震荡混合得到终浓度为1.2pM的上机文库。
提前将-20℃存放的上机试剂(方板)化冻1-2h，化冻后需拍打除掉底部小气泡。并且。提前将flowcell从4℃取出平衡至室温，使其干燥干净。
上机引物稀释：上机引物稀释成0.3μM的浓度。
选择选择Nextseq Mid试剂盒，Paired End双端测序程序，按测序仪指示步骤进行上机测序。
11)数据分析：
序列读取的处理和整合：将测序所得序列中，前6个碱基相同的序列，以及具有同样8个碱基的分子index标签的序列归一整合在一起，将其认定为来自同一个pcr处理前的样本。利用BWA-MEM软件程序，将整合后的序列比对到人类基因组参考序列(GrCh38)。
分析判定脱靶位点：将比对质量≥50的reads所起始的匹配位置保留并显示，采用10bp的滑动比对窗口将比对区域进行分组。分析出包含有整合的dsODN序列的reads，然后采用基于bin-consensus变异调用算法的分子index和SAMtools的计算机分析工具，在这些有dsODN序列的位置调用为SNPs和indels，然后将其比对参考基因组的非靶标序列，从而判断dsODN序列在细胞基因组上整合的具体参考坐标，从而判定为在靶或脱靶位点。
共电转的dsODN donor监测CRISPR-Cas9系统在HPV18 E7靶点的在靶与脱靶情况。图8显示当采用single E7-sgRNA1靶向切割HPV18 E7基因时，在靶位点可以检测到36505条reads，并且可以检测到1个脱靶位点。当采用single E7-sgRNA2靶向切割HPV18 E7基因时，在靶位点可以检测到75596条reads，并且可以检测到2个脱靶位点。
上述实施例只为说明本发明的技术构思及特点，其目的在于让熟悉此项技术的人士能够了解本发明的内容并据以实施，并不能以此限制本发明的保护范围。凡根据本发明精神实质所作的等效变化或修饰，都应涵盖在本发明的保护范围之内。

Claims (15)

  1. 非同源双链寡聚核苷酸片段在基因敲除系统中的应用，所述非同源双链寡聚核苷酸片段能够提高基因敲除系统对靶基因的切割效率并能够整合到切割断点处。
  2. 根据权利要求1所述的非同源双链寡聚核苷酸片段在基因敲除系统中的应用，其特征在于：所述非同源双链寡聚核苷酸片段能够激发细胞的非同源末端连接修复，引入更多插入或缺失。
  3. 根据权利要求1所述的非同源双链寡聚核苷酸片段在基因敲除系统中的应用，其特征在于：所述非同源双链寡聚核苷酸片段还可作为标记物用于监测基因敲除系统的脱靶效应。
  4. 根据权利要求1所述的非同源双链寡聚核苷酸片段在基因敲除系统中的应用，其特征在于：通过将用于敲除靶基因的CRISPR系统和非同源双链寡聚核苷酸片段共转染细胞。
  5. 根据权利要求4所述的非同源双链寡聚核苷酸片段在基因敲除系统中的应用，其特征在于：采用GUIDE-seq方法分析非同源双链寡聚核苷酸片段的整合位置判定脱靶位点。
  6. 根据权利要求1至5中任一项所述的非同源双链寡聚核苷酸片段在基因敲除系统中的应用，其特征在于：所述CRISPR系统为CRISPR/Cas9系统，和/或，所述非同源双链寡聚核苷酸片段的序列如SEQ ID NO.6所示，或与其具有至少80％及以上的同源性的序列，以及与SEQ ID NO.6所示序列具有至少80％及以上的同源性的序列反向互补配对的序列。
  7. 根据权利要求6所述的非同源双链寡聚核苷酸片段在基因敲除系统中的应用，其特征在于：所述非同源双链寡聚核苷酸片段的序列与SEQ ID NO.6所示序列具有至少85％及以上的同源性的序列，以及与SEQ ID NO.6所示序列具有至少85％及以上的同源性的序列反向互补配对的序列。
  8. 根据权利要求7所述的非同源双链寡聚核苷酸片段在基因敲除系统中的应用，其特征在于：所述非同源双链寡聚核苷酸片段的序列与SEQ ID NO.6所示序列具有至少90％及以上的同源性的序列，以及与SEQ ID NO.6所示序列具有至少90％及以上的同源性的序列反向互补配对的序列。
  9. 根据权利要求8所述的非同源双链寡聚核苷酸片段在基因敲除系统中的应用，其特征在于：所述非同源双链寡聚核苷酸片段的序列与SEQ ID NO.6所示序列具有至少95％及以上的同源性的序列，以及与SEQ ID NO.6所示序列具有至少95％及以上的同源性的序列反向互补配对的序列。
  10. 根据权利要求9所述的非同源双链寡聚核苷酸片段在基因敲除系统中的应用，其特征在于：所述非同源双链寡聚核苷酸片段的序列与SEQ ID NO.6所示序列具有至少98％及以上 的同源性的序列，以及与SEQ ID NO.6所示序列具有至少98％及以上的同源性的序列反向互补配对的序列。
  11. 根据权利要求10所述的非同源双链寡聚核苷酸片段在基因敲除系统中的应用，其特征在于：所述非同源双链寡聚核苷酸片段的序列为：5′-P-G*T*TTAATTGAGTTGTCATATGTTAATAACGGT*A*T-3′和5′-P-A*T*ACCGTTATTAACATATGACAACTCAATTAA*A*C-3′，其中，“P”表示5′磷酸化，“*”表示2个核苷酸之间的硫甙修饰连接。
  12. 根据权利要求6所述的非同源双链寡聚核苷酸片段在基因敲除系统中的应用，其特征在于：所述非同源双链寡聚核苷酸片段的5′端进行磷酸化修饰、5′端的三个核苷酸中相连两个核苷酸之间进行硫甙修饰、3′的三个核苷酸中相连两个核苷酸之间进行硫甙修饰。
  13. 根据权利要求1所述的非同源双链寡聚核苷酸片段在基因敲除系统中的应用，其特征在于：所述基因敲除系统针对人乳头瘤病毒18型E7基因的敲除。
  14. 根据权利要求13所述的非同源双链寡聚核苷酸片段在基因敲除系统中的应用，其特征在于：所述的CRISPR系统的sgRNA的序列如SEQ ID NO.1所示，或与其具有至少80％及以上的同源性的序列，或者如SEQ ID NO.2所示，或与其具有至少80％及以上的同源性的序列。
  15. 一种基因敲除系统，其特征在于：其包括权利要求1至14中任一项所述应用中的CRISPR系统和非同源双链寡聚核苷酸片段。
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