WO2020249111A1 - 一种基因组编辑检测方法、试剂盒及应用 - Google Patents

一种基因组编辑检测方法、试剂盒及应用 Download PDF

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WO2020249111A1
WO2020249111A1 PCT/CN2020/095927 CN2020095927W WO2020249111A1 WO 2020249111 A1 WO2020249111 A1 WO 2020249111A1 CN 2020095927 W CN2020095927 W CN 2020095927W WO 2020249111 A1 WO2020249111 A1 WO 2020249111A1
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base
dna
getpcr
editing
genome
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黄启来
李博
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山东大学
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Definitions

  • the present disclosure belongs to the field of gene editing detection, and specifically relates to a method for indirectly confirming the probability of genome editing by amplifying the proportion of wild-type DNA in a quantitative genome, and its application in genome editing efficiency evaluation and monoclonal screening.
  • CRISPR/cas9 is a currently mainstream genome editing technology, and its gene modification effect is related to guide RNA (sgRNA).
  • sgRNA guide RNA
  • the Cas9 nuclease is guided by sgRNA to the target DNA containing the adjacent motif of the original spacer sequence (PAM), and then cuts the two strands of the target DNA 3bp upstream of the PAM sequence and produces a double-strand break (DSB ).
  • PAM original spacer sequence
  • DSB double-strand break
  • HR homologous recombination
  • NHEJ non-homologous end joining
  • NHEJ involves the direct connection of the broken ends, does not require a homologous template and repairs DNA breaks in an error-prone way, usually leading to unpredictable base insertion or deletion at the DNA break, called indel, which can be applied to gene knockout. It has been widely used in gene function research and removal of disease-causing genes in clinical practice.
  • sgRNA In CRISPR-Cas9-mediated genome editing, pre-screening of excellent sgRNA is of great significance for obtaining good editing efficiency and specificity, and efficient and stable sgRNA can be used to obtain single-cell clones or progeny with expected changes.
  • Currently widely used methods are mainly based on DNA sequencing or mismatch-specific nucleases. For the Sanger sequencing method, before reading each DNA sequence separately, PCR amplification and cloning of the target region DNA is required. This multi-step method can provide detailed information about each mutation event induced by nucleases, but is very time-consuming, expensive and laborious.
  • the second-generation DNA sequencing (NGS) technology is also used to analyze DNA mutations mediated by Cas9 nuclease guided by sgRNA because of its powerful parallel analysis capabilities.
  • NGS second-generation DNA sequencing
  • a variety of online platforms have been developed to analyze NGS data, including CRISPR-GA, BATCH-GE, CRISPResso, Cas-analyzer and CRISPRMatch.
  • CRISPR-GA CRISPR-GA
  • BATCH-GE CRISPResso
  • Cas-analyzer CRISPRMatch
  • T7E1 T7 endonuclease 1
  • Surveyor nuclease to cut double strands containing mismatched bases formed between DNA strands with sequence differences DNA, and this difference between the two DNA strands is caused by nuclease cleavage, so that the editing efficiency can be detected.
  • T7E1 T7 endonuclease 1
  • Surveyor nuclease to cut double strands containing mismatched bases formed between DNA strands with sequence differences DNA, and this difference between the two DNA strands is caused by nuclease cleavage, so that the editing efficiency can be detected.
  • the advantage of this method is that only basic laboratory equipment is required, but it is not suitable for the detection of single nucleotide polymorphism regions, and it often misses single nucleotide mutations and large fragment deletions.
  • qEva-CRISPR21 engineered nuclease-induced translocation (ENIT), Cas9 nuclease-based restriction fragment length polymorphism Sex (RFLP) analysis, Indel detection (IDAA) and gene editing frequency digital PCR (GEF-dPCR) through amplicon analysis.
  • ENIT engineered nuclease-induced translocation
  • RFLP Cas9 nuclease-based restriction fragment length polymorphism Sex
  • IDAA Indel detection
  • GEF-dPCR gene editing frequency digital PCR
  • the present disclosure provides a method for detecting genome editing efficiency, which is hereinafter referred to as getPCR.
  • getPCR uses the selective amplification feature of Taq polymerase to amplify wild-type DNA in the genomic DNA to be tested, and determines the proportion of wild-type DNA by quantifying the wild-type DNA in the amplified product, and then determines the frequency of indels in the tested genome , The detection result is more accurate and has a wide range of application potential.
  • This method is applied to Cas9 endonuclease-induced indel detection with good accuracy, and can be applied to the detection of genome editing efficiency related to Cas9 nuclease technology, such as evaluation of sgRNA performance in CRISPR/cas9, HDR repair efficiency, Evaluation of the base editor; in addition, it can also be used to confirm and screen single-cell clones.
  • a method for detecting the frequency of indels induced by nuclease cleavage includes the following steps: adding primers and Taq DNA polymerase to a genomic sample to be tested; The type DNA is amplified, and the wild type DNA ratio is quantified by PCR to confirm the frequency of indel occurrence in the genome; the primer sequence matches the wild type DNA sequence and covers the nuclease cleavage site.
  • the nucleases include, but are not limited to, Cas9 nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR RNA guided FokI nucleases (RFNs), and paired cas9 nicks Enzyme.
  • the nuclease is Cas9 nuclease.
  • Zinc finger nucleases ZFNs
  • TALENS transcriptional activator-like effector nucleases
  • CRISPER-Cas9 system is common methods of modern genetic engineering technology, providing reliable and simple methods for evaluating the efficiency of the above genetic modification technologies. Significance. In the art, the frequency of occurrence of quantification of indels is usually used to evaluate the efficiency of CRISPR sgRNA. Real-time PCR technology is the most effective method for nucleic acid quantification. However, the diversity and unpredictability of indel occurrence makes it impossible to design indel-specific primers, so technicians cannot directly quantify indel frequency through real-time PCR.
  • the detection method described in the first aspect selectively amplifies wild-type DNA in the genome, and quantifies the proportion of wild-type DNA through the relative quantitative strategy of real-time PCR to bypass this obstacle.
  • Taq polymerase can specifically amplify the template that exactly matches the primer, but does not amplify the template that is mismatched with the primer, and Taq polymerase has a low tolerance for base mismatches between the primer and the complementary sequence.
  • the disclosed method utilizes the selective amplification of Taq polymerase to accurately quantify wild-type DNA, thereby obtaining the occurrence probability of indels.
  • the present disclosure takes Cas9 nuclease as an example to design a primer for a nuclease cleavage site with a directed cleavage function and optimize primer parameters to achieve a good detection effect. It proves that the research ideas and technical solutions of the present disclosure are feasible as detection methods for multiple gene editing technologies, and are expected to have good effects.
  • the PCR quantification is real-time PCR or ddPCR.
  • the amplification reaction is real-time PCR, and the annealing temperature of the amplification reaction is T m -T m +4°C.
  • the detection method further includes the following steps: introducing a control amplification at a position several hundred base pairs away from the cutting site, and calculating the percentage of wild-type DNA in the edited genomic DNA sample through the ⁇ Ct strategy.
  • the 3'end of the primer spans the Cas9 nuclease cleavage site.
  • the primer sequence includes a guarded base sequence
  • the guarded base is the sequence between the nuclease cleavage site and the 3'end of the primer, and the guarded base is 1-8 bp in length.
  • the primer is a nucleotide sequence
  • the length of the guard base is 3 to 5 bp.
  • the primer is a pair of forward and reverse sequences, and the length of the guard base is 4 bp.
  • the 3'terminal base of the guard base is an adenine base or cytosine or guanine base; more preferably, it is an adenine base.
  • the second aspect of the present disclosure provides a kit for detecting the frequency of indels induced by nuclease cleavage.
  • the kit includes primers, Taq DNA polymerase, and PCR detection reagents; On the one hand the detection method.
  • the third aspect of the present disclosure provides the application of the kit of the second aspect in evaluating genome editing efficiency and screening single cell clones.
  • the genome editing includes NHEJ-mediated indel, HDR-mediated gene modification and base editing generated by BE4.
  • the application further includes screening gRNA adapted to CRISPR.
  • a method for genotyping single cell clones includes the following steps: using wild-type DNA in the genome to be tested as a template, primers are designed for alleles, and the single cell to be tested is extracted The cloned genomic DNA is tested by the detection method described in the first aspect to detect whether indels have occurred in the alleles of the single-cell genomic DNA so as to realize the typing of single-cell genes.
  • a method for detecting the efficiency of HDR repair includes the following steps: designing primers for the genomic DNA repaired by HDR in the test genome, extracting the genomic DNA of the cell to be tested, and adopting the method described in the first aspect
  • the method detects the probability of occurrence of HDR; the percentage of DNA repaired by HDR is the HDR repair efficiency.
  • a method for detecting the editing efficiency of a base editor includes the following steps: using the genomic DNA to be tested as a template, designing primers for the target sequence after base editing, using the first aspect
  • the detection method detects the occurrence probability of base editing in the genome, which is the editing efficiency of the editor.
  • the published research takes the genome editing of 8 sgRNAs in 293T cells as an example.
  • the getPCR technology can accurately quantify genome editing efficiency in all genome editing cases, including NHEJ-induced indel, HDR and base editing.
  • this method has shown strong ability in single-cell clonal genotyping, because it can not only characterize whether the desired genome editing has occurred, but also inform that several alleles carry this specific editing.
  • Gene editing methods based on Cas nuclease digestion technology can all use the disclosed methods, including NHEJ-induced indel, HDR and base editing, and can also be applied to the screening of single cell clones.
  • the getPCR provided in the present disclosure can also be easily extended to genome editing experiments mediated by other types of genome cutting nucleases to evaluate the editing efficiency of a given cutting position, such as zinc finger nucleases (ZFNs), transcription activation Factor-like effector nucleases (TALENs) and CRISPR RNA-guided FokI nucleases (RFNs), as well as paired cas9 nickases, are expected to further promote the use of this technology in genome editing technology in molecular And extensive applications in cell biology research.
  • ZFNs zinc finger nucleases
  • TALENs transcription activation Factor-like effector nucleases
  • RNNs CRISPR RNA-guided FokI nucleases
  • Fig. 2 The principle diagram of getPCR primer design in embodiment 1;
  • Forward guarded bases include 3(a) or 4(b) forward guarded bases, or 3(c) or 4(d) reverse guarded bases.
  • (eh) Display of the amplification efficiency and selective amplification ability of guarded bases with different Tm values at different annealing temperatures in the PCR process.
  • three (e) or four (g) observation guards are used Guarded bases and reverse guarded bases with three (f) or four (h) guarded bases.
  • the PCR efficiency is characterized by the ⁇ Ct calculated relative to the Ct value at 65°C, and the selectivity is characterized by the ⁇ Ct between the use of the wild-type template and the indel template.
  • the watched base sequence is shown at the bottom.
  • the small circle refers to the best selectivity at the best amplification efficiency when it is decreased by 0.5 cycles (as shown by the dotted line).
  • (i-1) The effect of annealing temperature on PCR amplification efficiency and the linearity of the standard curve, characterized by R-squared value.
  • Example 4 shows the results of the genotyping application of getPCR in simulating single-cell cloning;
  • (a) Surveyor detection electrophoresis chromatogram, the detected samples contain a given percentage of indels, which are used to simulate genome-edited DNA;
  • Fig. 5 is a result graph of the editing frequency and genotype of single cell clones determined by getPCR in embodiment 5;
  • Fig. 6 is a result diagram of the application of getPCR in embodiment 6 to determine the HDR frequency and the genotype of single cell clones;
  • the bar graph shows the specificity of different watched base combinations for the PCR amplification of simulated indel plasmid, which is an alternative display graph of Figure 2e;
  • Figure 8 The performance of different DNA polymerase products in mismatch recognition
  • Electrophoresis chromatography shows the PCR amplification levels of different DNA polymerases.
  • the templates used in PCR have mismatched bases and mismatched bases, and the primers are forward and reverse guarded bases. ;
  • Figure 9 Use of a simulated indel plasmid for editing frequency determination and genotyping of single cell clones
  • the single-cell clones were genotyped by the getPCR method with two differently designed guarded bases.
  • the single-cell clones were derived from 293T cells with gRNA targeting the DYRK1A gene for genome editing.
  • the box plot shows the first quartile, median, and third quartile, the whiskers represent 1.5IQR, and the outliers are displayed separately;
  • Figure 12 Genotyping of a single cell clone obtained by base editing with a stop codon introduced into the HOXB13 gene;
  • the methods for detecting the efficiency of gene editing methods in the prior art have certain defects: such as Sanger, NGS, methods based on mismatch specific nucleases, etc., have complex operations, high costs, and detection accuracy. Not enough. It is of great significance to provide a method that can be quickly, simply and reliably applied to genome editing efficiency quantification and high-throughput genotyping, and does not require specific equipment.
  • the present disclosure provides a getPCR detection method that uses the specificity of Taq polymerase and uses wild-type DNA sequences as templates to design primer sequences covering nuclease cleavage sites, and quantify wild-type DNA in the genome through amplification. The percentage of type DNA indirectly determines the editing efficiency of the genome. After optimization and verification, the method has high detection accuracy and is easy to operate, and has a wide range of application values.
  • Plasmid and DNA fragment The plasmid containing the HOXB13 gene coding region on the vector pcDNA3.1 was presented by Professor Wei Gonghong from the University of Oulu.
  • the 26 DNA variants mimicking the potential different indels of HOXB13gRNA target 4 ( Figure 2a) and the other 15 variants containing mutations to introduce different types of primer-template mismatches were all constructed by site-directed mutagenesis.
  • the sgRNA expression plasmid was constructed by deleting the cas9 expression cassette from the pSpCas9(BB) vector (Addgene, #42230) by PCR. An annealed oligonucleotide pair with a 20-ntgRNA sequence was ligated between the BbsI sites of the sgRNA expression plasmid or pSpCas9(BB) vector.
  • High-fidelity CRISPR-Cas9 nuclease R661A/Q695A/Q926A/D1135E was obtained by site-directed mutagenesis based on pSpCas9 (BB).
  • the BE4-Gam plasmid (Addgene, #100806) was used for base editing experiments.
  • the 99-nt length single-stranded HDR template containing the EMX1-HindIII mutation was synthesized at Invitech (Shanghai).
  • the introduced HindIII site sequence is adjacent to the PAM sequence of EMX1gRNA target 5.
  • a plasmid containing the EMX1-HindIII mutation was constructed and used as a 100% homologous recombination repair efficiency.
  • the sequences of all primers and oligonucleotides used are shown in Table 1.
  • Cell culture The cell line Lenti-X293T (Cat#632180) was originally purchased from Clontech. Cell culture conditions are 37°C, 5% CO 2 concentration, Dulbecco's modified Eagle medium (Gibco, Cat#C11995500BT), supplemented with 10% (v/v) FBS (Gibco, Cat#10270-106) and'penicillin /Streptomycin (HyClone, Cat#SV30010). Refer to the product manual and use MycoBlueTM MycoplasmaDetector kit (Vazyme, Cat#D101-01) to check regularly for mycoplasma contamination.
  • MycoBlueTM MycoplasmaDetector kit Vazyme, Cat#D101-01
  • Lenti-X293T cells were seeded into a 24-well plate (Labserv, Cat#310109007) at a density of 120,000 cells per well. When the cell density reached about 70%, the cells were transfected with Lipofectamine 2000 (ThermoFisher Scientific, Cat#11668019) according to the manufacturer's instructions. 1 ⁇ g of plasmid co-expressing sgRNA and high-fidelity CRISPR-Cas9 was used in each transfection reaction to introduce indels. For base editing, 750 ng of BE4 plasmid and 250 ng of sgRNA expression plasmid were used for each transfection reaction.
  • HDR-mediated genome repair 600ng of plasmids co-expressing sgRNA and high-fidelity CRISPR-Cas9 and 10pmol HDR oligonucleotides were used for each transfection reaction. 48 hours after transfection, according to the manufacturer's instructions, genomic DNA was extracted with TIANamp Genomic DNA Kit (TIANGEN, Cat#DP304-03).
  • each qPCR reaction uses 0.1ng plasmid DNA or 2.5ng genomic DNA as a template, and AceQqPCRSYBRGreenMasterMix (Vazyme, Cat#Q111-02) is used.
  • the qPCR operation refers to the following conditions. Run the following procedure on the qPCR machine Rotor-GeneQ (Qiagen, Germany): 95°C pre-denaturation for 5 minutes; 95°C denaturation for 30 seconds, annealing at 65-69°C for 30 seconds, extension at 72°C for 10 seconds and detecting fluorescence signals Lasts 40 cycles.
  • each of these 26 plasmids was used to simulate single-cell clones with homozygous HOXB13 insertion deletion mutations; and each plasmid was mixed with the wild-type DNA plasmid in equal proportions to simulate the insertion on one allele. Missing heterozygous single cell clone.
  • the sequence of getPCR primers is shown in Table 2. For the frequency quantification of indels of genomic DNA samples, 2.5ng genomic DNA was used as a template, and the primers summarized in Table 3 were used for amplification.
  • b.getPCR is used to detect the indels of HOXB13 gRNA target 4
  • T100TM thermal cycler Bio-Rad
  • Bio-Rad was used to anneal 270 ng of the purified PCR product to obtain a heterologous duplex, which was then treated with SurveyorNuclease according to the instructions.
  • the DNA fragments were separated on a 2% agarose gel, and images were obtained using Quantum-ST5 (VILBERLOURMAT, France), and analyzed by QuantumST5Xpress software.
  • RFLP assay based on HindIII digestion.
  • a HindIII site was introduced near the PAM sequence, which was achieved by HindIII-based restriction fragment length polymorphism (RFLP) analysis to quantify the HDR repair efficiency.
  • PrimeSTARMax DNA polymerase was used to amplify a 639 bp fragment.
  • the HindIII site was 355 bp from the 5'end.
  • the primers used in PCR were the same as those in the Surveyor assay, as shown in Table 2a.
  • the PCR product was purified using Universal DNA Purification Kit (TIANGEN, Cat#DP214). Take the purified 270ng PCR product for HindIII enzyme digestion experiment, and separate it on a 2% agarose gel. The images were acquired using Quantum-ST5 (VILBERLOURMAT, France) and analyzed using QuantumST5Xpress software.
  • the NGS-based method will cover the DNA region near the genome editing site to construct an NGS amplicon library. After sequencing, count the NGS readings to calculate the editing efficiency.
  • genomic DNA as a template, two rounds of PCR amplification were performed to prepare a sequencing library.
  • a 250-280bp amplicon was designed, in which the Cas9 cleavage site was close to the middle part, and the binding sites of Illumina sequencing primers were introduced at both ends.
  • adaptor sequences were introduced for cluster generation during sequencing, and index sequences were also introduced. After the library DNA was purified and quantified, it was delivered to Genewiz for 150bp paired-end sequencing on the IlluminaHiSeqX-TEN platform.
  • Single cell cloning and genotyping approximately 48 hours after transfection single cells were isolated by limiting dilution and seeded in 96-well plates for growth. When the cells are overgrown in the 96-well plate, they are further transferred to the 24-well plate and continue to grow until healing.
  • the genomic DNA from the single cell clone was then isolated using the TIANamp Genomic DNA Kit (TIANGEN, Cat#DP304-03) according to the manufacturer's instructions. The genotype of each clone was determined by getPCR detection, and confirmed by Sanger sequencing of the amplicon covering the cutting site.
  • the primers used are as shown in Table 2a, and the high-fidelity PrimeSTARMax DNA polymerase (TaKaRa, Cat#R045B) was used for PCR amplification, and then the PCR products were subjected to Sanger sequencing (TsingKeBiologicalTechnology or GeneWiz).
  • Sanger sequencing TsingKeBiologicalTechnology or GeneWiz.
  • TIDEWeb tool https://tide.nki.nl/
  • the colonies were subjected to Sanger sequencing.
  • DNA polymerase products were used to compare the effects of primer mismatch amplification. They are Taq master mix (Vazyme, Cat#P111, Lot#511151), Premix Taq TM (TaKaRa, Cat#RR901, Lot#A3001A), NOVA Taq-Plus PCR Forest Mix (Yugong Biolabs, Cat#EG15139, Lot#1393216101) ), DreamTaq Green PCR Master Mix(ThermoFisher,Cat#K1081,Lot#00291017),Platinum TM Green Hot Start PCR Master Mix(Invitrogen,Cat#13001012,Lot#00401653), Max DNA Polymerase(TaKaRa,Cat#R045,Lot#AI51995A), Phusion Hot Start II high-Fidelity PCR Master Mix(ThermoFisher,Cat#F-565,Lot#00633307) as
  • the design rules of the guarded bases are studied in this embodiment. Since most indels appear near the nuclease cleavage site, and indels smaller than 15bp account for the main part. In addition, in order to better distinguish the indel sequence from the wild-type sequence, the number of bases is less in this example. The situation of insertion or deletion is investigated. In view of this, the inventors designed and constructed 26 plasmids with 1-15 bp indel mutants respectively to simulate genome editing induced by nuclease targeting the HOXB13 gene in vivo ( Figure 2a).
  • guarded bases two series of guarded bases were designed, they each have one to eight guarded bases (Figure 7a-c), from which representatives with ideal amplification efficiency (Figure 2b) were selected to further examine their identification Ability, that is, the ability to distinguish between indel and wild-type DNA sequences.
  • more guarded bases can increase the selectivity of guarded bases.
  • too many guarded bases will cause base mismatches to move from the 3'end to the 5'end of the primer, which will reduce the sensitivity of Taq polymerase.
  • 3 to 5 guarded bases can show superior ability to distinguish wild-type sequences from indel sequences.
  • the 3'end base type of the watched base further plays an important role in determining the discrimination ability of getPCR.
  • adenine bases show the best specificity and give the lowest non-specific amplification signal.
  • cytosine and guanine, and finally thymine Figure 2g.
  • the adenine base still shows the best specificity, and Taq polymerase has the lowest tolerance for mismatches between adenine and non-complementary paired bases ( Figure 2h).
  • the 3'end base type also determines the sensitivity of getPCR to upstream mismatches.
  • the adenine base is also the best choice, which can make getPCR amplification more sensitive to the penultimate base mismatches upstream of it. It is worth noting that if more than one mismatch occurs near the last base, no matter what the last base is, it will obviously destroy the amplification ability of PCR ( Figure 2i). In addition, the closer the mismatched base is to the 3'end, the more sensitive getPCR becomes ( Figure 7f-g, Figure 8a-b).
  • primers with mismatched bases at the 3'end and primers with mismatched bases deleted were used for PCR amplification and compared.
  • the primers with mismatched bases deleted can partially restore the amplification ability in qPCR and conventional PCR analysis ( Figure 7h-i, Figure 8a-b).
  • high-fidelity DNA polymerases such as Phusion and Q5 with proofreading activity, that is, 3'to 5'exonuclease activity, can also partially or completely restore PCR amplification ability.
  • the optimal parameters for getPCR operation is the optimal parameters for getPCR operation.
  • the annealing temperature during the getPCR reaction is studied.
  • the ability of getPCR to specifically amplify wild-type template DNA was significantly increased compared to indel templates containing mismatched bases ( Figure 3a-d) .
  • the PCR efficiency begins to decrease significantly. Since the best PCR efficiency is generally preferred for PCR amplification, this example systematically evaluates the selectivity of each guarded base under the best PCR efficiency ( Figure 3e-h).
  • the guarded base was designed to have a Tm value of about 65°C and getPCR was performed at an annealing temperature of 69°C. More importantly, even if raising the annealing temperature to exceed the Tm value may hinder PCR efficiency, for these four primers, the basis of real-time PCR quantification is the linear correlation between the Ct value and the amount of logarithmic template DNA , But it will not be affected at all. ( Figure 3i-1). DNA polymerase plays an important role in determining the discrimination ability of getPCR.
  • the plasmids shown in Figure 2a are used to simulate indel mutations (indels) caused by genome editing. First, they are used to evaluate the ability of getPCR to quantify genome editing efficiency.
  • twenty-six indel plasmids were mixed in equal parts, and then mixed with wild-type plasmids in a specific ratio to simulate 0%, 20%, 40%, 60%, 80%, and 100% indel frequency.
  • the indel frequency of the mixture was quantified and compared by getPCR and the classic Surveyor method. When the indel frequency is not higher than 20%, the quantitative result of the Surveyor method can truly reflect the expected value. However, as the frequency of indel increases further, the observed value gradually deviates from the expected value ( Figure 4a-b). On the contrary, whether it is a guarded base that carries 3, 4 or 5 guarded bases, all 12 getPCR strategies using different guarded bases can accurately quantify indel frequency ( Figure 4c, Figure 9a-c).
  • Cas9 and 8 different gRNAs targeting HOXB13, DYRK1A or EMX1 genes were used for genome editing in Lenti-X 293T cells, and getPCR was used to test the editing efficiency (Figure 5b).
  • the editing efficiency of each gRNA is determined by three different methods, namely getPCR, NGS-based amplicon sequencing, and Surveyor analysis.
  • the editing efficiency detected by the getPCR method is usually consistent with the results of the NGS method, and NGS is so far considered the most reliable method.
  • the editing efficiency value determined by the Surveyor method is significantly different from the other two methods, especially when the editing efficiency of target 6 and target 16 on the HOXB13 gene is higher ( Figure 5a).
  • cells that received genome editing of target 6 of the HOXB13 gene, target 1 and target 5 of the EMX1 gene, and target 1 of the DYRK1A gene were subjected to the isolation of single cell clones and propagation and expansion. After preparing a genomic DNA sample, genotyping was performed by getPCR, and Sanger sequencing was used for verification.
  • getPCR can not only detect cell clones carrying indels, but also successfully identify whether the cell clone is edited with one allele or both alleles ( Figure 5c-i, Figure 10a-b).
  • getPCR can not only detect cell clones carrying indels, but also successfully identify whether the cell clone is edited with one allele or both alleles ( Figure 5c-i, Figure 10a-b).
  • getPCR can not only detect cell clones carrying indels, but also successfully identify whether the cell clone is edited with one allele or both alleles.
  • the HDR frequency of repeated samples is approximately 25%.
  • the cells undergoing Cas9-mediated HDR repair at EMX1 target 5 single-cell clones were isolated and propagated and amplified, and 50 single-cell clones were obtained. They were genetically analyzed by getPCR using two value-keeping bases. Type, successfully selected 6 homozygous repair cell clones and 17 monoallelic repair cell clones (Figure 6d-e).
  • Example 7 GetPCR determines the editing frequency of the base editor and the genotype of the single cell clone
  • This embodiment describes the application of getPCR in base editor editing frequency and single cell clone genotype detection.
  • the gRNA of EMX1 target 6 or the gRNA of HOXB13 target 8 and the BE4 base editor were used for genome editing in Lenti-X 293T cells, and getPCR was used to detect the editing efficiency (Figure 6b).
  • the detection results of getPCR are highly consistent with the results of the NGS-based amplicon sequencing method ( Figure 6g-h).
  • EMX1 target 6 about 27% of "C" bases are converted to "T" at positions 5 and 6 in the gRNA targeting sequence.
  • the base editing at these two positions tends to occur simultaneously to generate the T5T6 genotype ( Figure 6g).
  • the base change frequency from C to T at position 8 is about 15%. This change can terminate the open reading frame prematurely by introducing a leading stop codon'TAG' ( Figure 6h).

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Abstract

本发明提供了一种确认基因组编辑效率的getPCR方法，其包括对待测基因组中的野生型DNA进行定量，通过计算野生型DNA的百分比来确认基因组编辑效率。

Description

一种基因组编辑检测方法、试剂盒及应用 技术领域
本公开属于基因编辑检测领域，具体涉及一种通过扩增定量基因组中野生型DNA比例间接确认基因组编辑概率的方法，及其在基因组编辑效率评价及单克隆筛选方面的应用。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本公开的总体背景的理解，而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
CRISPR/cas9是一种目前主流应用的基因组编辑技术，其基因修饰效果与向导RNA(sgRNA)相关。在CRISPR/cas9***中，Cas9核酸酶通过sgRNA引导至含有原间隔序列相邻基序(PAM)的靶DNA，然后在PAM序列上游3bp处切割靶DNA的两条链并产生双链断裂(DSB)。一旦细胞感知到DSB的存在，就会通过两种不同的内在机制对断裂的基因组DNA进行修复，即同源重组(HR)或非同源末端连接(NHEJ)。NHEJ涉及断裂末端的直接连接，不需要同源模板并且以易错的方式修复DNA断裂，通常导致DNA断裂处不可预测的碱基***或缺失，称之为indel，可应用于基因的敲除，已广泛用于基因功能研究和在临床中去除致病基因。
在CRISPR-Cas9介导的基因组编辑中，预筛选出优秀的sgRNA对于获得良好的编辑效率和特异性具有重要意义，高效稳定的sgRNA可用于获得具有预期改变的单细胞克隆或子代。目前广泛使用的方法主要基于DNA测序或错配特异性核酸酶。对于Sanger测序方法，在分别读取每个DNA序列之前，需要涉及到靶区域DNA的PCR扩增和克隆。这种需要多个步骤的方法可以提供核酸酶诱导的每个突变事件的详细信息，但是非常耗时，昂贵且费力。第二代DNA测序(NGS)技术也用于分析由sgRNA引导Cas9核酸酶介导的DNA突变，因为它具有强大的并行分析能力。目前已经开发出了多种在线平台来分析NGS数据，包括CRISPR-GA,BATCH-GE,CRISPResso,Cas-analyzer and CRISPRMatch等。然而，发明人认为上述在线分析平台仍然需要多步骤的实验操作，时间及经济成本较高。基于错配特异性核酸酶的方法是目前最流行的方法，利用T7核酸内切酶1(T7E1)或Surveyor核酸酶来切割含有序列差异的DNA链之间形成的含错配碱基的双链DNA，而这种两条DNA链之间的差异就是由核酸酶切割引起的，从而可实现编辑效率的检测。这种方法的优点是只需要基本的实验室设备，但不适用于单核苷酸多态性区域的检测，并且往往会遗漏单核苷酸突变以及大片段的缺失的情况。此外，科学家还开发了许多其他的替代方案，但仅在某些方面有所改进，诸如qEva-CRISPR21,工程化核酸酶诱导的易位(ENIT)、基于Cas9核酸酶的限制性片段长度多态性(RFLP)分析、通过扩增子分析进行Indel检测(IDAA)和基因编辑频率数字PCR(GEF-dPCR)。发明人认为上述实验步骤较为繁琐，并且它们使用基因组靶DNA区域的PCR扩增产物而不是直接用基因组DNA本身来定量编辑效率。众所周知的，PCR扩增过程中引入的序列和长度依赖性偏差将不可避免地影响检测的准确度。
发明内容
针对上述研究背景，发明人认为提供一种快速、简单并可靠的应用于基因组编辑效率定量和高通量基因分型、而且无需特定装置的方法具有重要意义。本公开提供了一种对基因组编辑效率进行检测的方法，以下文中称为getPCR。所述getPCR利用Taq聚合酶选择性扩增特点对待测基因组DNA中野生型DNA进行扩增，通过定量扩增产物中野生型DNA，确定野生型DNA比例，进而判断待测基因组中indel的发生频率，检测结果准确性更高，并且具有广泛的应用潜力。该方法应用于Cas9核酸内切酶诱导的indel检测具有良好的准确度，可适用于Cas9核酸酶技术相关的基因组编辑效率的检测，如用于CRISPR/cas9中sgRNA性能的评价、HDR修复效率、碱基编辑器的评价；除此之外，还可以用于单细胞克隆基因型的 确认和筛选。
为了实现上述技术效果，本公开提供以下技术方案：
本公开第一方面，提供一种用于检测核酸酶切诱导的indel发生频率的方法，所述方法包括以下步骤：向待测基因组样品中加入引物及Taq DNA聚合酶，对基因组样品中的野生型DNA进行扩增，通过PCR定量野生型DNA比例，从而确认基因组中indel发生的频率；所述引物序列与野生型DNA序列相匹配，并且覆盖核酸酶切割位点。
优选的，所述核酸酶包括但不限于Cas9核酸酶、锌指核酸酶(ZFNs)，转录激活因子样效应核酸酶(TALENs)和CRISPR RNA指导FokI核酸酶(RFNs)，以及成对的cas9切口酶。进一步的，所述核酸酶为Cas9核酸酶。
锌指核酸酶(ZFNs)、转录激活因子样效应物核酸酶(TALENS)及CRISPER-Cas9***为现代基因工程技术的常用手段，提供可靠、简便的方法对于上述基因修饰技术的效率进行评价，具有重要的意义。本领域内通常采用定量indel发生的频率来评估CRISPR sgRNA的效率，实时PCR技术是核酸定量中最有效的方法。然而，indel发生的多样性和不可预测性使得不可能设计indel特异性引物，因此技术人员无法通过实时PCR直接量化indel频率。第一方面所述的检测方法，即getPCR技术，对基因组中的野生型DNA进行选择性扩增，通过实时PCR的相对定量策略量化野生型DNA比例来绕过这一障碍。Taq聚合酶能够特异性的扩增与引物完全匹配的模板，而不去扩增与引物发生错配的模板，并且Taq聚合酶对于引物与互补序列之间的碱基错配容忍程度较低。本公开方法利用Taq聚合酶的选择性扩增，可以准确的定量野生型DNA，进而得到indel的发生概率。本公开以Cas9核酸酶为例，对于具有定向切割功能的核酸酶切位点进行引物设计并对引物参数进行优化，实现了良好的检测效果。证明本公开的研究思路及技术方案作为多种基因编辑技术的检测方法具有可行性，且有望具有良好的效果。
优选的，所述PCR定量为实时PCR或ddPCR。
进一步优选的，所述扩增反应为实时PCR，扩增反应退火温度为T m～T m+4℃。
优选的，所述检测方法还包括以下步骤：在距离切割位点数百碱基对的位置处引入对照扩增，通过ΔΔCt策略计算经过编辑后的基因组DNA样品中野生型DNA的百分比。
优选的，所述引物3'末端跨越Cas9核酸酶切割位点。
优选的，所述引物序列中包括值守碱基序列，所述值守碱基为核酸酶切位点与引物3'末端之间的序列，所述值守碱基长度为1～8bp。
进一步优选的，所述引物为一条核苷酸序列，所述值守碱基的长度为3～5bp。
进一步优选的，所述引物为正向和反向的一对序列组合，所述值守碱基的长度为4bp。
进一步优选的，所述值守碱基3'末端碱基为腺嘌呤碱基或胞嘧啶或鸟嘌呤碱基；更优选的，为腺嘌呤碱基。
本公开第二方面，提供一种用于检测核酸酶切诱导的indel发生频率的试剂盒，所述试剂盒中包括引物、Taq DNA聚合酶及PCR检测试剂；所述试剂盒的使用执行如第一方面所述检测方法。
本公开第三方面，提供第二方面所述试剂盒在评价基因组编辑效率、单细胞克隆筛选方面的应用。
优选的，所述基因组编辑包括NHEJ介导的indel，HDR介导的基因修饰及通过BE4产生的碱基编辑。
优选的，所述应用还包括筛选适配CRISPR的gRNA。
本公开第四方面，提供一种对单细胞克隆进行基因分型的方法，所述方法包括以下步骤：以待测基因组中野生型DNA为模板，针对等位基因设计引物，提取待测单细胞克隆的基因组DNA，通过第一方面所述的检测方法检测单细胞基因组DNA中等位基因是否发生了indel从而对单细胞基因实现分型。
本公开第五方面，提供一种HDR修复效率的检测方法，所述检测方法包括以下步骤：针对待测基因 组中HDR修复的基因组DNA设计引物，提取待测细胞基因组DNA，采用第一方面所述方法检测HDR的发生概率；HDR修复DNA所占百分比即HDR修复效率。
本公开第六方面，提供一种碱基编辑器编辑效率的检测方法，所述检测方法包括以下步骤，以待测基因组DNA为模板，针对碱基编辑后的靶序列设计引物，采用第一方面所述的检测方法检测基因组中碱基编辑的发生概率，即为编辑器的编辑效率。
本公开研究以8个sgRNA在293T细胞中的基因组编辑为例，getPCR技术可以在所有基因组编辑案例中准确地定量基因组编辑效率，包括NHEJ诱导的indel，HDR和碱基编辑。同时，该方法在单细胞克隆基因分型中表现出强大的能力，因为它不仅可以表征所期望的基因组编辑是否发生，而且还可以告知具体有几条等位基因携带了这种特定的编辑。
与现有技术相比，本公开的有益效果是：
1.随着CRISPR技术的快速发展和广泛应用，提供一种简单、准确可靠的基因组编辑效率评价方法，对于gRNA的筛选及实验方案的优化具有重要的意义。本公开提供的方法过程简单、定量结果可靠、节省时间和成本低、并且不涉及特定装置，仅需要一个qPCR步骤。针对CRISPR靶标上精准测定indel频率，检测的准确度与公认最准的NGS方法一致。
2.基于Cas核酸酶切技术的基因编辑方法均可以使用本公开方法，包括NHEJ诱导的indel，HDR和碱基编辑，同时还可以应用于单细胞克隆的筛选。
3.本公开提供的getPCR也可以很容易地扩展应用于由其它类型基因组切割核酸酶介导的基因组编辑实验，去评估给定切割位置的编辑效率，如锌指核酸酶(ZFNs)，转录激活因子样效应核酸酶(TALENs)和CRISPR RNA指导FokI核酸酶(RFNs)，以及成对的cas9切口酶等，通过进一步的确定值守碱基的设计规则，有望进一步推动该技术在基因组编辑技术在分子和细胞生物学研究中的广泛应用。
附图说明
构成本公开的一部分的说明书附图用来提供对本公开的进一步理解，本公开的示意性实施例及其说明用于解释本公开，并不构成对本公开的不当限定。
图1本公开getPCR原理及流程图；
(a)getPCR鉴别indel和野生序列的原理(b)getPCR策略概述
图2实施例1中getPCR引物设计原理图；
(a)26个质粒在HOXB13基因gRNA靶标4处模拟indel；
(b)16种具有不同值守碱基的getPCR值守碱基；评估分别使用反向引物(c)和正向引物(d)以及正向与反向引物组合使用；
(e)时能够区分indels与野生型序列的能力；
(f)正反向引物组合使用时自扩增背景信号的研究；
(g)引物3'末端的第一个碱基对扩增特异性的影响；
(h)不同类型的碱基错配对扩增效率的影响；
(i)3'末端碱基类型在确定getPCR对错配的敏感性中的作用。平均值±s.e.m，n＝3个独立技术重复)图3实施例2中运行getPCR的参数优化图；
(a-d)在不同退火温度下，使用四种值守碱基对indel/野生型序列DNA模板进行扩增的扩增曲线。正向值守碱基包含3(a)或4(b)个正向值守碱基，或3(c)或4(d)个反向的值守碱基。
(e-h)PCR过程中不同的退火温度下，不同Tm值的值守碱基的扩增效率以及选择性扩增能力的展示，其中使用到了具有三个(e)或四个(g)观察值守的值守碱基和具有三个(f)或四个(h)值守碱基的反向值守碱基。PCR效率表征为相对于65℃下的Ct值计算的ΔCt，选择性表征为使用野生型模板和*** 缺失模板之间的ΔCt。值守碱基序列显示在底部。小圆圈指在下降了0.5个循环时(如虚线所示)最佳扩增效率下的最佳选择性。
(i-l)退火温度对PCR扩增效率的影响以及标准曲线的线性，以R平方值为特征。在检测中使用的四个值守碱基分别具有三个(i)或四个(k)正向值守碱基，或分别具有三个(j)或四个(l)反向值守碱基。(平均值±s.e.m，n＝3个独立技术重复)
图4实施例4中getPCR在模拟单细胞克隆的基因分型应用结果图；(a)Surveyor检测电泳色谱图，检测的样品中含有给定百分比的***缺失，用来模拟基因组编辑后的DNA；
(b)Surveyor检测得到的编辑频率结果的量化；
(c)单独或组合使用正向和反向值守碱基运用getPCR方法检测Indel频率；
(d-f)使用三种不同设计的值守碱基运用getPCR对模拟的单细胞克隆的基因分型。(平均值±s.e.m，n＝3个独立技术重复，*P<0.05，**P<0.01，***P<0.001)
图5实施例5中getPCR确定单细胞克隆的编辑频率和基因型结果图；
在gRNA靶向HOXB13，DYRK1A和EMX1基因进行基因组编辑的293T细胞中进行Indel频率测定，并进行单细胞克隆的基因分型；
(a)在对8种gRNA介导的基因组编辑组合，getPCR对产生的indel频率进行定量，并与NGS和Surveyor方法相比较；
(b)getPCR中使用的gRNA序列和值守碱基的图示；来自编辑的靶向HOXB13基因(c，d)，EMX1基因(e，f，i)和DYRK1A基因(g，h)的293T细胞的单细胞克隆通过getPCR方法进行基因分型。箱形图分别展示了第一四分位数、中位数和第三四分位数，胡须表示1.5IQR，异常值单独显示。在基因分型(j-l)中评估两种不同设计的值守碱基的相关性和组合效果。(平均值±s.e.m，n＝3个独立技术重复，*P<0.05，**P<0.01，***P<0.001)
图6实施例6中getPCR应用于确定HDR频率和单细胞克隆的基因型结果图；
(a)getPCR在HDR和碱基编辑方面的量化原理示意图；
(b)靶向EMX1基因的HDR修复效率检测与靶向HOXB13基因的碱基编辑效率检测所用到的引物；
(c)使用getPCR进行HDR效率定量并与NGS和HindIII酶切方法的相比；
(d-f)单独或组合使用两种不同的值守碱基运用getPCR方法，对来自HDR实验的单细胞克隆的基因分型，箱形图分别展示了第一四分位数、中位数和第三四分位数，胡须表示1.5IQR，异常值单独显示；
(g，h)分别在靶向EMX1和HOXB13基因的碱基编辑实验中通过getPCR和NGS方法确定的每种基因型的频率，通过getPCR对来自EMX1基因碱基编辑的第5和第6位杂合的10个克隆进行详细的基因分型；
(i)通过getPCR对来自EMX1基因碱基编辑的第5和第6位杂合的10个克隆进行详细的基因分型；
(j，k)条形图和散点图在EMX1基因编辑实验中通过getPCR显示第5个核苷酸的单细胞克隆基因分型；
(l，m)相应的第6个核苷酸的单细胞克隆基因分型；
(n，o)条形图和散点图显示HOXB13基因的碱基编辑中的单细胞克隆基因分型。(平均值±s.e.m，n＝3个独立技术重复，*P<0.05，**P<0.01，***P<0.001)
图7设计getPCR引物和运行getPCR的注意事项；
(a，b)分别在正向和反向设计具有给定值守碱基但具有不同长度/Tm值的多个getPCR引物；
(c)这些getPCR引物在野生型模板上的扩增效率；
(d)条形图显示不同的值守碱基组合对模拟indel质粒PCR扩增的特异性，是图2e的替代展示图；
(e)显示在不添加模板的情况下值守碱基组合的PCR自扩增信号的条形图，是图2f的替代展示；
(f，g)相对于3'末端，单碱基错配的位置对PCR扩增的影响，分别展示正向和反向值守碱基的结果；
(h，i)比较3'末端碱基错配与3'末端碱基缺失对PCR扩增的阻碍能力，分别展示正向和反向值守碱基；
(j)多种qPCRSYBRGreenmix产品在getPCR应用中的适用性比较。(平均值±s.e.m，n＝3个独立技术重复)
图8不同DNA聚合酶产物在错配识别中的表现；
(a，b)电泳色谱显示不同DNA聚合酶的PCR扩增水平，PCR中使用到的模板分别有不含错配碱基、含错配碱基，引物分别为正向和反向值守碱基；
(c)来自a和b的PCR产物的Sanger测序色谱；
(d，e)条形图，说明在多个qPCR产品的扩增中，相对于3'末端，对在不同位置的单碱基错配的敏感性，分别使用了正向和反向值守碱基。(平均值±s.e.m，n＝3个独立技术重复)
图9使用模拟***缺失的质粒进行编辑频率测定和单细胞克隆的基因分型；
(a-c)使用正向和反向值守碱基组合的getPCR方法对模拟***缺失的DNA进行频率定量；
(d-f)通过组合两种不同设计的getPCR值守碱基对模拟单细胞克隆进行基因分型；参考图2a，以获得模拟***信息。(平均值±s.e.m，n＝3个独立技术重复)
图10针对gRNA靶向基因HOXB13，DYRK1A和EMX1进行基因组编辑以产生***缺失突变的单细胞克隆进行基因分型；
(a，b)分别用两种不同设计的值守碱基通过getPCR方法对单细胞克隆进行基因分型，单细胞克隆来自gRNA靶向DYRK1A基因进行基因组编辑的293T细胞。箱形图分别展示了第一四分位数、中位数和第三四分位数，胡须表示1.5IQR，异常值单独显示；
(c-g)散点图显示两种不同设计的值守碱基在基因分型中的相关性和组合效应；
(h-l)通过Sanger测序在单细胞克隆基因分型中确定的***缺失突变，分别针对gRNAHOXB13靶标6，EMX1靶标5，DYRK1A靶标1和EMX1靶标1；(平均值±s.e.m，n＝3个独立技术重复，*P<0.05，**P<0.01，***P<0.001)
图11针对gRNA靶向EMX1基因完成碱基编辑后分离得到的单细胞克隆进行基因分型；
(a)条形图，显示在EMX1基因编辑实验中通过getPCR在第5个核苷酸处进行单细胞克隆基因分型，即图6j用详细克隆编号注释；
(b)条形图，显示在EMX1基因编辑实验中通过getPCR在第6个核苷酸处进行单细胞克隆基因分型，即图6l用详细克隆编号注释；
(c)单细胞克隆基因分型的Sanger测序层析。(平均值±s.e.m，n＝3个独立技术重复)
图12由HOXB13基因上引入终止密码子的碱基编辑得到的单细胞克隆的基因分型；
(a)对HOXB13基因碱基编辑实验中得到的单细胞克隆，通过getPCR在第8个核苷酸处进行基因分型，即图6n用详细克隆编号注释；
(b)单细胞克隆基因分型的Sanger测序层析。(平均值±s.e.m，n＝3个独立技术重复)。
具体实施方式
应该指出，以下详细说明都是例示性的，旨在对本公开提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本公开所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本公开的示例性实施方式。如在这里所使用的，除非上下文另外明确指出，否则单数形式也意图包括复数形式，此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
正如背景技术所介绍的，现有技术中针对基因编辑方法效率进行检测的方法存在一定的缺陷：如Sanger、NGS、基于错配特异性核酸酶的方法等具有操作复杂、成本高、检测准确度不足等。提供一种能够快速、简单并可靠的应用于基因组编辑效率定量和高通量基因分型，而且无需特定装置的方法具有重要的意义。为了实现该技术目的，本公开提供了一种getPCR检测方法，利用Taq聚合酶的特异性，以野生型DNA序列为模板，设计覆盖核酸酶切位点的引物序列，通过扩增定量基因组中野生型DNA的百分比间接确定基因组的编辑效率。经优化和验证，该方法的检测准确度高并且操作方便，具有广泛的应用价值。
为了使得本领域技术人员能够更加清楚地了解本公开的技术方案，以下将结合具体的实施例与对比例详细说明本公开的技术方案。
以下实施例中所使用的试剂和材料来源如下：
质粒与DNA片段pcDNA3.1载体上含有HOXB13基因编码区的质粒由奥卢大学的卫功宏教授赠送。
模拟HOXB13gRNA靶标4的潜在不同***缺失(图2a)的26个DNA变体和包含为了引入不同类型的引物-模板错配突变的其他15种变体都是通过定点诱变构建得到的。通过用PCR方法从pSpCas9(BB)载体(Addgene，#42230)中删除cas9表达框构建得到了sgRNA表达质粒。将带有20-ntgRNA序列的退火寡核苷酸对连接到sgRNA表达质粒或pSpCas9(BB)载体的BbsI位点之间。基于pSpCas9(BB)通过定点诱变获得高保真CRISPR-Cas9核酸酶(R661A/Q695A/Q926A/D1135E)。
BE4-Gam质粒(Addgene，#100806)用于碱基编辑实验。
含有EMX1-HindIII突变的99-nt长度的单链HDR模板是在英潍捷基公司(上海)合成得到，引入的HindIII位点序列与EMX1gRNA靶标5的PAM序列相邻。构建得到含有EMX1-HindIII突变的质粒，并作为100％的同源重组修复效率。所有使用的引物和寡核苷酸的序列均显示在表1中。
表1、用于质粒构建和转染的寡核苷酸序列
a、用于构建HOXB13变体的引物
Figure PCTCN2020095927-appb-000001
Figure PCTCN2020095927-appb-000002
b、用于构建空白sgRNA表达质粒的引物
Figure PCTCN2020095927-appb-000003
c、通过定点诱变构建HF-Cas9(R661A，Q695A，Q926A，D1135E)的引物
Figure PCTCN2020095927-appb-000004
Figure PCTCN2020095927-appb-000005
表1d、用于构建给定靶标的sgRNA表达质粒的引物
Figure PCTCN2020095927-appb-000006
表1e、HDR模板序列(5'-3')
Figure PCTCN2020095927-appb-000007
细胞培养 细胞系Lenti-X293T(Cat#632180)最初购自Clontech公司。细胞培养条件为37℃，5％的CO 2浓度，使用Dulbecco改良Eagle培养基(Gibco，Cat#C11995500BT)，添加有10％(v/v)FBS(Gibco，Cat#10270-106)和'青霉素/链霉素(HyClone，Cat#SV30010)。参照产品手册，使用MycoBlueTMMycoplasmaDetector试剂盒(Vazyme，Cat#D101-01)定期检查是否有支原体污染。
细胞转染 在转染前一天，将Lenti-X293T细胞以每孔120,000个细胞的密度接种到24孔板(Labserv，Cat#310109007)中。在细胞密度达到约70％时，根据制造商的说明书，使用Lipofectamine2000(ThermoFisherScientific，Cat#11668019)转染细胞。在每个转染反应中使用1μg共表达sgRNA和高保真CRISPR-Cas9的质粒以引入***缺失。对于碱基编辑，每个转染反应使用750ng的BE4质粒和250ng的sgRNA表达质粒。对于HDR介导的基因组修复，每个转染反应使用600ng共表达sgRNA和高保真CRISPR-Cas9的质粒以及10pmolHDR寡核苷酸。转染后48小时，根据制造商的说明，用TIANamp基因组DNA试剂盒(TIANGEN，Cat#DP304-03)提取基因组DNA。
getPCR conditions.在15μL体积的反应体系中，每个qPCR反应使用0.1ng质粒DNA或2.5ng基因组DNA作为模板，使用AceQqPCRSYBRGreenMasterMix(Vazyme，Cat#Q111-02)，qPCR的运行参照以下条件。在qPCR仪Rotor-GeneQ(Qiagen，德国)上按照如下程序运行：95℃预变性5分钟；95℃变性30秒，65-69℃下退火30秒，72℃下延伸10秒并检测荧光信号，持续40个循环。使用
Figure PCTCN2020095927-appb-000008
仪(德国罗氏应用科学公司)时，使用以下条件：95℃变性15秒，65-69℃下退火20秒，72℃下延伸15秒并检测荧光信号，持续40个循环；然后进行标准的熔解曲线步骤。引物Tm值的计算使用了在线OligoCalc工具50。
使用getPCR对indel的频率进行量化将模拟不同类型的***缺失突变的26种质粒等比例混合，作为100％的***缺失(图2a)；以给定的比例与野生型DNA进一步混合以获得不同***缺失效率的DNA样品。使用getPCR方法评估indel发生的频率。在getPCR检测中，将0.1ng质粒DNA用作每个qPCR反应的模板。如图1b中所述计算混合物样品中的野生型DNA所占百分比和***缺失频率。同时，将这26个质粒中的每一个质粒用于模拟具有纯合HOXB13***缺失突变的单细胞克隆；并且将每个质粒与野生型DNA质粒等比例混合 以模拟在一条等位基因上携带***缺失的杂合单细胞克隆。getPCR引物的序列展示如表2所示。对于基因组DNA样品的indel的频率定量，使用2.5ng基因组DNA作为模板，并使用表3中所总结的引物进行扩增。
表2基因组编辑效率测定
a.Surveyor DNA扩增和sanger测序的引物
Figure PCTCN2020095927-appb-000009
b.getPCR用于检测HOXB13 gRNA靶标4的***缺失的引物
Figure PCTCN2020095927-appb-000010
Figure PCTCN2020095927-appb-000011
表3.细胞基因组编辑效率
a.用于indel效率定量的getPCR引物
Figure PCTCN2020095927-appb-000012
Figure PCTCN2020095927-appb-000013
b.getPCR引物用于碱基编辑效率定量
Figure PCTCN2020095927-appb-000014
c.用于HDR修复效率量化的getPCR引物
Figure PCTCN2020095927-appb-000015
Surveyor核酸酶分析 使用已经报道过的Surveyor核酸酶测定法测定Indel频率，用到了
Figure PCTCN2020095927-appb-000016
突变检测试剂盒(IntegratedDNATechnologies，Cat#706020)。过程简单陈述如下：根据产品手册，使用 TIANampGenomicDNAKit(TIANGEN，Cat#DP304-03)提取基因组DNA；然后使用高保真
Figure PCTCN2020095927-appb-000017
聚合酶(TaKaRa，Cat#R045B)扩增得到DNA片段，该片段的任一末端距离cas9的切割位点距离200-400bp，PCR所用的引物展示在表2a中。使用T100TM热循环仪(Bio-Rad)对270ng纯化的PCR产物进行退火，以得到异源的双链体，随后根据使用说明用SurveyorNuclease处理。用2％琼脂糖凝胶分离DNA片段，并使用Quantum-ST5(VILBERLOURMAT，France)获得图像，并用QuantumST5Xpress软件分析。
getPCR在HDR和BE4实验中的应用如表3中所总结的，在3'末端设计具有修饰核苷酸的修饰特异性getPCR引物。在getPCR分析中，使用2.5ng基因组DNA作为每个反应的模板。使用如图6a中所示的公式计算基因组修饰效率。
基于HindIII酶切的RFLP测定。在针对EMX1基因的HDR实验中，在PAM序列附近引入一个HindIII位点，这通过基于HindIII酶切的限制性片段长度多态性(RFLP)分析来实现HDR修复效率的定量。简言之，使用PrimeSTARMaxDNA聚合酶扩增639bp片段，HindIII位点距离5'末端有355bp的长度，PCR用到的引物与Surveyor测定法相同，如表2a中所示。使用UniversalDNA纯化试剂盒(TIANGEN，Cat#DP214)将PCR产物纯化。取纯化的270ngPCR产物进行HindIII酶切实验，并在2％琼脂糖凝胶上分离。使用Quantum-ST5(VILBERLOURMAT，France)获取图像并使用QuantumST5Xpress软件进行分析。
基于NGS的方法 将覆盖基因组编辑位点附近的DNA区域进行NGS扩增子文库的构建，进行测序后通过计数NGS的读数来计算编辑效率。使用基因组DNA作为模板，进行两轮PCR扩增以制备测序文库。在第一轮PCR中，设计250-280bp的扩增子，其中Cas9切割位点靠近中间部分，两端引入Illumina测序引物的结合位点。在第二轮PCR中，引入了接头序列用于测序过程中的簇生成，同时还引入了索引序列。文库DNA进行纯化和定量后，交付Genewiz公司在IlluminaHiSeqX-TEN平台上进行150bp双末端测序。对于NHEJ介导的***缺失，用野生型DNA的特征序列获得每个文库中的野生型读数计数，并使用公式“编辑效率＝1-野生型读数/总读数*100％”计算***缺失编辑效率。关于碱基编辑和HDR实验中的编辑效率，获得文库中预期DNA序列的读数计数，并使用等式“效率＝预期DNA序列的读数/总读数*100％”计算编辑效率。有关文库制备和计数方法的详细信息，请参见表4。
表4通过NGS进行基因组编辑效率定量
a.用于文库制备的引物
Figure PCTCN2020095927-appb-000018
Figure PCTCN2020095927-appb-000019
1st round PCR,take 50ng gDNA as template,28 cycles,15μl system,set NTC control, anealed@60℃,using
Figure PCTCN2020095927-appb-000020
Max DNA Polymerase(TaKaRa)
Figure PCTCN2020095927-appb-000021
Figure PCTCN2020095927-appb-000022
2nd round PCR,take 1ng of purified DNA from 1st round PCR as template,10cycles,15μl system,anealed@65℃,using
Figure PCTCN2020095927-appb-000023
Max DNA Polymerase(TaKaRa)
b.R程序读取计数的特征序列
Figure PCTCN2020095927-appb-000024
c.R读取计数程序
library(ShortRead)
reads＝readFastq("libraryName")
reads
total_counts＝length(reads)
total_counts
sequences＝sread(reads)
dict＝DNAStringSet(substr(sequences,1,150))
hits＝vcountPattern("Wild Type characteristic sequence",dict,max.mismatch＝0,with.indels＝FALSE)
wild_type_counts＝sum(hits)
wild_type_counts
library(ShortRead)
reads＝readFastq("libraryName")
reads
total_counts＝length(reads)
total_counts
sequences＝sread(reads)
dict＝DNAStringSet(substr(sequences,1,150))
hits＝vcountPattern("expected_characteristic sequence",dict,max.mismatch＝0,with.indels＝FALSE)
expected_sequence_counts＝sum(hits)
expected_sequence_counts
单细胞克隆和基因分型转染后约48小时，通过有限稀释法分离单细胞并接种到96孔板中生长。当细胞长满96孔板时，将其进一步转移到24孔板中继续生长至愈合。然后根据制造商的说明用TIANamp基因组DNA试剂盒(TIANGEN，Cat#DP304-03)分离来自单细胞克隆的基因组DNA。通过getPCR检测确定每个克隆的基因型，并通过覆盖切割位点的扩增子的Sanger测序确认。使用到的引物如表2a中所示，用高保真PrimeSTARMaxDNA聚合酶(TaKaRa，Cat#R045B)进行PCR扩增，然后对PCR产物进行Sanger测序(TsingKeBiologicalTechnology或GeneWiz)。为了确定杂合细胞的每个等位基因的确切序列，使用TIDEWeb工具(https://tide.nki.nl/)直接分析Sanger测序ab1文件，或者将扩增子克隆到载体中之后，再对菌落进行Sanger测序。
不同DNA聚合酶对错配的敏感性使用多种商业DNA聚合酶产物来比较引物错配对扩增的影响。它们是Taq master mix(Vazyme,Cat#P111,Lot#511151),Premix Taq TM(TaKaRa,Cat#RR901,Lot#A3001A),NOVA Taq-Plus PCR Forest Mix(Yugong Biolabs,Cat#EG15139,Lot#1393216101),DreamTaq Green PCR Master Mix(ThermoFisher,Cat#K1081,Lot#00291017),Platinum TM Green Hot Start PCR Master Mix(Invitrogen,Cat#13001012,Lot#00401653),
Figure PCTCN2020095927-appb-000025
Max DNA Polymerase(TaKaRa,Cat#R045,Lot#AI51995A),Phusion Hot Start II high-Fidelity PCR Master Mix(ThermoFisher,Cat#F-565,Lot#00633307)as well as 
Figure PCTCN2020095927-appb-000026
Hot Start high-Fidelity DNA Polymerase(NEB,Cat#M0493)。在20μl反应体系中，使用10ng质粒DNA作为模板，并按照产品手册所建议的程序进行热循环。然后将PCR产物直接进行2.0％琼脂糖凝胶电泳和Sanger测序。使用Quantum-ST5(VILBERLOURMAT，France)获得凝胶图像，并用QuantumST5Xpress软件分析。
不同qPCR SYBR Green产品在getPCR中的比较 为了测试getPCR的广泛可用性，将多种qPCRSYBRmix产品应用于getPCR，包括AceQ qPCR SYBR Green Master Mix(Vazyme,Cat#Q111-02),SYBR TM Select Master  Mix(Applied Biosystems TM,Cat#4472908),Power SYBR Green PCR Master Mix(Applied Biosystems TM,Cat#4367659),QuantiNova SYBR Green PCR Kit(QIAGEN,Cat#208054),FastStart Essential DNA Green Master(Roche,Cat#06402712001),
Figure PCTCN2020095927-appb-000027
SYBR One-Step qRT-PCR SuperMix(novoprotein,Cat#E092-01A),2×T5 Fast qPCR Mix(TSINGKE,Cat#TSE202),UltraSYBR Mixture(CWBIO,Cat#CW0957),SYBR Premix Ex Taq(TaKaRa,Cat#RR420,A5405-1)。实时定量PCR在热循环仪Rotor-GeneQ(Qiagen，德国)或
Figure PCTCN2020095927-appb-000028
仪(德国罗氏应用科学公司)上运行。根据制造商的说明书和设定的退火温度确定qPCR条件。
统计分析 基于Levene测试的结果应用学生t检验(双尾)以评估使用IBMSPSSStatistics版本的单细胞克隆基因分型的getPCR结果的统计学显着性。使用Pearson测试评估两种不同getPCR策略之间的相关性，用到了IBMSPSSStatistics软件的第21个版本。
实施例1 getPCR中值守碱基的设计
为了使getPCR技术能更好的应用，本实施例中针对值守碱基的设计规则进行研究。由于大多数indel出现在核酸酶切割位点附近，且小于15bp的indel占主要部分，此外，要想使indel序列与野生型序列更好的区分开来，本实施例中针对碱基数目较少的***或缺失的情况进行考察。鉴于此，发明人了设计构建了26个质粒，分别带有1-15bp的***缺失突变体，以模拟在体内靶向HOXB13基因的核酸酶诱导的基因组编辑(图2a)。
本实施例中设计了两个系列的值守碱基，它们分别具有一至八个值守碱基(图7a-c)，从中筛选出具有理想扩增效率的代表(图2b)以进一步检查它们的鉴别能力，即区分indel和野生型DNA序列的能力。从理论上讲，更多的值守碱基可以增加值守碱基的选择性。然而，过多的值守碱基会使碱基错配从引物的3'末端向5'末端方向移动，反而会降低Taq聚合酶的敏感性。单独使用单向的值守碱基时，不论是对于反向(图2c)和正向(图2d)引物，3至5个值守碱基就能表现出优越的区分野生型序列和indel序列的能力。当正向和反向值守碱基组合使用时，总共4到6个值守碱基可成功区分***缺失(图2e，图7d)。然而，由于引物自扩增，累计5到6个值守碱基的时候就会显示出更高的背景信号(图2f，图7e)。因此，累计4个值守碱基的设计组合是getPCR引物的理想选择。
本实施例进一步对值守碱基的3'末端碱基类型在决定getPCR的鉴别能力中起重要作用。当与模板中非互补配对碱基形成错配时，腺嘌呤碱基显示出最佳的特异性并且给出最低的非特异扩增信号。接下来是胞嘧啶和鸟嘌呤，最后是胸腺嘧啶(图2g)。当错配位于倒数第二位置时，腺嘌呤碱基仍显示出最佳的特异性，并且Taq聚合酶对腺嘌呤与非互补配对碱基之间的错配容忍度最低(图2h)。此外，3'末端碱基类型也决定了getPCR对上游错配的敏感性。在这里，腺嘌呤碱基同样也是最佳选择，它能够使getPCR扩增对其上游的倒数第二位的碱基错配更为敏感。值得注意的是，如果在最后一个碱基附近发生多于一个错配，则无论最后一个碱基是什么，都将明显破坏PCR的扩增能力(图2i)。此外，不匹配碱基越接近3'端，getPCR变得越敏感(图7f-g，图8a-b)。
当探讨getPCR对错配敏感的潜在机制时，本实施例中将3'末端带有错配碱基的引物与删除了该错配碱基的引物分别去进行PCR扩增并进行比较。有趣的是，删除了错配碱基的引物在qPCR以及常规PCR分析中都能部分恢复扩增能力(图7h-i，图8a-b)。此外，具有校对活性即3'至5'核酸外切酶活性的高保真DNA聚合酶如Phusion和Q5也可部分或完全恢复PCR扩增能力。PCR产物的Sanger测序结果显示引物3'末端的错配核苷酸在聚合过程中能够被3'至5'核酸外切酶活性除去。相反，缺乏3'至5'核酸外切酶活性的Taq DNA聚合酶可以容忍并直接绕过错配(图8c)。这表明，错配一方面阻碍了引物与模板配对，同时由错配引起的空间几何障碍也进一步阻碍了Taq聚合酶合成反应的起始。
实施例2运行getPCR的参数
另一个需要确定的因素是getPCR运行的最佳参数，本实施例中针对getPCR反应时的退火温度进行研究。对于实施例1中设计的四组值守碱基，随着退火温度的升高，对比于含错配碱基indel模板，getPCR特异性扩增野生型模板DNA的能力明显增加(图3a-d)。然而，当退火温度升高到高于Tm值4℃以上时，PCR效率开始显著下降。由于PCR扩增通常优选最佳PCR效率，因此本实施例在最佳PCR效率下***地评估每种值守碱基的选择性(图3e-h)。有趣的是，无论引物具有多少个值守碱基或者总碱基数，在退火温度比其Tm值高约4℃时通常能观察到最佳的选择性(图3e-h)。由于固定的值守碱基数目，通过在引物的5'末端添加更多碱基来增加引物Tm值不会显著改变区分***缺失的能力。四种类型引物中的三种表现出稳定的鉴别***缺失的能力(图3e-g)。只有一种类型的引物显示略微增加的能力，并在65.8℃左右的Tm值达到最佳值(图3h)。因此，在随后的实验中，值守碱基被设计为具有约65℃的Tm值并且在退火温度为69℃下进行getPCR。更重要的是，即使升高退火温度至超过Tm值可能会阻碍PCR效率，但对于这四种引物来讲，实时PCR定量的基础也就是Ct值和对数模板DNA量之间的线性相关性，却根本不会受到影响。(图3i-1)。DNA聚合酶在决定getPCR的辨别能力中起着重要作用。
本实施例中针对多种商业Taq酶进行测试，其性能表现不尽相同，但几乎全都表现出可观的足以区分***缺失与野生型序列的能力(图7j)。然而，当评估对单碱基错配的敏感性时，9种SYBR Green qPCR商品中的7种都显示出较高的适用性能(图8d-e)。
实施例3 getPCR定量基因组编辑准确性的研究
图2a中所示的是用来模拟基因组编辑导致的***缺失突变(indel)的质粒，首先用它们来评估getPCR定量基因组编辑效率的能力。本实施例中将二十六个indel质粒等份混合，然后再按特定比例与野生型质粒混合，以模拟0％，20％，40％，60％，80％和100％的***缺失频率。通过getPCR以及经典的Surveyor方法对混合物进行***缺失频率定量并进行比较。当indel频率不高于20％时，Surveyor方法的量化结果可以真实地反映预期值。然而，随着indel频率的进一步增加，观察值便逐渐偏离预期值(图4a-b)。相反，无论是值守碱基上携带3,4或5个值守碱基，使用不同值守碱基的所有12种getPCR策略都可以准确地定量***缺失频率(图4c，图9a-c)。
实施例4 getPCR在模拟单细胞克隆的基因分型中的应用
基因组编辑实验中的单细胞克隆筛选或子代基因分型是getPCR技术的另一个重要应用。单独使用图2a中所示的每个indel质粒，或将每个indel质粒与野生型质粒等比例混合，来模拟两个等位基因或一个等位基因发生编辑的单细胞克隆基因组DNA。所有三种getPCR策略不仅可以确定是否发生了***缺失，而且还可以准确地确定是一个等位基因还是两个等位基因发生了***缺失突变(图4d-f)。此外，当将任何两种getPCR策略进行组合分析时，它们的检测值还表现出极高的相关性，Pearson相关系数等于或高于0.995。有趣的是，两种getPCR策略的组合可以显著提高鉴定基因型的性能(图9d-f)。
实施例5 getPCR确定单细胞克隆的编辑频率和基因型
本实施例在Lenti-X 293T细胞中用Cas9和靶向HOXB13，DYRK1A或EMX1基因的8条不同的gRNA进行了基因组编辑，并应用getPCR检测编辑效率(图5b)。每种gRNA的编辑效率分别通过三种不同的方法确定，即getPCR、基于NGS的扩增子测序以及Surveyor分析。
对于设计的所有值守碱基，getPCR方法检测的编辑效率通常与NGS方法的结果相一致，而NGS是迄今为止被认为是最可靠的方法。相比之下，Surveyor方法测定出的编辑效率值与其它两种方法有明显的偏差，特别是当HOXB13基因上目标6和目标16的编辑效率较高时(图5a)。本实施例中对接受HOXB13基因的靶标6，EMX1基因的靶标1和靶标5以及DYRK1A基因的靶标1进行了基因组编辑的细胞，进行分离单细胞克隆并繁殖扩增。制备基因组DNA样品后通过getPCR进行基因分型，同时使用Sanger测序进行验证。总体而言，用这四种gRNA靶标进行基因组编辑实验的所有单细胞克隆都可以通过getPCR进行准确的基因分型。值得注意的 是，getPCR不仅可以检测出携带***缺失的细胞克隆，而且同时可以成功鉴定出该细胞克隆是一条等位基因发生编辑还是两条等位基因均发生了编辑(图5c-i，图10a-b)。对于在HOXB13基因靶标6处进行的基因组编辑，使用分别含有3或4个值守碱基的两种不同设计的getPCR引物，从总共42个细胞克隆中准确鉴定出了24个双等位基因编辑的细胞和5个单等位基因编辑的细胞(图5c-d，图10h)。类似地，在EMX1基因靶标5处基因组编辑，分别使用带有4个值守碱基的正向和反向引物通过getPCR鉴定出8个双等位基因编辑的细胞和5个单等位基因编辑的细胞(图5e-f，图10i)。在DYRK1A基因的靶标1处，使用getPCR从总共53个单克隆细胞中筛选出11个双等位基因编辑的细胞，5个单等位基因编辑的细胞，用到了四条不同设计的值守碱基，其中三条是正向引物携带有3,4或5个值守碱基，一条为反向引物携带有4个值守碱基(图5g-h，图10a-b，j)。对于EMX1基因的靶1处，使用携带4个值守碱基的引物的getPCR成功鉴定了来自45个克隆的1个双等位基因编辑的细胞克隆和9个单等位基因编辑的细胞克隆(图5i，图10k)。值得注意的是，任何两种不同设计的getPCR策略都显示出高度相关的检测值，并且当组合分析时可以帮助进行基因分型(图5j-1，图10c-g)。
实施例6 getPCR应用于确定HDR频率和单细胞克隆的基因型
本实施例中针对getPCR应用于HDR的基因组编辑修复效率的测定进行说明(图6a)。在Lenti-X 293T细胞中进行Cas9介导的基因组编辑实验，其中用到了EMX1基因的靶标5 gRNA和HDR模板，在PAM序列相邻的位置引入了HindIII位点序列(图6b)。应用getPCR方法以及基于NGS的扩增子测序和HindIII介导的限制性片段长度多态性(RFLP)分析来确定修复效率。结果表明，分别使用正向和反向的两条值守碱基的getPCR法都可以确定HDR频率，检测结果与基于RFLP和NGS方法的检测结果高度一致(图6c)，根据评估，来自三个生物重复样品的HDR频率约为25％。此外，对在EMX1靶标5处进行Cas9介导的HDR修复的细胞，进行分离单细胞克隆并繁殖扩增，得到了50个单细胞克隆，分别使用两条值守碱基通过getPCR对它们进行基因分型，成功地挑选出6个纯合修复的细胞克隆和17个单等位基因修复的细胞克隆(图6d-e)。此外，这两个值守碱基的检测值具有高度一致性即强相关性(r＝0.982，P＝1.207×10-36)，并且二者的组合分析可以明显更好的实现基因分型，特别是对于杂合的细胞克隆(图6f)。
实施例7 getPCR确定碱基编辑器的编辑频率和单细胞克隆基因型
本实施例针对getPCR在碱基编辑器的编辑频率和单细胞克隆基因型检测方面的应用进行说明。本实施例在Lenti-X 293T细胞中用EMX1靶标6的gRNA或HOXB13靶标8的gRNA以及BE4碱基编辑器进行了基因组编辑，并应用getPCR检测编辑效率(图6b)。在碱基编辑频率的定量中，getPCR的检测结果与基于NGS的扩增子测序方法的结果高度一致(图6g-h)。对于EMX1靶标6，在gRNA靶向序列中第5位和第6位将约27％的“C”碱基转化为“T”。有趣的是，这两个位置的碱基编辑倾向于同时发生以产生T5T6基因型(图6g)。对于HOXB13靶标8的gRNA的碱基编辑，第8位的C到T的碱基变化频率约为15％，该变化能够通过引入一个前置的终止密码子'TAG'来提前终止开放阅读框(图6h)。
在Lenti-X 293T细胞中用EMX1靶标6或HOXB13靶标8进行碱基编辑后，进一步分离单细胞克隆，并用getPCR方法进行基因分型。通过getPCR分析，对于EMX1靶标6的碱基编辑，确定46个克隆中的25个在第5位发生了C到T的转变(图6j-k)，46个克隆中的22个克隆在第6位被证实携带C到T的转变(图6l-m)。getPCR检测结果中丢失的碱基组成百分比显示，其中有三个克隆，E01，E29和E70可能在第5位含有除C和T以外的碱基，还有一个克隆E24可能在第6位携带这样的碱基。这些克隆的Sanger测序显示在E01和E29的第5位，E24的第6位发生了C到G的碱基编辑(图11a-c)。特别的，E70克隆在第5个核苷酸处不携带C到T之外的碱基转变，但在其一条等位基因上，在gRNA靶向序列的-8位核苷酸处具有A到T的突变(图11c)。该A到T的突变位于值守碱基的3'末端第14个核苷酸处，它阻止了引物退火到该等位基因上，并最终导致了丢失的getPCR信号。而Lenti-X 293T是一种基于HEK 293的细胞系，据报道其基因组接近三倍体，每个细胞有62-70 个染色体。于此一致地，在getPCR分析中，杂合克隆的每个等位基因的百分比通常为33％或66％左右，而不是50％(图6j，l)。
此外，在Sanger测序分析中这些三倍体特征得到了进一步验证，两个杂合等位基因峰图的高度通常具有两倍而不是对等的相互关系(图11c)。例如，在getPCR分析中，E11克隆的第5个核苷酸处的T和C碱基的百分比分别被确定为28.8％和62.9％，并且在Sanger测序中，C碱基的峰高几乎是T的两倍。然而，即使拿到了Sanger测序结果，仍有10个克隆的等位基因特异性基因型是未知的，仅仅知道它们在第5和第6核苷酸处均是杂合的(图11c)。本实施例设计了四个值守碱基以通过getPCR方法对这些克隆进行进一步基因分型(图6b)，并且成功确定了这些克隆的确切等位基因特异性基因型(图6i)。克隆E02和E15定义为C5C6/C5C6/T5T6，并且E33，E39，E40以及E49被证明是C5C6/T5T6/T5T6。发现克隆E01和E29均为C5C6/T5T6/G5C6，并且E24，E34克隆最终分别确定为C5C6/T5C6/T5G6和C5C6/T5T6/T5C6。
对于在HOXB13靶标8处进行的碱基编辑以引入框内终止密码子，本实施例从49个细胞单克隆中确定了14个细胞克隆在sgRNA的第8位碱基处发生了C到T的转化，这将带来一个提前终止密码子(图6n-o)。值得注意的是，getPCR检测结果中碱基组成丢失的百分比表明，S37克隆可能在该位置携带除C和T碱基之外的额外碱基，Sanger测序显示在gRNA的第8位核苷酸处，三条等位基因之一的C碱基转变为了G碱基(图12a-b)。同样的，getPCR也可以确定杂合子克隆的精准基因型，这也被Sanger测序所证实。例如，在HOXB13 gRNA靶标8序列的第8个核苷酸处，对6个克隆S15，S47，S44，S18，S02和S35进行基因分型为C/C/T。
以上所述仅为本公开的优选实施例而已，并不用于限制本公开，对于本领域的技术人员来说，本公开可以有各种更改和变化。凡在本公开的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内。

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  1. 一种用于检测核酸酶切诱导的indel发生频率的方法，其特征在于，所述方法包括以下步骤：向待测基因组样品中加入引物及Taq DNA聚合酶，对基因组样品中的野生型DNA进行扩增，通过PCR定量野生型DNA比例，从而确认基因组中indel发生的频率；所述引物序列与野生型DNA序列相匹配，并且覆盖核酸酶切割位点；优选的，所述PCR定量为实时PCR或ddPCR；优选的，所述检测方法还包括以下步骤：在距离切割位点数百碱基对的位置处引入对照扩增，通过ΔΔCt策略计算经过编辑后的基因组DNA样品中野生型DNA的百分比。
  2. 如权利要求1所述的检测方法，其特征在于，所述核酸酶包括但不限于Cas9核酸酶、锌指核酸酶，转录激活因子样效应核酸酶和CRISPR RNA指导FokI核酸酶，以及成对的cas9切口酶；进一步的，所述核酸酶为Cas9核酸酶；所述引物3'末端跨越Cas9核酸酶切割位点。
  3. 如权利要求2所述的检测方法，其特征在于，所述引物序列中包括值守碱基序列，所述值守碱基为核酸酶切位点与3'末端之间的序列，所述值守碱基长度为1～8bp；优选的，所述引物为一条核苷酸序列，所述值守碱基的长度为3～5bp；或所述引物为正向和反向的一对核苷酸序列，所述值守碱基的长度为4bp。
  4. 如权利要求3所述的检测方法，其特征在于，所述值守碱基3'末端碱基为腺嘌呤碱基或胞嘧啶或鸟嘌呤碱基；优选的，为腺嘌呤碱基。
  5. 如权利要求2所述的检测方法，其特征在于，所述扩增反应的退火温度为T m～T m+4℃。
  6. 一种用于检测核酸酶切诱导的indel发生频率的试剂盒，所述试剂盒中包括引物、Taq DNA聚合酶及PCR检测试剂。
  7. 权利要求6所述的试剂盒在评价基因组编辑效率、单细胞克隆筛选方面的应用；优选的，所述基因组编辑包括NHEJ介导的***缺失，HDR介导的基因修饰及通过BE4产生的碱基编辑；优选的，所述应用还包括对筛选适配CRISPR的gRNA。
  8. 一种对单细胞克隆进行基因分型的方法，其特征在于，所述方法包括以下步骤：以待测基因组中野生型DNA为模板，针对等位基因设计引物，提取待测单细胞克隆的基因组DNA，通过权利要求1-5任一项所述的检测方法检测单细胞基因组DNA中等位基因是否发生了indel从而对单细胞基因实现分型。
  9. 一种HDR修复效率的检测方法，其特征在于，所述检测方法包括以下步骤：针对待测基因组中HDR修复的基因组DNA设计引物，提取待测细胞基因组DNA，采用权利要求1-5任一项所述的检测方法检测HDR的发生概率；HDR修复DNA所占百分比即HDR修复效率。
  10. 一种碱基编辑器编辑效率的检测方法，其特征在于，所述检测方法包括以下步骤，以待测基因组DNA为模板，针对碱基编辑后的靶序列设计引物，采用权利要求1-5任一项所述的检测方法检测基因组中碱基编辑的发生概率，即为编辑器的编辑效率。
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