Method for detecting CRISPR/Cas-induced gene mutation and gene editing frequency based on ddPCR
阅读说明：本技术 一种基于ddPCR检测CRISPR/Cas诱导的基因突变和基因编辑频率的方法 (Method for detecting CRISPR/Cas-induced gene mutation and gene editing frequency based on ddPCR ) 是由 彭城 徐俊锋 丁霖 陈笑芸 汪小福 于 2020-12-28 设计创作，主要内容包括：本发明提供了一种基于ddPCR检测CRISPR/Cas诱导的基因突变和基因编辑频率的方法。所述方法包括根据CRISPR/Cas靶序列的突变位置,设计一对变异位点特异性PCR扩增引物和变异位点特异性探针,以及一对内源参考基因特异性PCR扩增引物和内源参考基因特异性探针；其中,所述变异位点特异性探针设置在突变位点区域,并且探针的5’端位于PAM区且连接有第一荧光基团,3’端连接有第一淬灭基团；所述内源参考基因特异性探针的5’端连接有第二荧光基团,3’端连接有第二淬灭基团。所述方法对于基因突变检测灵敏度高,对基因编辑频率的检测下限(LOD)更低,更接近预期的编辑频率值。(The invention provides a method for detecting CRISPR/Cas-induced gene mutation and gene editing frequency based on ddPCR. The method comprises the steps of designing a pair of mutation site specific PCR amplification primers and a mutation site specific probe, and a pair of endogenous reference gene specific PCR amplification primers and an endogenous reference gene specific probe according to mutation positions of a CRISPR/Cas target sequence; the mutation site specific probe is arranged in a mutation site region, the 5 'end of the probe is positioned in a PAM region and is connected with a first fluorescent group, and the 3' end of the probe is connected with a first quenching group; the 5 'end of the endogenous reference gene specific probe is connected with a second fluorescent group, and the 3' end of the endogenous reference gene specific probe is connected with a second quenching group. The method has high sensitivity for detecting gene mutation, has lower limit of detection (LOD) on gene editing frequency, and is closer to expected editing frequency value.)
1. A method for detecting CRISPR/Cas-induced gene mutation based on ddPCR, comprising:
designing a pair of mutation site specific PCR amplification primers and a mutation site specific probe, and a pair of endogenous reference gene specific PCR amplification primers and an endogenous reference gene specific probe according to the mutation position of the CRISPR/Cas target sequence;
the mutation site specific probe is arranged in a mutation site region, the 5 'end of the probe is positioned in a PAM region and is connected with a first fluorescent group, and the 3' end of the probe is connected with a first quenching group; the 5 'end of the endogenous reference gene specific probe is connected with a second fluorescent group, the 3' end of the endogenous reference gene specific probe is connected with a second quenching group, and the first fluorescent group and the second fluorescent group are different.
2. The method of claim 1, further comprising:
performing ddPCR reaction using the primer and probe of claim 1;
the results are based on the fluorescent signal of the fluorescent droplet: droplets in which both the first and second fluorescent signals are positive are wild-type droplets, and droplets in which the second fluorescent signal is positive but the first fluorescent signal is negative are mutant droplets.
3. A method for detecting CRISPR/Cas-induced gene editing frequency based on ddPCR, which is characterized in that the gene editing mutation frequency is calculated by calculating the ratio of the copy number of mutant microdroplets to the copy number of wild type microdroplets based on the result of fluorescent signal of fluorescent microdroplets obtained by the method of claim 2.
4. The method of claim 1 or 2, wherein the first quencher group is selected from BHQ or MGB; the first fluorophore and the second fluorophore are selected from any one of FAM, HEX and TEX; preferably, the first fluorophore is FAM and the second fluorophore is HEX.
5. The method according to any one of claims 1 to 3, wherein the CRISPR/Cas target sequence is a sequence of rice TGW6 gene, and the nucleotide sequence is shown as SEQ ID NO. 17.
6. The method according to claim 5, wherein the sequences of the upstream and downstream primers of the mutation site-specific PCR amplification primer are shown in SEQ ID No.1 and SEQ ID No.2, respectively, and the sequence of the mutation site-specific probe is shown in SEQ ID No. 3.
7. The method according to claim 5, wherein the sequences of the upstream and downstream primers of the PCR amplification primer specific for the endogenous reference gene are shown as SEQ ID No.4 and SEQ ID No.5, respectively, and the sequence of the probe specific for the endogenous reference gene is shown as SEQ ID No. 6.
8. The method as claimed in claim 7, further comprising extracting a plant template DNA, and then preparing a reaction mixture using the template DNA together with mutation site-specific PCR amplification primers and mutation site-specific probes and endogenous reference gene-specific PCR amplification primers and endogenous reference gene-specific probes, and performing ddPCR reaction using the reaction mixture.
9. The method as claimed in claim 8, wherein the concentration of the primer in the ddPCR reaction is 400-600nM, preferably the concentration of the primer is 450-500 nM; the concentration of the probe is 200-400 nM; preferably, the concentration of the probe is 250-300 nM.
10. The method as claimed in claim 8, wherein the ddPCR reaction is programmed to 93-96 ℃ for 5-15min, 93-95 ℃ for 5-15s, 55-60 ℃ for annealing and extension for 50-70s, 35-40 cycles, and 95-100 ℃ for 8-12min to terminate the reaction.
Technical Field
The invention relates to the technical field of biomedicine, in particular to a method for detecting CRISPR/Cas-induced gene mutation and gene editing frequency based on ddPCR.
Background
CRISPR/Cas systems have been widely used for genome editing and other targeted modifications in organisms. In contrast to traditional agrobacterium-mediated T-DNA transgene approaches, which rely on random recombination or integration of plants, the site-specific double-strand break (DSB) produced by Cas nucleases is repaired primarily by error-prone non-homologous end joining (NHEJ) machinery. The repair results in a variety of substitution, insertion or deletion (indels) mutations, most commonly deletion mutations with only 1bp variation.
Several methods for detecting CRISPR/Cas system-induced mutations of target genes have been reported, including Polymerase Chain Reaction (PCR) -based detection, T7 endonuclease I recognition cleavage, high-resolution melting-curve analysis (HRM), and NGS-based methods. However, these methods are semi-quantitative, and it is difficult to detect a variation in a processed food sample containing a low initial concentration of DNA, and it is also difficult to accurately quantify the gene editing frequency. Furthermore, existing methods are also difficult to detect well for very low frequency mutations in the genome of complex polyploid plants.
Droplet digital pcr (ddpcr) is a breakthrough technique that relies on dividing a single amplification into different compartments and detecting its end amplification products. It provides ultrasensitive and absolute nucleic acid quantitation without a standard curve. To date, there have been few reports on ddPCR technology to detect gene-edited plants and processed foods derived therefrom.
The present invention has been made in view of the above.
Disclosure of Invention
The invention aims to provide a method for detecting CRISPR/Cas-induced gene mutation and gene editing frequency based on ddPCR. The method solves the technical problems that the prior art is difficult to quantitatively detect the CRISPR/Cas induced plant gene mutation and accurately quantify the plant gene editing frequency.
The technical scheme provided by the invention is as follows:
a method for detecting CRISPR/Cas-induced gene mutation based on ddPCR, the method comprising:
designing a pair of mutation site specific PCR amplification primers and a mutation site specific probe, and a pair of endogenous reference gene specific PCR amplification primers and an endogenous reference gene specific probe according to the mutation position of the CRISPR/Cas target sequence;
the mutation site specific probe is arranged in a mutation site region, the 5 'end of the probe is positioned in a PAM region and is connected with a first fluorescent group, and the 3' end of the probe is connected with a first quenching group; the 5 'end of the endogenous reference gene specific probe is connected with a second fluorescent group, and the 3' end of the endogenous reference gene specific probe is connected with a second quenching group. The first fluorophore and the second fluorophore are different.
The variant site specific PCR amplification primers were designed to span the mutation site. The primers were screened using conventional PCR to determine that a single product of the correct fragment size could be amplified. The CRISPR/Cas 9-induced mutations were predictable, with most mutations occurring at 3 bases upstream of the 5' end of the protospacer motif adjacent motif (PAM). The variant site-specific probes should be located in the PAM region. In order to maintain the sensitivity of the probe to mutations, it is preferable to label the PAM region as the 5' end of the probe.
The endogenous reference gene is a validated reference gene, the detection region of the endogenous reference gene does not contain a mutation site for gene editing, and the copy number of the selected endogenous reference gene needs to be the same as the copy number of the edited target gene in the plant sample. Mutations caused by gene editing will cause the mutation site-specific probes to fail to bind to the target sequence, but will not affect the binding of endogenous reference gene probes, which are mainly used to assess the number of mutated genes, while endogenous reference gene probes are mainly used to analyze the total number of alleles in a sample.
In one embodiment, the method further comprises: carrying out ddPCR reaction by adopting the mutation site specificity PCR amplification primer and the mutation site specificity probe as well as the endogenous reference gene specificity PCR amplification primer and the endogenous reference gene specificity probe;
the results are based on the fluorescent signal of the fluorescent droplet: droplets in which both the first and second fluorescent signals are positive are wild-type droplets, and droplets in which the second fluorescent signal is positive but the first fluorescent signal is negative are mutant droplets.
Wild type microdroplets and homozygous mutant microdroplets can be clearly distinguished by a two-dimensional view of ddPCR analysis.
A method for detecting CRISPR/Cas-induced gene editing frequency based on ddPCR, which calculates gene editing mutation frequency by calculating the ratio of mutant microdroplet copy number to wild-type microdroplet copy number based on the result of fluorescent signal of fluorescent microdroplet obtained according to the aforementioned method.
In one embodiment, in the above ddPCR-based method for detecting CRISPR/Cas-induced gene mutation and gene editing frequency, the first quencher group is selected from BHQ or MGB; the first fluorophore and the second fluorophore are selected from any one of FAM, HEX and TEX; preferably, the first fluorophore is FAM and the second fluorophore is HEX.
The endogenous reference gene probe is a 5 'end HEX marker, and the mutation site specific probe is a 5' end FAM marker.
In one embodiment, the CRISPR/Cas target sequence is a stretch of rice TGW6 gene (Os06g0623700), and its nucleotide sequence is shown in SEQ ID No. 17.
In one embodiment, the sequences of the upstream and downstream primers of the variation site specific PCR amplification primer are shown as SEQ ID NO.1 and SEQ ID NO.2, respectively, and the sequence of the variation site specific probe is shown as SEQ ID NO. 3.
In one embodiment, the upstream and downstream primer sequences of the PCR amplification primer specific for the endogenous reference gene are shown as SEQ ID NO.4 and SEQ ID NO.5, respectively, and the sequence of the probe specific for the endogenous reference gene is shown as SEQ ID NO. 6.
The primer pair and the probe are used for detecting CRISPR/Cas induced rice TGW6 gene mutation and gene editing frequency by ddPCR technology, and the primer pair consists of the sequence of the mutation site specific PCR amplification primer and the sequence of the mutation site specific probe as well as the sequence of the endogenous reference gene specific PCR amplification primer and the sequence of the endogenous reference gene specific probe.
In one embodiment, the method further comprises extracting a plant template DNA, and then preparing a reaction mixture by combining the template DNA with the mutation site-specific PCR amplification primers and the mutation site-specific probes and the endogenous reference gene-specific PCR amplification primers and the endogenous reference gene-specific probes, and performing ddPCR reaction using the reaction mixture.
In one embodiment, the amount of the plant template DNA in the ddPCR reaction system is 0.116-10 ng/. mu.L.
In one embodiment, the concentration of the primer in the ddPCR reaction is 400-600nM, preferably, the concentration of the primer is 450-500 nM; the concentration of the probe is 200-400 nM; preferably, the concentration of the probe is 250-300 nM.
In a specific embodiment, in the ddPCR reaction, the concentration of the primer is 450 nM; the concentration of the probe was 250 nM.
In one embodiment, the ddPCR reaction system further comprises ddPCR Supermix and sterile water. The PCR reaction solution is put into a droplet generation card, and droplet generation oil is added to generate droplets in a droplet generator. The droplets were transferred to a 96-well PCR plate for PCR reaction.
In one embodiment, the procedure for the ddPCR reaction is 93-96 ℃ for 5-15min, 93-95 ℃ for 5-15s, 58 ℃ or 68 ℃ for annealing and extension for 50-70s, 35-40 cycles, and 95-100 ℃ for 8-12min to stop the reaction.
In a specific embodiment, the ddPCR reaction is programmed to anneal and extend at 95 ℃ for 10min, 94 ℃ for 10s, 58 ℃ or 68 ℃ for 60s, 40 cycles, stop the reaction at 98 ℃ for 10min, and cool to 4 ℃.
In one embodiment, the method specifically comprises the steps of:
(1) extracting gene editing plant DNA to obtain a DNA template;
(2) diluting heterozygote gene editing DNA samples into different concentration gradients, preferably, the DNA concentrations are respectively 10, 5, 0.4, 0.08 and 0.016 ng/mu L;
(3) designing a mutation site specificity PCR amplification primer and a mutation site specificity probe according to the mutation site; the 5 'end of the probe is positioned in the PAM region and is connected with a first fluorescent group (preferably FAM), and the 3' end of the probe is connected with a first quenching group; selecting specific primers and probes of the endogenous reference gene, wherein the 5' end of the probe is connected with a second quenching group (preferably HEX);
(4) performing digital PCR in microdroplet format and performing data analysis, analyzing the concentration of mutant and wild-type microdroplets and determining a threshold to distinguish between positive and negative microdroplets;
(5) the mutation frequency of gene editing is determined by the ratio of mutant droplets (e.g., only HEX-positive droplets) to wild-type droplets (e.g., HEX/FAM double-positive droplets).
Has the advantages that:
the method for detecting CRISPR/Cas-induced gene mutation and gene editing frequency based on the droplet-type digital PCR has higher sensitivity for different types of gene mutations induced by gene editing technology. Compared with the qPCR and NGS based methods, the ddPCR method has a lower limit of detection (LOD) for edit frequency, closer to the expected edit frequency value.
The method for detecting CRISPR/Cas-induced gene mutation based on the droplet-type digital PCR has higher accuracy and precision, can accurately detect the mutation frequency of a gene editing mutant, and can become a powerful tool for detecting and evaluating the plant gene editing frequency.
The method provided by the invention can detect the gene mutation in the processed food sample containing the DNA with low initial concentration, is simultaneously suitable for detecting the low-frequency gene mutation in the complex polyploid plant genome, and has lower error rate.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic diagram of primer and probe design provided in the present invention and a two-dimensional distribution diagram of droplets for detecting wild-type rice samples, homozygous and heterozygous gene mutations by ddPCR;
FIG. 2 is a graph showing the results of Sanger sequencing-verified homozygous rice samples of different mutation types detected by ddPCR according to an embodiment of the present invention;
FIG. 3 is a graph showing the results of sensitivity measurement of ddPCR method for detecting different initial DNA concentrations of the same gene-edited sample according to the present invention;
FIG. 4 is a comparison of the results of analyzing the frequency of gene editing by different methods (ddPCR, qPCR and NGS) provided in the embodiments of the present invention;
FIG. 5 is a graph showing the results of verifying the gene editing frequency in a polyploid organism (Brassica napus) by the ddPCR method.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Examples
1. Plant material
The CRISPR/Cas9 gene editing rice mutant is edited by using CRISPR/Cas9 gene from an inventor group, and the number of a mutant strain generated by independent research and development is 1-8.
The sgRNA is designed by utilizing a network tool CRISPR-P, and the nucleotide sequence of the target site region is shown as SEQ ID NO.17
(CCTCGTGCTGCTCTCGCCGTCCCCTACTGCCGCCGCCACAGCCACAACGAGAATGTTCAA, where TCC is a PAM site).
The gene editing rape strain (S1-14, S1-18, S1-24, S1-53, S1-104) is CRISPR/Cas9 gene editing crop, and the nucleotide sequence of the target site region is shown as SEQ ID NO.18 (GGCCACTTGCCCATGCTGGCTAAGTACGGCCCTGAC, wherein CCA is PAM site). Is provided by Dr Zhengming, oil institute of agricultural academy of China.
DNA extraction
Plant DNA is extracted by using a Tiangen kit, the concentration of the DNA is determined by using NanoDrop 1000, and the integrity of the DNA extraction is further identified by agarose gel electrophoresis. All DNA templates were diluted to 10 ng/. mu.L and all DNA templates were stored in a freezer at-20 ℃.
3. Sample preparation
To analyze DNA at a low initial concentration, a heterozygote gene-editing DNA sample was diluted with water and used2.0(Life technologies, USA) and DNA concentrations were 10, 5, 0.4, 0.08, 0.016 ng/. mu.L, respectively.
4. Primer and probe design
Most mutations induced by CRISPR/Cas9 occurred at 3 bases upstream of the 5' end of the protospacer motif adjacent motif (PAM). In the digital PCR system of the droplet type, two pairs of primers (a primer pair for the endogenous reference gene and a primer pair for the mutation site-specific gene) and respective detection probes (a probe for the endogenous reference gene and a mutation site-specific probe) were designed (see panel a in fig. 1): the amplification region of one pair of primers comprises a mutation site, and the probe is positioned in the mutation site region and is detected by a mutation site specific probe (modified by FAM); another pair of primers and probes can select an endogenous reference gene (HEX modified) that has been validated, and the detection region does not contain a mutation site for gene editing. The selected endogenous reference gene copy number needs to be the same as the edited target gene copy number in the plant sample. The mutation frequency of gene editing was calculated using the ratio of the copy number of mutant droplets (FAM positive droplets only) to the copy number of wild type droplets (HEX/FAM double positive droplets).
Primers and probes for mutation sites were designed using Primer Express software 3.0. The design also follows the following principles: (1) the primers span the mutation sites, (2) the probes should be located in the PAM region, and (3) to maintain the sensitivity of the probes to mutations, the PAM region is labeled as the 5' end of the probe. The primers were screened using conventional PCR to determine that a single product of the correct fragment size could be amplified.
The reference gene probe is a 5 'end HEX marker, and the mutation site specific probe is a 5' end FAM marker.
The primers and probes used in this study are listed in table 1 below.
TABLE 1 primers and fluorescent probes used in ddPCR
aProbes modified with MGB as reference gene
5. Droplet digital PCR
The droplet digital PCR assay comprises the following components:
20 μ L of total reaction: mu.L ddPCR Supermix (without dUTP) (Bio-Rad), 450nM for each primer pair (primer pair comprising: a primer pair for the endogenous reference gene and a primer pair for the mutation site-specific gene), 250nM for each probe (probe comprising: a probe for the endogenous reference gene and a mutation site-specific probe), sterile water was added to adjust the final volume to 19. mu.L, and 1. mu.L of DNA template was then added.
mu.L of the mixture was placed into the microdroplet card of BioRad DG8 and 70. mu.L of microdroplet oil was added. The droplet generation card was placed in a QX200 droplet generator (Bio-Rad) to generate droplets. The droplets were transferred to a 96-well PCR plate. After sealing and heat sealing with aluminum foil, the PCR plate was placed in a 7500 real-time PCR system and amplified under the following cycling conditions: 95 ℃ 10min, 40 cycles 94 ℃ 10s, 58 or 68 ℃ annealing and extension 60s, reaction termination at 98 ℃ 10min and cooling to 4 ℃. After amplification, the 96-well PCR plate was placed in a QX200 microdroplet reader (Bio-Rad) for data analysis.
Wild type and homozygous mutant can be clearly distinguished by a two-dimensional view of ddPCR analysis.
The concentration of mutant and wild-type droplets was analyzed using Bio-Rad QuantaSoft and a threshold was determined to distinguish between positive and negative droplets. The mutation frequency of gene editing was determined by the ratio of mutant droplets (only HEX positive droplets) to wild type droplets (HEX/FAM double positive droplets).
6. Real-time quantitative PCR
Quantitative real-time PCR was performed on a 7500 real-time PCR instrument (Life Technologies AB) using FastStart Universal Probe Master (Roche) and ROX reference dyes as indicated.
7. NGS-based sequencing
PCR primers (table 2) were designed to amplify the products flanking the target mutation site using nested PCR methods and unique sample-specific barcodes attached to the PCR products. High throughput sequencing was performed by the Illumina HiSeq platform (Illumina, usa) used by beijinuo and the institute for genetic bioinformatics, china. Initial use2.0(Life Technologies, USA) the concentration of the library was measured. Analysis of the frequency of gene editing mutations was performed using the hitom program for decoding of high throughput mutated sequences (http:// www.hi-tom. net/hi-tom /).
TABLE 2 primers used in NGS of the present invention
And (4) analyzing results:
1. gene editing rice based on ddPCR detection
The performance of the ddPCR-based gene editing plant mutation detection method was evaluated by using rice samples (rice TGW6 wild-type gene, CRISPR-Cas9 gene editing-induced homozygous mutant and CRISPR-Cas9 gene editing-induced heterozygous mutant).
ddPCR detection was performed using the primer pair (SEQ ID NO.1 and SEQ ID NO.2) and the mutation site-specific probe (SEQ ID NO.3) for the mutation site-specific gene designed for the mutation site of the rice (Oryza sativa) TGW6 gene (Os06g0623700) in Table 1 and the primer pair (SEQ ID NO.4 and SEQ ID NO.5) and the probe (SEQ ID NO.6) for the endogenous reference gene of rice (Oryza sativa) in Table 1 (LOC _ Os2g42314), and it was shown that three rice samples could be clearly distinguished by two-dimensional view of ddPCR analysis (shown in FIG. 1B).
In the two-dimensional view of the ddPCR analysis, the microdroplets containing both fluorescent signals were wild-type amplicons, while the microdroplets containing the HEX-positive but FAM-negative signals were mutant amplicons. Amplification of heterozygotic mutations comprises either mutant microdroplets or wild-type microdroplets.
2. The ddPCR platform can effectively identify different mutation types induced by gene editing
Since the mutation caused by Cas is usually only 1bp variation, it is very important that the newly developed method can effectively detect these tiny gene mutations.
To verify whether the ddPCR method is sensitive to different types of mutations (e.g., single nucleotide index), we tested sequencing-verified different types of homozygous mutated CRISPR/Cas9 gene edited TGW6 rice samples using the ddPCR platform. These mutations include not only single nucleotide deletions but also single nucleotide mutations (panel a in fig. 2).
ddPCR results indicated that in all of these samples, the droplets contained a HEX-positive signal, but a FAM-negative signal, and that when there were thousands of HEX-positive droplets, the number of FAM-positive droplets was almost zero (B, C panel in FIG. 2). Therefore, the ddPCR-based detection method can efficiently recognize various mutations caused by gene editing.
3. Sensitivity determination of Gene editing samples
To evaluate the sensitivity of ddPCR to detect gene-edited samples, we used low initial concentrations of DNA samples, even processed food samples, to evaluate the performance of ddPCR.
A series of heterozygous samples (10, 5, 0.4, 0.08, 0.016 ng/. mu.L) of DNA concentration were prepared for detection using ddPCR.
The results show that ddPCR method can accurately detect samples as low as 0.08 ng/. mu.L, while DNA samples of 0.016 ng/. mu.L are not recognized due to too much background noise. For processed food samples, the ddPCR method also showed good detection performance (FIG. 3, panel A), indicating that ddPCR has broad application prospects.
To determine the limit of detection (LOD) of the mutation frequency of ddPCR method, we mixed the mutant template (homozygous mutant DNA) and the wild-type template in different ratios (mutant DNA between 50% and 0.1%). As the amount of mutant template decreased, the concentration of mutant droplets in the mixed sample gradually decreased while the concentration of wild-type droplets remained essentially unchanged (panel B in FIG. 3).
The results showed that the mutant template could be detected at a mutation frequency of 0.1% based on the ddPCR detection method. Therefore, ddPCR is an ultrasensitive gene editing detection and evaluation method, and can be used for detecting samples with low initial concentration of DNA, such as processed food samples.
4. Comparison of ddPCR with qPCR and NGS methods
At present, qPCR and NGS based methods have been demonstrated to be useful for quantitative detection of plant mutation frequency induced by gene editing. We further detected and analyzed the same pooled samples using qPCR and NGS methods.
The results of the qPCR and NGS based methods compared to the ddPCR method showed no positive signal when the mutation template rate was below 5% (a-C in fig. 4, table 3), indicating that the LOD of the qPCR and NGS based methods was about 5% (B-C in fig. 4). The result detected by ddPCR method is linear with the expected editing frequency (R)2>0.999) and qPCR-based and NGS-based methods are R and NGS, respectively20.982 and 0.998. Furthermore, by using the NGS-based approach, we found that there was an unexpected mismatch (T->C) This may be caused by NGS sample preparation. In summary, ddPCR provides a more accurate and quantitative method for the detection and assessment of gene editing frequency than other methods.
TABLE 3 detection of Gene editing frequency by NGS method
5. Detection of homologous tetraploid rape gene editing mutation by utilizing ddPCR platform
To further confirm whether the ddPCR method can accurately detect the gene editing frequency in polyploid organisms, samples of Brassica napus (Brassica napus l., AACC, 2n ═ 38) were selected for analysis. We edited Brassica napus based on previously established genes with gene editing targets on different chromosomes, where one target was heterozygous (A or C genome) and the other was homozygous mutant or wild type for Sanger sequencing (panel A in FIG. 5), and theoretically these samples should have an editing frequency of 75% for one homozygous mutant and another homozygous mutant, or one homozygous wild type and 25% for another heterozygous mutant. When analyzed using ddPCR, the editing frequency of S1-14 (Aaccc), S1-18(aaCC) and S1-24 (Aaccc) was 75% and that of S1-53 (Aaccc) and S1-104(AACc) was 25% as expected, consistent with the previous results (panel B in FIG. 5), indicating the stability of our ddPCR method in detecting polyploid biological mutations.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> Zhejiang province academy of agricultural sciences
<120> method for detecting CRISPR/Cas-induced gene mutation and gene editing frequency based on ddPCR
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