阅读说明：本技术 一种基因组编辑检测方法、试剂盒及应用 (Genome editing detection method, kit and application ) 是由 黄启来 李博 于 2019-06-14 设计创作，主要内容包括：本公开属于基因组编辑效率检测领域,具体涉及一种基因组编辑检测方法、试剂盒及应用。针对现有技术中基因编辑效率检测方法的缺陷,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、检测准确度不足等缺陷,本公开提供了一种新方法,称为getPCR,基于Taq DNA聚合酶特异性和实时PCR技术,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。本公开研究提供了相应值守碱基的设计规则及优化的getPCR运行参数,经验证该方法具有良好的检测准确性,并且操作简便,可应用于所有基因组编辑方法定量基因组编辑效率,还可以应用于单细胞克隆的筛选。(The disclosure belongs to the field of genome editing efficiency detection, and particularly relates to a genome editing detection method, a kit and application. Aiming at the defects of gene editing efficiency detection methods in the prior art, such as Sanger, NGS, a method based on mismatch specific nuclease and the like, which have the defects of complex operation, high cost, insufficient detection accuracy and the like, the disclosure provides a new method, namely getPCR, which quantifies wild type DNA in a genome to be detected based on Taq DNA polymerase specificity and a real-time PCR technology, and confirms the genome editing efficiency by calculating the percentage of the wild type DNA. The open research provides the design rule of the corresponding on duty base and the optimized getPCR operation parameter, and the method is proved to have good detection accuracy and simple and convenient operation, can be applied to all genome editing methods for quantifying the genome editing efficiency and can also be applied to the screening of single cell clone.)

1. A method for detecting the frequency of occurrence of nuclease-induced indels, comprising the steps of: adding a primer and Taq DNA polymerase into a genome sample to be detected, amplifying wild type DNA in the genome sample, and quantifying the proportion of the wild type DNA by PCR (polymerase chain reaction) so as to confirm the frequency of indels in the genome; the primer sequence matches the wild-type DNA sequence and covers the nuclease cleavage site; preferably, the PCR quantification is real-time PCR or ddPCR; preferably, the detection method further comprises the following steps: control amplification was introduced hundreds of base pairs from the cleavage site and the percent of wild type DNA in the edited genomic DNA sample was calculated by the Δ Δ Ct strategy.

2. The assay of claim 1, wherein the nucleases include, but are not limited to, Cas9 nuclease, zinc finger nuclease, transcription activator-like effector nuclease and CRISPR RNA guide fokl nuclease, and paired Cas9 nickase; further, the nuclease is a Cas9 nuclease; the 3' end of the primer spans the Cas9 nuclease cleavage site.

3. The detection method according to claim 2, wherein the primer sequence comprises a guard base sequence, the guard base is a sequence between a nuclease cutting site and a 3' end, and the length of the guard base is 1-8 bp; preferably, the primer is a nucleotide sequence, and the length of the guard base is 3-5 bp; or the primer is a pair of nucleotide sequences in the forward direction and the reverse direction, and the length of the guard bases is 4 bp.

4. The detection method according to claim 3, wherein the base at the 3' -end of the guard base is an adenine base or a cytosine or guanine base; preferably, it is an adenine base.

5. The detection method according to claim 2, wherein the annealing temperature of the amplification reaction is T_m～T_m+4℃。

6. A kit for detecting the occurrence frequency of indels induced by nuclease digestion comprises a primer, Taq DNA polymerase and a PCR detection reagent.

7. The use of the kit of claim 6 for evaluating genome editing efficiency, single cell clone screening; preferably, the genome editing comprises NHEJ-mediated indels, HDR-mediated gene modification and base editing by BE 4; preferably, the use also includes the selection of CRISPR-adapted grnas.

8. A method for genotyping a single cell clone, said method comprising the steps of: using wild type DNA in a genome to be detected as a template, designing a primer aiming at allele, extracting the genome DNA of the single cell clone to be detected, and detecting whether indel occurs to the allele in the single cell genome DNA by the detection method of any one of claims 1-5 so as to realize typing of the single cell gene.

9. A method for detecting HDR repair efficiency is characterized in that the method comprises the following steps: designing a primer aiming at the genome DNA for repairing HDR in a genome to be detected, extracting the genome DNA of a cell to be detected, and detecting the occurrence probability of HDR by adopting the detection method of any one of claims 1-5; the percentage of HDR repair DNA is the HDR repair efficiency.

10. A method for detecting the editing efficiency of a base editor, which is characterized by comprising the following steps of using a genome DNA to be detected as a template, designing a primer aiming at a target sequence after base editing, and detecting the occurrence probability of base editing in a genome by using the detection method of any one of claims 1 to 5, namely the editing efficiency of the editor.

Technical Field

The disclosure belongs to the field of gene editing detection, and particularly relates to a method for indirectly confirming genome editing probability by amplifying the proportion of wild group DNA in a quantitative genome, and application of the method in the aspects of genome editing efficiency evaluation and monoclonal screening.

Background

The information in this background section is only for enhancement of understanding of the general background of the disclosure and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

CRISPR/cas9 is a currently mainstream genome editing technology, and its gene modification effect is related to guide rna (sgrna). In the CRISPR/Cas9 system, Cas9 nuclease is directed through the sgRNA to the target DNA containing the original spacer adjacent motif (PAM), and then cleaves both strands of the target DNA 3bp upstream of the PAM sequence and generates a Double Strand Break (DSB). Once the cell senses the presence of DSB, repair of the fragmented genomic DNA occurs through two distinct intrinsic mechanisms, Homologous Recombination (HR) or non-homologous end joining (NHEJ). NHEJ involves direct ligation of cleaved ends, does not require a homologous template and repairs DNA breaks in an error-prone manner, often resulting in unpredictable base insertions or deletions at DNA breaks, called indels, which can be used to reduce gene expression, and has been widely used in gene function studies and in clinical settings to eliminate pathogenic genes.

The excellent sgRNA is pre-screened in CRISPR-Cas9 mediated genome editing, so that the method has important significance for obtaining good editing efficiency and specificity, and the sgRNA with stable effect can be used for obtaining single cell clone or filial generation with expected change. The methods widely used today are mainly based on DNA sequencing or mismatch-specific nucleases. For the Sanger sequencing method, PCR amplification and cloning involving the target region DNA is required before each DNA sequence is read separately. This approach, which requires multiple steps, can provide detailed information for each mutation event induced by a nuclease, but is very time consuming, expensive and laborious. Second generation DNA sequencing (NGS) technology is also used to analyze sgRNA-guided Cas9 nuclease-mediated DNA mutations because it has powerful parallel analysis capabilities. A variety of online platforms have been developed to analyze NGS data, including CRISPR-GA, BATCH-GE, CRISPRSOS, Cas-analyzer and CRISPRMATCH, and others. However, the inventors believe that the above-described on-line analysis platform still requires multi-step experimental operations, which are costly in terms of time and economic cost. The mismatch-specific nuclease-based method is the most popular method at present, and double-stranded DNA containing mismatched bases formed between DNA strands having sequence differences, which are caused by nuclease cleavage, is cleaved using T7 endonuclease 1(T7E1) or Surveyor nuclease, so that the detection of editing efficiency can be achieved. 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 single nucleotide mutations and deletions of large fragments are often missed. In addition, scientists have developed many other alternatives, but only improved in certain respects, such as qEva-CRISPR21, engineered nuclease-induced translocation (ENIT), Restriction Fragment Length Polymorphism (RFLP) analysis based on Cas9 nuclease, Indel detection by amplicon analysis (IDAA), and gene editing frequency digital PCR (GEF-dPCR). The inventors consider that the above experimental procedures are cumbersome and they use PCR amplification products of the genomic target DNA region rather than directly using the genomic DNA itself to quantify the editing efficiency. It is well known that sequence and length dependent variations introduced during PCR amplification will inevitably affect the accuracy of the detection.

Disclosure of Invention

Against the background of the above-mentioned research, the inventors considered it important to provide a method that is fast, simple and reliable for genome editing efficiency quantification and high-throughput genotyping, and that does not require special equipment. The present disclosure provides a method for detecting genome editing efficiency, hereinafter referred to as getPCR. The getPCR is used for amplifying the wild type DNA in the genome DNA to be detected by using the selective amplification characteristic of Taq polymerase, the occurrence frequency of indels in the genome to be detected is judged by quantifying the wild type DNA in an amplification product and quantifying the proportion of the wild type DNA, and the detection result has higher accuracy and wide application. The method has good accuracy when applied to detection of an indel induced by a Cas9 endonuclease, and can be applied to detection of genome editing efficiency related to a Cas9 nuclease technology, such as evaluation of sgRNA performance, HDR repair efficiency and base editor in CRISPR/Cas 9; besides, the method can also be used for confirming and screening the single cell clone genotype.

In order to achieve the technical effects, the present disclosure provides the following technical solutions:

in a first aspect of the present disclosure, there is provided a method for detecting the frequency of occurrence of nuclease-induced indels, the method comprising the steps of: adding a primer and Taq DNA polymerase into a genome sample to be detected, amplifying wild type DNA in the genome sample, and quantifying the proportion of the wild type DNA by PCR (polymerase chain reaction) so as to confirm the frequency of indels in the genome; the primer sequence matches the wild-type DNA sequence and covers the nuclease cleavage site.

Preferably, the nucleases include, but are not limited to, Cas9 nucleases, Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR RNA guide FokI nucleases (RFNs), and paired Cas9 nickases. Further, the nuclease is Cas9 nuclease.

Zinc Finger Nucleases (ZFNs), Transcription Activator Like Effector Nucleases (TALENS) and CRISPER-Cas9 systems are common means of modern precise genetic engineering technology, provide a reliable and simple method for evaluating the efficiency of the genetic modification technology, and have important significance. The efficiency of CRISPR sgrnas is typically assessed in the art using the frequency of occurrence of quantitative indels, with real-time PCR technology being the most effective method in nucleic acid quantification. However, the diversity and unpredictability of indel occurrence makes it impossible to design indel-specific primers, and so the skilled person cannot directly quantify indel frequency by real-time PCR. The detection method described in the first aspect, i.e. the getPCR technique, selectively amplifies wild-type DNA in a genome, circumventing this obstacle by quantifying the proportion of wild-type DNA by a relative quantification strategy of real-time PCR. Taq polymerase is capable of specifically amplifying a template that is perfectly matched to a primer without amplifying a template that is mismatched to the primer, and Taq polymerase is less tolerant to base mismatches between the primer and the complementary sequence. The method disclosed by the invention can accurately quantify the wild type DNA by utilizing the selective amplification of Taq polymerase, so as to obtain the occurrence probability of indels. The method takes Cas9 nuclease as an example, carries out primer design on the nuclease cutting sites with the directional cutting function and optimizes primer parameters, thereby realizing good detection effect. The research idea and the technical scheme disclosed by the invention are proved to have feasibility as detection methods of various gene editing technologies, and are expected to have good effects.

Preferably, the PCR quantification is real-time PCR or ddPCR.

Further preferably, the amplification reaction is real-time PCR, and the annealing temperature of the amplification reaction is T_m～T_m+4℃。

Preferably, the detection method further comprises the following steps: control amplification was introduced hundreds of base pairs from the cleavage site and the percent of wild type DNA in the edited genomic DNA sample was calculated by the Δ Δ Ct strategy.

Preferably, the primer 3' end spans the Cas9 nuclease cleavage site.

Preferably, the primer sequence comprises a guard base sequence, the guard base is a sequence between a nuclease cutting site and the 3' end of the primer, and the length of the guard base is 1-8 bp.

Further preferably, the primer is a nucleotide sequence, and the length of the guard base is 3-5 bp.

Further preferably, the primer is a pair of sequence combinations of forward and reverse directions, and the length of the guard base is 4 bp.

Further preferably, the 3' terminal base of the conserved base is an adenine base or a cytosine or guanine base; more preferably, it is an adenine base.

In a second aspect of the disclosure, a kit for detecting the occurrence frequency of indels induced by nuclease digestion is provided, wherein the kit comprises a primer, Taq DNA polymerase and PCR detection reagent; use of the kit performs the detection method as described in the first aspect.

In a third aspect of the disclosure, the kit of the second aspect is provided for evaluating genome editing efficiency and screening single cell clones.

Preferably, the genome editing comprises NHEJ-mediated indels, HDR-mediated gene modification and base editing by BE 4.

Preferably, the use further comprises screening for CRISPR-adapted grnas.

In a fourth aspect of the present disclosure, there is provided a method of genotyping a single cell clone, the method comprising the steps of: using wild type DNA in a genome to be detected as a template, designing a primer aiming at allele, extracting the genome DNA cloned by the single cell to be detected, and detecting whether indels occur in the allele in the genome DNA of the single cell by the detection method of the first aspect so as to realize typing of the single cell gene.

In a fifth aspect of the present disclosure, a method for detecting HDR repair efficiency is provided, where the method includes the following steps: designing a primer aiming at the HDR repaired genome DNA in a genome to be detected, extracting the genome DNA of a cell to be detected, and detecting the occurrence probability of HDR by adopting the method in the first aspect; the percentage of HDR repair DNA is the HDR repair efficiency.

In a sixth aspect of the present disclosure, a method for detecting editing efficiency of a base editor is provided, where the method includes the following steps, using a genomic DNA to be detected as a template, designing a primer for a target sequence after base editing, and detecting occurrence probability of base editing in a genome by using the detection method of the first aspect, that is, editing efficiency of an editor.

Taking genome editing of 8 sgrnas in 293T cells as an example for the study of the present disclosure, the getPCR technique can accurately quantify genome editing efficiency in all genome editing cases, including NHEJ-induced indels, HDR, and base editing. At the same time, this method shows a strong ability in single cell clone genotyping, since it can not only characterize whether the desired genome editing has occurred, but also inform that a particular number of alleles carry this specific editing.

Compared with the prior art, the beneficial effect of this disclosure is:

1. with the rapid development and wide application of the CRISPR technology, a simple, accurate and reliable genome editing efficiency evaluation method is provided, and the method has important significance for the screening of gRNAs and the optimization of experimental schemes. The method provided by the present disclosure is simple in process, reliable in quantitative results, time-saving and low in cost, does not involve a specific device, and only requires one qPCR step. Aiming at accurately measuring indel frequency on a CRISPR target, the detection accuracy is consistent with the most accurate NGS method.

2. The gene editing method based on the Cas nuclease technology can be used in the method disclosed by the invention, comprises NHEJ-induced indel, HDR and base editing, and can also be applied to screening of single cell clone.

3. The getpcrs provided by the present disclosure can also be easily extended to be applied to genome editing experiments mediated by other types of genome-cleaving nucleases to evaluate the editing efficiency of a given cleavage position, such as Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR RNA-directed FokI nucleases (RFNs), and paired cas9 nickases, etc., and by further determining the design rules of guard bases, it is expected to further promote the wide application of the technology in genome editing technology in molecular and cell biology research.

Drawings

The accompanying drawings, which are included to provide a further understanding of the disclosure, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure and are not to limit the disclosure.

FIG. 1 illustrates the principles and flow diagram of the disclosed getPCR;

(a) principle of identification of indels and wild sequences by getPCR (b) strategy overview of getPCR

FIG. 2 schematic diagram of the design of the getPCR primer in example 1;

(a)26 plasmids mimic indels at HOXB13 gene gRNA target 4;

(b)16 species of getPCR guard bases having different guard bases; evaluation was performed using a reverse primer (c) and a forward primer (d), respectively, and a forward and reverse primer in combination;

(e) the ability to distinguish indels from wild-type sequences;

(f) research of self-amplification background signals when forward and reverse primers are used in combination;

(g) influence of amplification specificity of the first base pair at the 3' end of the primer;

(h) the effect of different types of base mismatches on amplification efficiency;

(i) role of 3' terminal base type in determining the susceptibility of getPCR to mismatches. Mean ± s.e.m, n ═ 3 independent technical repeats)

FIG. 3 is a graph of the parameter optimization for the implementation of getPCR in example 2;

(a-d) amplification curves for amplification using four guard base pair indel/wild type sequence DNA templates at different annealing temperatures. The forward conserved bases comprise 3(a) or 4(b) forward conserved bases, or 3(c) or 4(d) inverted conserved bases.

(e-h) display of amplification efficiency and selective amplification ability of the guard bases of different Tm values at different annealing temperatures during PCR, wherein the guard bases having three (e) or four (g) observational guard bases and the reverse guard bases having three (f) or four (h) guard bases are used. PCR efficiency was characterized as Δ Ct calculated relative to Ct value at 65 ℃ and selectivity as Δ Ct between wild-type template and indel template used. The conserved base sequence is shown at the bottom. The small circles indicate the best selectivity at the best amplification efficiency at a drop of 0.5 cycles (as indicated by the dashed line).

(i-l) the effect of annealing temperature on PCR amplification efficiency and the linearity of the standard curve, characterized by the R-squared value. The four conserved bases used in the assay have three (i) or four (k) forward conserved bases, respectively, or three (j) or four (l) reverse conserved bases, respectively. (mean ± s.e.m, n ═ 3 independent technical repeats)

FIG. 4 is a graph of the results of the genotyping application of getPCR in example 4 to mock single cell clones; (a) detecting an electrophoresis chromatogram map by Surveyor, wherein a detected sample contains insertion deletion with given percentage and is used for simulating DNA after genome editing;

(b) quantifying an editing frequency result obtained by Surveyor detection;

(c) detecting Indel frequency using getPCR method using either forward and reverse guard bases alone or in combination;

(d-f) genotyping of mock single cell clones using getPCR using three different designed guard bases. (mean ± s.e.m, n ═ 3 independent technical repeats, # P <0.05, # P <0.01, # P <0.001)

FIG. 5 results of the determination of editing frequency and genotype of single cell clones by getPCR in example 5;

indel frequency determination was performed in 293T cells genomically edited with gRNA targeting HOXB13, DYRK1A and EMX1 genes, and genotyping of single cell clones was performed;

(a) in the 8 gRNA-mediated genome editing combinations, getPCR quantified the indel frequencies generated and compared to NGS and Surveyor methods;

(b) graphical representation of the gRNA sequence and the conserved bases used in getPCR; single cell clones from 293T cells edited to target the HOXB13 gene (c, d), EMX1 gene (e, f, i) and DYRK1A gene (g, h) were genotyped by the getPCR method. The box plot shows the first quartile, median and third quartile, respectively, the beard represents 1.5IQR, and the outlier is displayed separately. The relevance and combinatorial effect of two differently designed guard bases was evaluated in genotyping (j-l). (mean ± s.e.m, n ═ 3 independent technical repeats, # P <0.05, # P <0.01, # P <0.001)

FIG. 6 results of the application of getPCR in example 6 to determine HDR frequency and genotype of single cell clones;

(a) schematic diagram of quantification principle of getPCR in HDR and base editing;

(b) the primers used for HDR repair efficiency detection of the target EMX1 gene and base editing efficiency detection of the target HOXB13 gene;

(c) HDR efficiency quantification using getPCR and comparison to NGS and HindIII enzymatic methods;

(d-f) genotyping single cell clones from HDR experiments using getPCR method with two different on-duty bases alone or in combination, boxplots showing the first quartile, median and third quartile, respectively, beard representing 1.5IQR, outliers displayed alone;

(g, h) frequency of each genotype determined by the getPCR and NGS methods in base editing experiments targeting EMX1 and HOXB13 genes, respectively, detailed genotyping of 10 clones heterozygous at positions 5 and 6 from EMX1 gene base editing by getPCR;

(i) detailed genotyping of 10 clones heterozygous at positions 5 and 6 from base editing of the EMX1 gene by getPCR;

(j, k) bar and scatter plots single cell clone genotyping at nucleotide 5 was shown by getPCR in the EMX1 gene editing experiment;

(l, m) genotyping a single cell clone corresponding to nucleotide 6;

the (n, o) bar and scatter plots show single cell clone genotyping in base editing of the HOXB13 gene. (mean ± s.e.m, n ═ 3 independent technical repeats, # P <0.05, # P <0.01, # P <0.001)

FIG. 7 design of getPCR primers and notes on running getPCR;

(a, b) designing a plurality of getPCR primers having a given value of nucleobase pair but different length/Tm values in forward and reverse directions, respectively;

(c) the amplification efficiency of these getPCR primers on wild-type template;

(d) bar graphs showing the specificity of different combinations of the conserved bases for PCR amplification of a mock indel plasmid are an alternative display to figure 2 e;

(e) a bar graph showing PCR self-amplified signals of the combinations of the conserved bases without the addition of template, is an alternative display of figure 2 f;

(f, g) effect of position of single base mismatch on PCR amplification relative to 3' end, showing results for forward and reverse guard bases, respectively;

(h, i) comparing the inhibition ability of 3 'terminal base mismatch and 3' terminal base deletion on PCR amplification, and respectively displaying forward and reverse guard bases;

(j) and the applicability of various qPCRSYBRGreenmix products in the application of getPCR is compared. (mean ± s.e.m, n ═ 3 independent technical repeats)

FIG. 8 the performance of different DNA polymerase products in mismatch recognition;

(a, b) displaying PCR amplification levels of different DNA polymerases by electrophoresis chromatography, wherein templates used in PCR respectively contain a base without mismatch and a base with mismatch, and primers respectively adopt a forward direction and a reverse direction guard base;

(c) sanger sequencing chromatography of PCR products from a and b;

(d, e) bar graph illustrating sensitivity to single base mismatches at different positions relative to the 3' end in the amplification of multiple qPCR products using forward and reverse guard bases, respectively. (mean ± s.e.m, n ═ 3 independent technical replicates) figure 9 was used to perform editing frequency determination and genotyping of single cell clones using plasmids mimicking indels;

(a-c) frequency quantifying the DNA mimicking the indels using the getPCR method with combinations of forward and reverse conserved bases; (d-f) simulating unicellular cloning by combining two differently designed getPCR guard base pairs for genotyping; refer to fig. 2a to obtain analog insertion information. (mean ± s.e.m, n ═ 3 independent technical repeats)

Figure 10 genotyping single cell clones that were genomically edited for gRNA targeting genes HOXB13, DYRK1A and EMX1 to generate indel mutations;

(a, b) genotyping single cell clones from 293T cells targeted by gRNA to the DYRK1A gene for genome editing by the getPCR method using two differently designed conserved bases, respectively. The box chart respectively shows a first quartile, a median and a third quartile, the beard represents 1.5IQR, and an abnormal value is displayed independently;

(c-g) scatter plots showing the correlation and combinatorial effect of two differently designed guard bases in genotyping;

(h-l) indel mutations determined in single cell clone genotyping by Sanger sequencing against gRNAHOXB13 target 6, EMX1 target 5, DYRK1A target 1 and EMX1 target 1, respectively; (mean ± s.e.m, n ═ 3 independent technical repeats, # P <0.05, # P <0.01, # P <0.001)

FIG. 11 genotyping a single cell clone isolated after base editing of the gRNA-targeted EMX1 gene is completed; (a) bar graph showing single cell clone genotyping at nucleotide 5 by getPCR in EMX1 gene editing experiments, i.e. figure 6j is annotated with detailed clone numbers;

(b) bar graph showing single cell clone genotyping at nucleotide 6 by getPCR in EMX1 gene editing experiments, i.e. figure 6l annotated with detailed clone numbers;

(c) sanger sequencing chromatography for genotyping single cell clones. (mean ± s.e.m, n ═ 3 independent technical repeats)

FIG. 12 genotyping of single cell clones resulting from base editing of the HOXB13 gene with the introduction of a stop codon;

(a) genotyping the single cell clone obtained in the HOXB13 gene base editing experiment at nucleotide 8 by getPCR, i.e. figure 6n is annotated with the detailed clone number;

(b) sanger sequencing chromatography for genotyping single cell clones. (mean ± s.e.m, n ═ 3 independent technical replicates).

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

As introduced in the background art, the methods for detecting the efficiency of gene editing methods in the prior art have certain disadvantages: such as Sanger, NGS, mismatch-specific nuclease-based methods, etc., have complex operation, high cost, insufficient detection accuracy, etc. It is important to provide a method that can be applied quickly, easily and reliably to genome editing efficiency quantification and high throughput genotyping, without the need for special equipment. In order to realize the technical purpose, the disclosure provides a getPCR detection method, which utilizes the specificity of Taq polymerase, takes a wild type DNA sequence as a template, designs a primer sequence covering a nucleic acid enzyme cutting site, and indirectly determines the editing efficiency of a genome by amplifying and quantifying the percentage of the wild type DNA in the genome. Through optimization and verification, the method has high detection accuracy, is convenient to operate and has wide application value.

In order to make the technical solutions of the present disclosure more clearly understood by those skilled in the art, the technical solutions of the present disclosure will be described in detail below with reference to specific examples and comparative examples.

The reagents and material sources used in the following examples are as follows:

plasmid and DNA fragment pcDNA3.1 the plasmid containing the coding region of the HOXB13 gene on the vector was given by professor Weigong macro of Oulu university.

Both 26 DNA variants mimicking potential different indels of HOXB13gRNA target 4 (fig. 2a) and 15 other variants containing mutations for the introduction of different types of primer-template mismatches were constructed by site-directed mutagenesis. sgRNA expression plasmids were constructed by deleting the cas9 expression frame from the pSpCas9(BB) vector (adddge, #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 the base editing experiments.

A99-nt length single-stranded HDR template containing the EMX1-HindIII mutation was synthesized by EnxWeiji corporation (Shanghai), and the introduced HindIII site sequence was adjacent to the PAM sequence of the EMX1gRNA target 5. A plasmid containing the EMX1-HindIII mutation was constructed and used as 100% homologous recombination repair efficiency. The sequences of all primers and oligonucleotides used are shown in table 1.

TABLE 1 oligonucleotide sequences for plasmid construction and transfection

a. Primers for the construction of an amorphous HOXB13 variant

b. Primer for constructing blank sgRNA expression plasmid

c. Primers for the construction of HF-Cas9(R661A, Q695A, Q926A, D1135E) by site-directed mutagenesis

Table 1d primers used to construct sgRNA expression plasmids for given targets

TABLE 1e HDR template sequence (5'-3')

Cell culture cell line Lenti-X293T (Cat #632180) was originally purchased from Clontech. The cell culture conditions were 37 ℃ and 5% CO₂Concentrations, using Dulbecco's modified Eagle Medium (Gibco, Cat # C11995500BT) supplemented with 10% (v/v) FBS (Gibco, Cat #10270-106) and' penicillin/streptomycin (HyClone, Cat # SV 30010). The MycoBlueTMMycoplasma Detector kit (Vazyme, Cat # D101-01) was used to periodically check for mycoplasma contamination, according to the product manual.

Cell transfection Lenti-X293T cells were seeded into 24-well plates (Labserv, Cat #310109007) at a density of 120,000 cells per well the day before transfection. When the cell density reached about 70%, the cells were transfected using Lipofectamine2000(ThermoFisher scientific, Cat #11668019) according to the manufacturer's instructions. 1 μ g of plasmid co-expressing sgRNA and high fidelity CRISPR-Cas9 was used in each transfection reaction to introduce indels. For base editing, 750ng of the BE4 plasmid and 250ng of the sgRNA expression plasmid were used per transfection reaction. For HDR-mediated genome repair, 600ng of plasmid co-expressing sgRNA and high fidelity CRISPR-Cas9 and 10pmol HDR oligonucleotide were used per transfection reaction. At 48 hours post transfection, genomic DNA was extracted using the TIANAmp genomic DNA kit (TIANGEN, Cat # DP304-03) according to the manufacturer's instructions.

getpcrs conditions in a 15 μ L volume reaction system, 0.1ng plasmid DNA or 2.5ng genomic DNA was used as template for each qPCR reaction, aceqqpcrsrbrbreenmastermix (Vazyme, Cat # Q111-02) was used, and qPCR was run under the following conditions. The following procedure was followed on a qPCR instrument Rotor-GeneQ (Qiagen, germany): pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30 seconds, annealing at 65-69 ℃ for 30 seconds, extension at 72 ℃ for 10 seconds and detection of the fluorescence signal, for 40 cycles. Use of96qPCR instruments (Roche applied sciences, Germany) used the following conditions: denaturation at 95 ℃ for 15 seconds, annealing at 65-69 ℃ for 20 seconds, extension at 72 ℃ for 15 seconds and detection of fluorescent signals for 40 cycles; a standard melting curve procedure is then performed. The primer Tm values were calculated using the in-line OligoCalc tool 50.

Quantification of indel frequency using getPCR an equal proportion of 26 plasmids mimicking different types of indel mutations were mixed as 100% indels (fig. 2 a); further mixed with wild-type DNA at a given ratio to obtain DNA samples of different indel efficiencies. The frequency of indels occurring was evaluated using the getPCR method. In the getPCR assay, 0.1ng of plasmid DNA was used as template for each qPCR reaction. The percentage of wild type DNA and the frequency of indels in the mixture samples were calculated as described in figure 1 b. At the same time, each of these 26 plasmids was used to mimic a single cell clone with the homozygous HOXB13 indel mutation; and each plasmid was mixed with the wild-type DNA plasmid in equal proportions to mimic a heterozygous single-cell clone carrying an indel on one allele. The sequence of the getPCR primers is shown in table 2. For frequency quantification of indels for genomic DNA samples, amplification was performed using 2.5ng of genomic DNA as template and primers summarized in table 3.

TABLE 2 genome editing efficiency assay

Primers for Surveyor DNA amplification and sanger sequencing

Primer for detecting insertion deletion of HOXB13gRNA target 4 by adopting getPCR

TABLE 3 cellular genome editing efficiency

a. getPCR primer for indel efficiency quantification

Use of getPCR primers for base editing efficiency quantification

c. GetPCR primer for HDR repair efficiency quantification

Surveyor nuclease assay reported Surveyor nuclease assay for determining Indel frequency, usingMutation detection kit (IntegratedDNAtechnologies, Cat # 706020). The process is briefly stated as follows: extracting genomic DNA using TIANPGENOMICDNAkit (TIANGEN, Cat # DP304-03) according to the product manual; then using high fidelityThe DNA fragments amplified by MaxDNA polymerase (TaKaRa, Cat # R045B) were 200-400bp apart from the cleavage site of cas9 at either end, and the primers used for PCR are shown in Table 2 a. 270ng of the purified PCR product was annealed using a T100TM thermal cycler (Bio-Rad) to give a heteroduplex, which was subsequently treated with SurveyorNuclean according to the instructions. DNA fragments were separated on a 2% agarose gel and images were obtained using Quantum-ST5(VILBERLOURMAT, France) and analyzed using Quantum ST5Xpress software.

Application of getpcrs in HDR and BE4 experiments modification specific getPCR primers with modified nucleotides were designed at the 3' end as summarized in table 3. In the getPCR assay, 2.5ng of genomic DNA was used as template for each reaction. The efficiency of genome modification was calculated using the formula as shown in figure 6 a.

RFLP assay based on HindIII digestion. In HDR experiments with EMX1 gene, a HindIII site was introduced near the PAM sequence, which allows quantification of HDR repair efficiency by Restriction Fragment Length Polymorphism (RFLP) analysis based on HindIII cleavage. Briefly, 639bp fragments were amplified using PrimeSTARMaxDNA polymerase, with a HindIII site 355bp in length from the 5' end and the primers used for PCR identical to the Surveyor assay, as shown in Table 2 a. The PCR product was purified using the Universal DNA purification kit (TIANGEN, Cat # DP 214). The purified 270ng PCR product was subjected to HindIII digestion and separated on a 2% agarose gel. Images were acquired using Quantum-ST5(VILBERLOURMAT, France) and analyzed using Quantum ST5Xpress software.

The NGS-based method constructs NGS amplicon libraries covering DNA regions near the editing site of the genome, and calculates editing efficiency by counting NGS reads after sequencing. Two rounds of PCR amplification were performed to prepare sequencing libraries using genomic DNA as template. In the first round of PCR, 250-280bp amplicons were designed, in which the Cas9 cleavage site was near the middle and binding sites for Illumina sequencing primers were introduced at both ends. In the second round of PCR, linker sequences were introduced for cluster generation during sequencing, along with an index sequence. After purification and quantification of the library DNA, Genewiz was delivered for 150bp double-ended sequencing on the IlluminaHiSeqX-TEN platform. For NHEJ-mediated indels, wild-type read counts in each library were obtained using the signature sequence of wild-type DNA and indel editing efficiencies were calculated using the formula "editing efficiency 1-wild-type reads/total reads 100%". For base editing and editing efficiency in HDR experiments, read counts of the expected DNA sequences in the library were obtained and the editing efficiency was calculated using the equation "efficiency ═ reads of expected DNA sequences/total reads 100%". For details on the library preparation and enumeration methods, see table 4.

TABLE 4 genome editing efficiency quantification by NGS

a. Primers for library preparation

1st round PCR,take 50ng gDNA as template,28 cycles,15μl system,set NTC control,[email protected]℃, usingMax DNA Polymerase(TaKaRa)

2nd round PCR,take 1ng of purified DNA from 1st round PCR as template,10cycles,15μl system,[email protected]℃, usingMax DNA Polymerase(TaKaRa)

b.R characteristic sequence of program read counts

c.R reading counting program

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

About 48 hours after single cell cloning and genotyping transfection, single cells were isolated by limiting dilution and seeded into 96-well plates for growth. When the cells grew out of the 96-well plate, they were further transferred to a 24-well plate and continued to grow until they healed. Genomic DNA from single cell clones 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 the getPCR assay and confirmed by Sanger sequencing of the amplicon covering the cleavage site. The primers used were PCR amplified with high fidelity PrimeSTARMaxDNA polymerase (TaKaRa, Cat # R045B) as shown in Table 2a, followed by Sanger sequencing of the PCR products (TsingKebiologicals technology or GeneWiz). To determine the exact sequence of each allele of the heterozygous cells, Sanger sequencing ab1 files were analyzed directly using the TIDEWeb tool (https:// tide. nki. nl /), or colonies were Sanger sequenced after cloning the amplicons into the vector.

Sensitivity of different DNA polymerases to mismatches a variety of commercial DNA polymerase products were used to compare the effect 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 well asHot Start high-Fidelity DNA Polymerase (NEB, Cat # M0493). In a 20. mu.l reaction, 10ng of plasmid DNA was used as template and thermal cycling was performed according to the procedures suggested in the product manual. The PCR products were then directly subjected to 2.0% agarose gel electrophoresis and Sanger sequencing. Gel images were obtained using Quantum-ST5(VILBERLOURMAT, France) and analyzed using Quantum ST5Xpress software.

Comparison of different qPCR SYBR Green products in getpcrs to test the widespread availability of getpcrs, various qPCR ybrmix products were applied to getpcrs, including 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),SYBR One-Step qRT-PCR Supermix (novoprotein, Cat # E092-01A),2 XT 5Fast qPCR Mix (TSINGKE, Cat # TSE202), UltraSYBR Mix (CWBIO, Cat # CW0957), SYBR Premix Ex Taq (TaKaRa, Cat # RR420, A5405-1). Real-time quantitative PCR in a thermal cycler Rotor-GeneQ (Qiagen, Germany) or96qPCR instruments (Roche applied sciences, Germany). qPCR conditions were determined according to the manufacturer's instructions and annealing temperature settings.

Statistical analysis student's t-test (two-tailed) was applied based on the results of the Leven test to assess the statistical significance of the getPCR results of genotyping single cell clones using versions of IBMSPSSSstatics. The correlation between the two different getPCR strategies was evaluated using the Pearson test, with the 21 st version of the ibms pssstatistics software.