CN114395555A

CN114395555A - Method for improving ctDNA detection sensitivity by using CRISPR (clustered regularly interspaced short palindromic repeats) shearing technology

Info

Publication number: CN114395555A
Application number: CN202111338700.8A
Authority: CN
Inventors: 李小花; 刘朝煜; 姚旭梅
Original assignee: Shenzhen Sinang Yiyun Technology Co ltd
Current assignee: Shenzhen Sinang Yiyun Technology Co ltd
Priority date: 2021-11-12
Filing date: 2021-11-12
Publication date: 2022-04-26

Abstract

The method for highlighting low-frequency mutation by shearing a non-mutation target by using a CRISPR technology comprises the steps of constructing a library of DNA molecules, designing specific sgRNA, adding the sgRNA and Cas9 for in-vitro treatment, combining the sgRNA and Cas9 protein in vitro, and adding the combined product into sample DNA; the CRISPR technology is utilized to cut the non-mutation target so as to highlight the low-frequency mutation, and NGS sequencing is used to confirm that the method can be applied to the detection of low-frequency ctDNA (circulating tumor DNA) or applied to an FFPE sample with low tumor cell ratio, so that the combined detection of 2 or more low-frequency mutation sites is realized.

Description

Method for improving ctDNA detection sensitivity by using CRISPR (clustered regularly interspaced short palindromic repeats) shearing technology

Technical Field

The application belongs to the field of gene detection, and particularly relates to the field of gene target cutting and detection.

Background

As part of an accurate medical protocol, fluid biopsies can be somewhat effective in making advanced diagnoses, gaining time for patients while allowing for instructional medication and monitoring post-operative recovery in real time, etc. (Snyder, M.W.et. al. copy-number variation and false positive diagnosis systems. the New England of medicine 372,1639, 1645, doi:10.1056/NEJMoa1408408 (2015)). In fluid biopsy, circulating cell-free DNA (cfDNA) detection is an important component. cfDNA is a fragmented DNA free In plasma or serum, Of about 170bp In size, and has been shown to be strongly correlated with transcription factors (Snyder, M.W., Kircher, M., Hill, A.J., Daza, R.M. & Shuend, J.cell-free DNA compositions an In Vivo nucleic acids focus print information items Tissues-Of-origin.cell 164,57-68, doi:10.1016/j.cell.2015.11.050 (2016)). In cancer patients, cfDNA derived primarily from apoptotic and necrotic tumor cells is called circulating tumor DNA (ctDNA) (Diehl, F.et al. circulating mutant DNA to assessment tumor dynamics. Nature media 14,985-990, doi:10.1038/nm.1789 (2008)). Because ctDNA mainly comes from tumor necrotic cells and contains epigenetic information and genetic information related to tumors, the ctDNA can be used for detecting the tumor state in real time, thereby avoiding the pain of patients caused by puncture surgery.

When the liquid biopsy is used for detection, the problem that the low-frequency mutation is difficult to solve needs to be solved. When the low-frequency mutation sites are detected by next generation gene sequencing (NGS), the signal flooding problem is caused due to the absolute high amount of non-mutant site templates, so that the low-frequency mutation sites cannot be really identified (Diaz, L.A., Jr. & Bardelli, A.liquid biologicals: genetic cloning of tumor DNA. journal of Clinical analysis: of the American Society of Clinical analysis 32, 579. 586, doi:10.1200/JCO.2012.45.2011 (2014)). Although ddPCR can detect low-frequency mutations, it has the problem of low throughput, and cannot fully utilize rare samples, and also has certain limitations (Pan, W., Gu, W., Nagpal, S., Gephart, M.H. & Quake, S.R. brain tissue failure in cellular specific fluid. clinical chemistry 61, 514. 522. doi:10.1373/clinchem.2014.235457 (2015)).

Crispr (clustered regulated short palindromic repeats) and Cas nucleases have been widely used in vitro and in vivo experiments to precisely cleave DNA sequences in targeted regions. In 2107, W.Gu et al introduced CRISPR into RNA sequencing and detection against the single gene site KRAS (c.35G > A, p.G12D) (GU, W.et al.deletion of Absolute Sequences by Hybridization (DASH): using Cas9 to remove high-absorbance spectra in sequencing libraries and molecular counting applications. genome biology 17,41, doi:10.1186/s 13059-016-4-5 (2016)). However, in this project, W.Gu et al cleaved at only one site and analyzed. Mutations with an extremely low frequency cannot be identified if they are lower than the detection limit of the prior art, which is a pain point in the field of gene detection (insufficient detection sensitivity),

disclosure of Invention

The purpose of the invention is as follows: the method solves the problems that the low-frequency mutation of ctDNA and the real mutation of a tissue sample with poor quality are difficult to detect.

The technical scheme is as follows:

1. specific sgRNA sequences, wherein the DNA sequences of the sgrnas are shown in table 1.

2. The use of specific sgrnas for the detection of low-frequency ctDNA (circulating tumor DNA) or for FFPE samples with a low tumor cell fraction is described.

3. The method for highlighting low-frequency mutation by shearing a non-mutation target by using a CRISPR technology comprises the steps of constructing a library of DNA molecules, designing specific sgRNA, adding the sgRNA and Cas9 for in-vitro treatment, combining the sgRNA and Cas9 protein in vitro, and adding the sgRNA and the Cas9 protein into sample DNA, wherein any one or more of the sequences of the sgRNA are selected from the table 1, and the method is characterized by comprising the following steps of:

a) extracting gDNA from the FFPE sample;

b) performing enzyme digestion interruption on the gDNA obtained in the step a) and then performing end repair;

c) connecting the two ends of the fragmented DNA molecules treated in the step b) with linkers containing molecular tags;

d) purifying the DNA molecule obtained in the step c), and adding Cas9 and sgRNA for cutting; wherein sgRNA is sg-KRAS-GU-chr12:25398284 and sg-EGFR-chr7: 55249010;

e) purifying the DNA molecules obtained in step d); performing PCR amplification;

f) hybridizing the DNA library obtained in the step e), and then performing deep sequencing to obtain a sequencing result;

g) and f) identifying and classifying the sequencing result of the step f) through a sample index and a molecular tag, removing or reducing the system error introduced in the PCR amplification and sequencing stage in the NGS sequencing, and analyzing the change of the frequency of the specific site before and after the Crispr treatment.

Has the advantages that: the CRISPR system is used for accurately identifying a target region by combining Cas9 and sgRNAs, and the research of W.Gu et al is combined, so that multiple sgRNAs are used in the same NGS reaction, multiple sites can be simultaneously targeted in one reaction, and the cost is reduced and the efficiency is improved. Due to the sampling reason and the existence of the problems of tumor heterogeneity, the tumor cell tissues of the obtained tissue samples can not be guaranteed to meet the detection standard. Therefore, the invention can be used for processing low-quality tissue samples to obtain useful information, thereby guiding the treatment of patients more accurately.

In the invention, 12 sgRNAs are designed, can be freely combined in the project, and can cut 12 targeting sequences at most simultaneously. Gu's scheme and ddPCR etc. have had a substantial improvement over the detection of only one site. The invention also provides a site selection principle, a design method and a specific experimental operation method, and sgRNA can be designed and cut aiming at more sites as required so as to identify more sites, further improve the occupied ratio of the sgRNA and obtain accurate information. According to the invention, amplification of mutation signals of key sites (closely related to clinical medication guidance) of 2 key genes (strongly related to lung cancer) is realized by a CRISPR (clustered regularly interspaced short palindromic repeats) shearing method, so that ultralow-frequency mutation can be detected, the gene detection sensitivity of the key sites is substantially improved, and the method is expected to benefit clinical diagnosis and treatment. Meanwhile, the detection also has the creative problem of an application scene, the technicians for early diagnosis and screening of tumors have difficulty in the combined monitoring of ultralow frequency, basically rely on PCR template quantity and ultra-deep sequencing to accumulate data for analysis, and rely on a quantity-winning strategy, the application hopes to realize the detection of the key sites of the two genes with ultralow frequency (directly related to the medication guidance of lung cancer) by shearing the target sites by the CRISPR technology

The application also provides a technical scheme for migration, and the method can be applied to other sites of other cancer species or can be popular. The invention can be used for not only blood samples in vitro but also tissue samples in vitro with poor quality. The application prospect comprises the aspects of tumor liquid biopsy early screening, medication guidance and the like. The method has the advantages of high detection sensitivity, high flux, high efficiency and the like.

Drawings

Fig. 1.CRISPR principle schematic;

FIG. 2A shows that the templates of EGFR-chr7:55249010 and KRAS-chr12:25398284 are reduced after CRISPR treatment. The amount of EGFR template treated was 1/10 for the control group and the amount of KRAS template treated was 1/5 for the control group;

FIG. 2B statistics of template reads after sequencing revealed a significant reduction in reads for KRAS-chr12:25398284 and EGFR-chr7:55249010 and sites surrounding them.

Detailed Description

While the following is a description of the preferred embodiments of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.

In the examples of the present invention, unless otherwise specified, reagents and consumables used therein are commercially available.

In the examples of the present invention, if specifically described, the sequencing library construction is described in reference to Roche, illumina or ABI high throughput sequencing library construction instructions.

The invention principle is as follows: the NGS-combined CRISPR technology can be used for carrying out combined detection on two or more potential low-frequency mutation sites. The present invention is based on the principle that the CRISPR system requires Cas9 to bind to sgrnas in order to accurately recognize the targeted region. sgrnas are complementary to the targeting region, and Cas9 needs to bind to pam (ngg) after the targeting region before it can function. When PAM (NGG) is mutated, the target sequence cannot be cleaved particularly when G at the second position is mutated. Sgrnas designed to bind to these sites are also one of the important protection items of the present invention.

The deep sequencing method of the invention employs sequencing equipment including, but not limited to, Roche/454, Illumina sequencer (NextSeq series, Hiseq series, MiSeq series, XTen, and subsequent sequencer series), BGI (Huada corporation, BGI500 series, and subsequent sequencer series), Life Tech sequencing instruments (Ion, Proton, and subsequent sequencing instruments series), PacBio sequencing instruments (RSII, sequence, and subsequent sequencing instruments), or Nanopore-based sequencing instruments (Genia, Nanopore, and similar third generation sequencers).

The invention provides a method for cutting and sequencing non-mutant DNA by using CRISPR (clustered regularly interspaced short palindromic repeats), so that the ratio of the mutant DNA is increased. The invention designs specific sgRNA, and adds the sgRNA and Cas9 protein after in vitro combination into sample DNA. The sgRNA can recognize and cut non-mutant DNA, so that fragmentation of the non-mutant DNA cannot be sequenced, and the mutation frequency of low-frequency DNA is highlighted. The invention is suitable for detecting low-frequency ctDNA (circulating tumor DNA) and can also be applied to FFPE samples with low tumor cell ratio. The present invention provides a technique which can excise a plurality of non-mutant DNAs simultaneously and can flexibly adjust the type of excision as required. In one embodiment of the present invention, the detection of an FFPE sample with a low tumor cell ratio comprises the following steps:

a) extracting gDNA from the FFPE sample;

d) purifying the DNA molecule obtained in the step c), and adding Cas9 and sgRNA for cutting; wherein the sgRNA is sg-KRAS-GU-chr12:25398284 and

sg-EGFR-chr7:55249010；

In one embodiment of the present invention, the CRISPR system specifically recognizes pam (ngg), and when the second G is mutated, the CRISPR system cannot recognize, bind to and excise the sequence, thereby preserving it. Whereas non-mutants are cleaved. In a specific embodiment of the invention, sgRNA is designed according to PAM and mutation sites. In the present invention, a site of definite clinical significance or potential clinical significance is targeted. Referring to the results of a design website (http:// criprp. mit. edu/batch), sgRNA is adopted according to the given fraction, and the sequence of the sgRNA applied in the project is shown in Table 1 and is one of the important protection directions of the patent of the invention. Table 1 shows the DNA sequence of sgRNA used in this example, and RNA molecules were formed by reverse transcription in actual use. In sgRNA, the first 20nt specifically recognizes the targeting region.

TABLE 1 original DNA sequence reverse transcribed into sgRNA

In one embodiment of the invention, two sgRNAs sg-KRAS-GU-chr12:25398284 and sg-EGFR-chr7:55249010 are used to treat the FFPE sample. The targeted targets were chr12:25398284 and chr7:55249010, respectively (yellow region in table 1).

By applying the technical scheme of the invention, multiple sgRNAs are added at one time, so that multiple non-mutant DNA molecules can be cut in one reaction at the same time. In the present invention, DNA molecules are first pooled, sgRNA and Cas9 are added for in vitro treatment, and the targeting strand is cleaved into two strands. Since both strands do not have the complete P5/P7 ends, they cannot be amplified efficiently in the next PCR process, and the ratio is reduced. Meanwhile, due to the deletion of the PAM box, the Cas9 cannot identify a mutation target region, and the region can completely reserve a linker part, so that the effective amplification can be realized in the PCR process, and the occupied ratio is increased. The detection principle of the present invention is schematically shown in fig. 1.

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention and the accompanying drawings, and the described embodiments are only a part of the embodiments of the present invention, but not all of the embodiments. 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.

Example 1

Method for detecting cancer gene from peripheral blood free DNA by combining molecular tag with deep sequencing method

Method for highlighting low-frequency mutation by shearing non-mutation target by CRISPR technology

1. Sample extraction

FFPE samples are used in this example. The DNA extraction method was according to QIAamp DNA FFPE Tissue Handbook (Qiagen). The FFPE sample is dewaxed, cracked, heated, column-bound with DNA, eluted, dissolved and the like to obtain the DNA sample. The Qubit is quantified.

2. DNA fragmentation

The reaction system was prepared according to the following formula (for example, KAPAHyperPrep Kit library-building Kit)

Reagent	Volume of
		DNA (200 ng/EDTA-free) + ddH2O	35ul
Fragmentbuffer (cover)	5ul
		Fragment enzyme (lid)	10ul
Total of	50ul

And (4) adding samples on ice, mixing uniformly and placing on the ice. The PCR machine was programmed in advance to the following conditions to ensure that the reaction was immediately carried out after the sample was mixed.

After the reaction was completed, the subsequent steps were immediately performed.

3. End repair and base addition to the 3' end

Reagent	Volume of
		Extracting free DNA template	50μl
End repair and buffer A	7μl
		End repairing and A enzyme adding mixed solution	3μl
Total of	60μl

The prepared reaction system is put into a PCR instrument for reaction according to the reaction conditions of the following table

Step (ii) of	Temperature of	Time
			Tip repair	20 deg.C (without lid and hot lid)	30min
Addition of base A	65 deg.C (hot lid 85 deg.C)	30min
			Heat preservation	4 deg.C (20 deg.C if connected immediately)	∞

4. Joint connection

The product A obtained in step 3	60μl
		Nuclease-free water	6μl
Ligation buffer	30μl
		DNA ligase	10μl
The joint obtained in the step 2	4μl
		Total of	110μl

And placing the prepared reaction system on a PCR instrument, and reacting for 15min-4h at 20 ℃. After completion of the reaction, the reaction mixture was purified using 0.8X AMPure XP magnetic beads (Beckmann Co.), 31ul dd H2O was dissolved, and 30ul DNA was collected.

5. CRISPR processing

Reaction system:

a CRISPR system (NEB, M0386) is prepared in advance, and the reaction system:

after 10min at 25 ℃ 30ul DNA was added, after 15min at 37 ℃ 1.0. mu.l protease K (NEB, P8107s) was added and incubated for 10min at room temperature.

After completion of the reaction, the reaction mixture was purified using 1X AMPure XP magnetic beads (Beckmann Co.), 24ul dd H2O was dissolved, and 23ul DNA was collected.

6. Amount of template for PCR amplification

Reaction system:

reagent	Volume of
		Purified DNA product	23μl
P5Index(25μM)	1.0μl
		P7Index(25μM)	1.0μl
KAPAHiFiHotstartReadyMix(2x)	25μl
		Total of	50μl

Reaction conditions are as follows:

7. hybridization of

500ng of DNA was taken, and the mixture was dried at 56 ℃ after adding the reagent in the following system

Reagent	Volume of
		Mixed hybridization samples: (<5ug)	n
Cot-1Human(1ug/ul)	5ul
		xGenUniversalBlockers-TSMix	2ul

Using IDT probes for hybridization, the dried DNA was dissolved by adding reagents in the following manner

Reagent	Volume of
		Dried pooled hybridization samples: (<5ug)	-
ddH2O	1.8ul
		hybridizationbufferEnhancer	2.7ul
2xhybridizationbuffer	8.5ul
		Total	13ul

The redissolved hybridization samples were transferred to 0.2ml PCR tubes and incubated at 95 ℃ for 10min, followed immediately by the addition of 4ul xGen Lockdown Probe pool (0.75 pmol/ul). After mixing, 65 ℃ immediately (hot lid 75 ℃) was used for 16 h.

8. Magnetic bead capture probe hybridization products

Mixing the pretreated Dynabeads M-270 magnetic beads with the probe hybridization product according to the specification, washing according to the product specification, and finally adding 20ul ddH₂And O, resuspending the magnetic beads.

The DNA product with M-270 magnetic beads was subjected to PCR amplification. The reaction is as follows:

reagent	Volume of
		IDT Probe hybridization Capture product (with magnetic bead)	23
Universal P5 primer(25p)	1ul
		Universal P7 primer(25p)	1ul
KAPA HiFi Hotstart ReadyMix (2x) (Green cover)	25ul
		Total	50ul

9. Library detection and sequencing on machine

After completion of the reaction, the reaction mixture was purified by using 1X AMPure XP magnetic beads (Beckmann Co., Ltd.) to obtain 41. mu. ldd H₂O dissolved, and 40. mu.l was taken. Mu.l were used for detection with a qubit (life) and Agilent 2100Bioanalyzer, respectively. And sequencing according with the computer quality.

10. Analysis of sequencing results

After the library was constructed, the amount of DNA template was first detected using Q-PCR and the amount of CRISPR-treated template was determined to be reduced by amplification of the template with specific primers (see Table 2 for sequences). qPCR assay showed that as a result of CRISPR treated samples, EGFR template amount was 1/10 for control and KRAS template amount was 1/5 for control (see fig. 2A).

TABLE 2 primer sequences for detection of EGFR-chr7:55249010 and KRAS-chr12:25398284 in this example

Primer	Seq
		EGFR-chr7:55249010-F	ccaggaagcctacgtgatgg
EGFR-chr7:55249010-R	ggtggaggtgaggcagatg
		KRAS-Gu-chr12:25398284-F	tagctgtatcgtcaaggcac
KRAS-Gu-chr12:25398284-R	ggcctgctgaaaatgactga

The total amount of data is required according to the captured region size. In this example, the capture region is 29kb, and the total data size of the off-hook region is 10G. In this example, again using the laboratory patented technology, identically labeled reads can be grouped into a cluster by adding molecular tags, which are then combined into a template strand. Analysis of the template reads revealed that the resulting processed template, KRAS-chr12:25398284 and EGFR-chr7:55249010 and the sites around it, demonstrating that the CRISPR system can cut the DNA template where these two sites are located efficiently (fig. 2B).

And the data qualified in quality control can enter mutation detection analysis. Analysis of the results showed that sample 1 of the control group had KRAS-chr12:25398284 and EGFR-chr7:55249010 mutation frequencies (AF) were 31.82% and 0%, respectively; KRAS-chr12 for sample 2:25398284 and EGFR-chr7:55249010 mutation frequencies (AF) were 28.82% and 0%, respectively. And KRAS-chr12:25398284 and EGFR-chr7:55249010 mutation frequency (AF) was raised to 50.0% and 0.35%, respectively, KRAS-chrl 2:25398284 and EGFR-chr7:55249010 mutation frequency (AF) was elevated to 50.0% and 0.21%, respectively, especially against EGFR-chr7:55249010 this low frequency mutation site, CRISPR treatment can greatly increase its mutation frequency (Table 3).

Table 3 CRISPR treatment was performed on both samples and the mutated EGFR-chr7 was found in the treated samples: 55249010 and KRAS-chr12:25398284 site ratio increase

	Untreated KRAS	DASH treated KRAS	Untreated EGFR	DASH treated EGFR
						AF consensus	AF consensus	AF consensus	AF consensus
Sample
	1	31.82％	50.00％	0.00％	0.35％
Sample 2						28.82％	50.00％	0.00％	0.21％

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Sequence listing

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Claims

1. Specific sgRNA sequences, characterized in that the original DNA sequences reverse transcribed into sgrnas are shown in table 1.

2. The application of the specific sgRNA is characterized by being applied to the detection of low-frequency ctDNA or applied to an FFPE sample with low tumor cell ratio.

a) extracting gDNA from the FFPE sample;

d) purifying the DNA molecule obtained in the step c), and adding Cas9 and sgRNA for cutting;

4. The method application of utilizing CRISPR technology to cut a non-mutant target and further highlight low-frequency mutation according to claim 3, wherein the sgRNA is sg-KRAS-GU-chr12:25398284 and sg-EGFR-chr7: 55249010.