CN109207571B

CN109207571B - Method for detecting endonuclease cleavage site

Info

Publication number: CN109207571B
Application number: CN201710522336.8A
Authority: CN
Inventors: 李卓坤; 徐东洋; 杨晋; 顾颖; 黎宇翔; 陈奥; 徐崇钧; 章文蔚
Original assignee: BGI Shenzhen Co Ltd
Current assignee: BGI Shenzhen Co Ltd
Priority date: 2017-06-30
Filing date: 2017-06-30
Publication date: 2022-01-04
Anticipated expiration: 2037-06-30
Also published as: CN109207571A

Abstract

The invention discloses a method for detecting endonuclease cleavage sites. The invention utilizes billions of nucleic acid sequences on a chip to systematically detect the recognition cutting sites and core sequence characteristics of DNA by endonuclease in high flux, respectively obtains base sequences and normal double-stranded DNA by twice sequencing, and obtains the recognition cutting sites of the endonuclease by applying a biological information analysis method according to the change of fluorescence signals before and after enzyme digestion. Through the research on the recognition and cutting sites of the endonuclease, the tendency of the recognition and cutting sites of the endonuclease can be detected, the star activity of the endonuclease and the off-target effect of the endonuclease in the genome editing technology can be predicted in advance, and meanwhile, the method can also be used for screening the PAM sequence required to be recognized by the endonuclease used in a novel genome editing system in a large scale. Compared with the sequencing method in the prior art, the detection method has the advantages of high speed and high flux.

Description

Method for detecting endonuclease cleavage site

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for detecting endonuclease cutting sites.

Background

Endonucleases are one type of nucleic acid hydrolases that hydrolyze phosphodiester bonds within a nucleic acid molecule chain to produce oligonucleotides corresponding to exonucleases. They can be classified into endonucleases and endonucleases according to their catalytic substrates. In general, most of endonucleases do not have base specificity, but a few endonucleases have been widely used in molecular biology because they can recognize and cleave a specific base or base sequence, for example: restriction enzymes used in genetic engineering (Meselson M., Nature,217(134):1110-4.1968.), and endonucleases commonly used in gene editing techniques (Ran FA., Nat Protoc.8(11): 2281-308.2013), and the like. These endonucleases capable of recognizing and cleaving specific base sequences are central in molecular biology techniques, particularly in molecular cloning, genetic disease diagnosis, paternity testing, forensics, genomics (e.g., human genome project), epigenetics, transgenic organisms and even synthetic life. The ability of endonucleases used in these techniques to recognize and cleave a specific base sequence plays a key role.

Restriction enzymes, which are widely used in molecular biology at present, depend on their ability to recognize and cleave a specific base sequence. For example, in molecular cloning, restriction enzymes are often used to cleave specific sites of a DNA molecule to form sticky ends (sticky ends) or blunt ends (blunt ends), and to join different DNA molecules together under the action of a ligase to form a new DNA molecule. In gene editing techniques, the endonuclease used also depends on its ability to recognize and cleave a specific base sequence. For example, in the CRISPR/Cas9 system, the genome editing capacity of Cas9 is achieved by a short DNA sequence called "PAM" (pro-spacer adjacent motif).

The currently used restriction enzymes can generate star activity under non-standard conditions, which leads to the cutting of non-specific sites, and if the star activity needs to be determined, the recombinant DNA molecules after molecular cloning need to be sequenced for determination; meanwhile, the endonuclease used in the genome editing technology depends on the PAM sequence when acting, and different systems recognize different PAM sequences (for example, the PAM sequence used by Cas9 is "-NGG-" and the PAM sequence used by Cpf1 is "-TTTN-"), when a novel CRISPR-Cas system is screened and developed, the PAM sequence of the endonuclease used by the system needs to be detected in a large scale; in addition, off-target effects can occur when genome editing is performed, identification of off-target effects likewise requires sequencing, and the sites at which off-target may occur cannot be predicted in advance. When the strain containing the recombinant DNA or the cell subjected to gene editing is sequenced, the DNA is extracted firstly, then a primer is designed for PCR, and then the sequencing can be carried out, so that the process is complicated, the time consumption and the flux are low, and the price is high.

Disclosure of Invention

An object of the present invention is to provide a method for detecting an endonuclease cleavage site.

The method for detecting the endonuclease cleavage site provided by the invention comprises the following steps (1) or (2):

the (1) comprises the following steps:

(a1) constructing a library to be sequenced, and fixing the library on a chip to obtain the chip to be sequenced;

(a2) performing first sequencing on the chip to be sequenced to obtain the chip subjected to the first sequencing, and obtaining sequence information and an ID number of each nucleic acid sequence;

(a3) eluting the chip subjected to the first sequencing, and eluting a new chain generated by the first sequencing to obtain an eluted chip;

(a4) performing second sequencing on the eluted chip to obtain a chip subjected to second sequencing, and photographing to obtain a fluorescent signal and an ID number of a corresponding DNA sequence site;

(a5) carrying out enzyme digestion on the DNA sequence on the chip after the second sequencing by using endonuclease, carrying out elution after enzyme digestion reaction, removing the cut DNA sequence to obtain the chip after enzyme digestion, and photographing to obtain a fluorescent signal and an ID number of a corresponding DNA sequence site;

(a6) comparing the fluorescent signals obtained in the step (a4) and the step (a5) to obtain the ID of the DNA sequence subjected to the enzyme digestion reaction;

(a7) analyzing the DNA sequence subjected to the enzyme digestion reaction according to the ID of the DNA sequence subjected to the enzyme digestion reaction to obtain sequence information of a cutting site;

the (2) comprises the following steps:

(b1) constructing a library to be sequenced, and fixing the library on a chip to obtain the chip to be sequenced;

(b2) performing first sequencing on the chip to be sequenced to obtain the chip subjected to the first sequencing, and obtaining sequence information and an ID number of each nucleic acid sequence;

(b3) eluting the chip subjected to the first sequencing, and eluting a new chain generated by the first sequencing to obtain an eluted chip;

(b4) carrying out enzyme digestion on the DNA sequence on the eluted chip by using endonuclease, carrying out elution after enzyme digestion reaction, and removing the cut DNA sequence to obtain the enzyme digested chip; photographing to obtain a fluorescent signal and an ID number of a corresponding DNA sequence site;

(b5) performing second sequencing on the chip subjected to enzyme digestion to obtain a chip subjected to second sequencing, and photographing to obtain a fluorescent signal and an ID number of a corresponding DNA sequence site;

(b6) comparing the fluorescent signals obtained in the step (b4) and the step (b5) to obtain the ID of the DNA sequence subjected to the enzyme digestion reaction;

(b7) and analyzing the DNA sequence subjected to the enzyme digestion reaction according to the ID of the DNA sequence subjected to the enzyme digestion reaction to obtain sequence information of the cleavage site.

In the above method, the methods in step (a1) and step (b1) are sequencing platforms based on sequencing-by-synthesis technology.

In the method, the first sequencing and the second sequencing both adopt a sequencing method of sequencing while synthesizing.

In the method, the last two cycles of the second sequencing are conventional sequencing reactions, and the dNTPs added in the sequencing reactions of the other cycles do not carry fluorescent groups and only carry reversible blocking groups (i.e., cold dNTPs).

In the above method, the number of cycles of the second sequencing is not greater than the number of cycles of the first sequencing. The number of cycles may be slightly greater than the number of bases required for the cleavage site to the maximum length that can be sequenced by the method. In the specific embodiment of the present invention, the restriction enzyme with the recognition site of 6 bases is used for enzyme digestion, so that the first sequencing and the second sequencing can perform 10-300 cycles of sequencing reaction.

In the method, the first sequencing is performed for 10-300 cycles of routine sequencing reaction, and dNTP added in the routine sequencing reaction simultaneously carries a fluorescent group and a reversible blocking group. Preferably, the first sequencing is performed for 50 cycles of a conventional sequencing reaction.

In the method, the second sequencing is performed for 10-300 cycles of sequencing reaction, wherein the last two cycles are routine sequencing reaction, and the dNTPs added in the sequencing reaction of the other cycles do not carry fluorescent groups but only carry reversible blocking groups. Preferably, the second sequencing is performed for 50 cycles, 48 cycles of sequencing reaction are performed first, in which dNTPs not carrying a fluorophore but only carrying a reversible blocking group are added, and then 2 cycles of conventional sequencing reaction are performed.

In the method, the reagent for eluting the new chain is 100 percent formamide.

In the above method, the endonuclease is EcoRI.

In the method, the temperature of the enzyme digestion reaction is 37 ℃; the time of the enzyme digestion reaction is at least 1 hour.

In the above method, the library is a library of e.coli; coli E.coli has ATCC number 8739.

In the method of the present invention, the base sequence of each nucleic acid sequence can be obtained by the first conventional sequencing, after the new strand is washed away, the second sequencing reaction can be performed first, after the reaction, the normal natural double-stranded DNA is obtained, and then the enzyme digestion reaction is performed on the double-stranded DNA, as described in the method (1); it is also possible to perform an endonuclease reaction on the single-stranded DNA on the eluted chip, and then perform a second sequencing reaction, as described in method (2). In both the method (1) and the method (2), the ID of the DNA sequence subjected to the enzyme digestion reaction can be obtained by changing the fluorescent signal generated by the enzyme digestion reaction, so that the characteristics of the sequenced DNA sequence on the nucleic acid sequence subjected to the enzyme digestion reaction are researched, and the information of the recognition cutting site, the tendency and the like of the endonuclease is analyzed and counted.

It is another object of the present invention to provide a novel use of the above method.

The present invention provides the use of the above method in any one of the following 1) to 4):

1) detecting the propensity of the endonuclease to recognize a cleavage site;

2) predicting the star activity of the endonuclease;

3) predicting the off-target effect of endonuclease in the genome editing technology;

4) PAM sequences required to be recognized by endonucleases used in novel genome editing systems are screened on a large scale.

The invention utilizes billions of nucleic acid sequences on a chip to systematically detect the recognition cutting sites and the core sequence characteristics of DNA by endonuclease in high flux, respectively obtains base sequences and normal double-stranded DNA by twice sequencing, and simultaneously analyzes the cutting conditions of the added endonuclease on the nucleic acid sequences by using a biological information analysis method according to the change of fluorescence signals before and after enzyme cutting, thereby further obtaining the recognition cutting sites of the endonuclease. Through the research on the recognition and cutting sites of the endonuclease, the tendency of the recognition and cutting sites of the endonuclease can be detected, the star activity of the endonuclease and the off-target effect of the endonuclease in the genome editing technology can be predicted in advance, and meanwhile, the method can also be used for screening the PAM sequence required to be recognized by the endonuclease used in a novel genome editing system in a large scale. Compared with the sequencing method in the prior art, the detection method has the advantages of high speed and high flux.

Drawings

FIG. 1 shows the short sequences contained in the nucleic acid sequences in which the fluorescence signal is altered before and after cleavage.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

In the quantitative tests in the following examples, three replicates were set up and the results averaged.

The sequencing platform used in the following examples: illumina HiSeq 2000, sequencing type SE50 (unidirectional sequencing 50 nt).

Wild-type E.coli (E.coli) in the examples below has ATCC number 8739.

Example 1 method for detecting endonuclease cleavage site

The method for detecting the endonuclease restriction enzyme cutting sites is based on a sequencing platform of a sequencing technology while synthesis. In the specific embodiment of the invention, the recognition and cutting ability of EcoRI to double-stranded DNA was tested by using commercial EcoRI restriction endonuclease (NEB, R0101) as exemplified by Illumina HiSeq 2000 platform and E.Coli standard DNA library. The method comprises the following specific steps:

1. preparation of E.coli standard library and preparation chip

By using

The DNA PCR-Free Library Prep Library construction kit (the company illumina) constructs an E.coli standard Library from the genome of wild type Escherichia coli (E.coli) (ATCC No. 8739) according to the Library construction process of the illumina, and fixes the constructed Library on a chip to obtain the chip to be sequenced.

The library construction specific steps refer to Illumina official network library construction method (https:// support.illumina. com/content/dam/Illumina-support/documents/documentation/chemistry _ documentation/sampleprep _ tresseq/trescdna _ pcr/tresseq-dna-pcr-library-prep-15036187-d.pdf).

2. First sequencing

And (3) carrying out first sequencing (SBS sequencing while synthesizing) on the chip to be sequenced by adopting a HiSeq SBS kit V4 kit to obtain the chip subjected to the first sequencing, acquiring the base sequence corresponding to each nucleic acid sequence, and recording the ID number corresponding to each nucleic acid sequence. The first sequencing was performed for a total of 50 cycles of the conventional SE50 sequencing reaction, each conventional sequencing cycle comprising the steps of: adding dNTP with a fluorescent group and a reversible blocking group- > to obtain a fluorescent signal- > to cut off the fluorescent group and the blocking group- > to add the dNTP with the fluorescent group and the blocking group again to start a second cycle.

3. Elution of the chip

And (3) adding 100% Formamide (Formamide) into the chip subjected to the first sequencing obtained in the step (2) for treatment for 15min, wherein the treatment temperature is 37 ℃, and eluting the newly generated sequencing chain to obtain the eluted chip.

4. Second sequencing

And (3) performing second sequencing on the eluted chip obtained in the step (3) to obtain a chip subjected to second sequencing, and photographing to obtain a fluorescent signal and an ID number of a corresponding DNA sequence site. And (2) carrying out 50 cycles of sequencing reaction in the second sequencing reaction, adding dNTP (namely cold dNTPs, a Woha gene BGISEQ-500RS high-throughput sequencing kit (SE50), PartNumber:30-05219-00) only carrying reversible blocking groups and not carrying fluorescent groups in the first 48 cycles of sequencing reaction, and then carrying out 2 cycles of conventional sequencing reaction (the dNTP added in the conventional sequencing reaction simultaneously carries the reversible blocking groups and the fluorescent groups, and the specific steps of the conventional sequencing reaction of each cycle are the same as the step 2).

5. Enzyme digestion reaction

And (3) carrying out enzyme digestion reaction on the DNA sequence on the chip subjected to the second sequencing by using a matched reaction Buffer solution (NE Buffer EcoRI, B0101S) carried by EcoRI enzyme at 37 ℃, wherein the reaction time is 1 hour, eluting by using an elution reagent (HiSeq SBS kit V4), removing the cut DNA sequence to obtain the chip subjected to the enzyme digestion reaction, and photographing to obtain a fluorescent signal and an ID number of a corresponding DNA sequence site.

6. Comparison of fluorescence signals

And (3) performing two cycles of conventional sequencing reactions before the enzyme digestion reaction to obtain a fluorescence signal of the nucleic acid sequence, and obtaining a fluorescence signal of a corresponding sequence again after the enzyme digestion reaction, wherein the fluorescence signal of the nucleic acid sequence subjected to the enzyme digestion reaction is changed, and comparing the fluorescence signal of the chip subjected to the second sequencing before the enzyme digestion reaction (step 4) with the fluorescence signal of the chip subjected to the enzyme digestion reaction (step 5) to obtain the ID of the DNA sequence subjected to the enzyme digestion reaction.

7. Biological information analysis

According to the comparison results of two photographing before and after the enzyme digestion reaction, biological information analysis is carried out on the DNA sequence subjected to the enzyme digestion reaction, the characteristics of the sequenced DNA sequence on the nucleic acid sequence subjected to the enzyme digestion reaction are researched through the change of the fluorescent signal, and then information such as the recognition cutting site and the tendency of the endonuclease is analyzed and counted.

The results are shown in FIG. 1 (the ordinate represents the percentage: the ratio of the number of reads containing the corresponding short sequences in the sets of reads left after filtration to the total number of reads; the abscissa represents the partial six-base sequence). As can be seen from the figure: the EcoRI recognition sequence GAATTC is significantly higher than other sequences, and similar sequences, i.e., AATT-containing sequences, are also higher than completely unrelated sequences. The method of the invention can accurately detect the standard recognition sequence (GAATTC) of EcoRI, and finds that the sequence similar to the standard recognition sequence of EcoRI is also partially cut (the four-base sequence of the recognition center is AATT), but the number of the cutting sites is obviously lower than that of the cutting sites of the standard recognition sequence of EcoRI.

Claims

1. A method for detecting endonuclease cutting sites comprises the following steps:

the last two cycles of the second sequencing are conventional sequencing reactions, and dNTPs added in the sequencing reactions of the other cycles only carry reversible blocking groups and do not carry fluorescent groups;

and the first sequencing and the second sequencing both adopt a sequencing method of synthesizing and sequencing.

2. The method of claim 1, wherein: the methods in step (a1) are all sequencing platforms based on sequencing-by-synthesis techniques.

3. The method according to claim 1 or 2, characterized in that: the number of cycles of the second sequencing is no greater than the number of cycles of the first sequencing.

4. The method of claim 3, wherein: the first sequencing is carried out for 10-300 cycles of routine sequencing reaction, and the second sequencing is carried out for 10-300 cycles of sequencing reaction.

5. The method of claim 4, wherein: the number of cycles performed for the first sequencing reaction and the number of cycles performed for the second sequencing reaction were both 50.

6. The method according to claim 1 or 2, characterized in that: the reagent used for eluting the new strand is 100% formamide.

7. The method according to claim 1 or 2, characterized in that: the endonuclease is EcoRI.

8. Use of the method of any one of claims 1 to 7 in any one of the following 1) to 4):

1) detecting the propensity of the endonuclease to recognize a cleavage site;

2) predicting the star activity of the endonuclease;