CN116286787A - Nucleic acid enrichment, separation and purification method based on CRISPR-Cas system - Google Patents

Abstract

The invention discloses a method for enriching, separating and purifying nucleic acid based on a CRISPR-Cas system, which comprises the steps of preparing a complex 1 of Cas effect protein and sgRNA, crRNA or crRNA/tracrRNA; forming a complex 1 and a complex 2 of the target nucleic acid by the nucleic acid sample to be treated and the complex 1; capturing the complex 2 by using the magnetic beads coated with avidin when the complex 1 is marked by biotin, so as to form a complex 3 of the magnetic beads and the complex 2; capturing the complex 2 by using magnetic beads modified by antibodies corresponding to the Cas effect proteins when the complex 1 is not labeled by biotin, so as to form a complex 3 of the magnetic beads and the complex 2; the complex 3 is magnetically separated. The invention utilizes the characteristic that the complex formed by the Cas effect protein can target and combine nucleic acid, and combines a magnetic bead method to realize enrichment, separation and purification of target nucleic acid in a biological sample to be detected, and is specific, rapid, simple and efficient.

Description

Nucleic acid enrichment, separation and purification method based on CRISPR-Cas system

Technical Field

The invention belongs to the field of separation of target nucleic acid in biological samples, and particularly relates to a method for enriching, separating and purifying nucleic acid based on a CRISPR-Cas system.

Background

Nucleic acids are basic genetic material, and are important carriers of genetic information, and research or detection thereof is widely involved in the fields of scientific research, medical diagnosis, environmental microorganism detection, side epidemic prevention detection, and the like. In general, two preconditions are required for the correct investigation or detection of nucleic acids, first the purity of the nucleic acids. In the scientific research field, a molecular cloning technology is one of research means for researching functions or characteristics of target genes, and if a template fails to clone due to pollution, a research result is seriously influenced; contamination with nucleic acids is also highly likely to lead to diagnostic errors during medical diagnostics. Secondly, the concentration of nucleic acid is low, and the current method for detecting nucleic acid has the problem of low sensitivity, so that the application field of the method is limited. Since the actual test substances are rarely nucleic acid samples with high purity and high concentration at present, the extraction and separation of nucleic acids meeting requirements from the actual samples are particularly important. At present, the principle is basically similar for different biological samples, although the methods for extracting and separating DNA are different. Treating the sample with protein denaturant (sodium dodecyl sulfonate, cetyl trimethyl ammonium bromide, sodium hydroxide solution, etc.) and protease, or physically and mechanically destroying cell wall to release DNA, separating protein and DNA by phenol-chloroform, saturated salting out, etc., and purifying the separated DNA with ethanol with a certain concentration to meet the requirement of research or detection. However, the existing method still has the defects of complicated extraction steps, long operation process, toxic reagents and the like, so that the novel simple, convenient, rapid and safe DNA extraction or extraction method has important application value.

The nucleic acid is combined to the surface of the magnetic beads by modifying the groups of the affinity nucleic acid on the surface of the magnetic beads by a common method of magnetic bead enrichment and separation, and then the nucleic acid on the surface of the magnetic beads is separated from other interference components by external magnetic field force so as to achieve the aim of separation and purification, such as silicon hydroxyl magnetic beads and nano magnetic beads. The method has the characteristics of rapidness, simplicity, convenience, high efficiency and the like. However, it has the disadvantage that targeted nucleic acid DNA cannot be directionally enriched and isolated.

CRISPR-Cas systems are the acquired immune system that exists in most bacteria or archaea to resist foreign nucleic acid invasion. Depending on the effector protein, the system can be divided into multiple types. Effector proteins in the system can target exogenous nucleic acid substances or combine with other auxiliary elements and act as nucleases to cut or degrade the exogenous nucleic acid substances for defense purposes. Currently, the best studied for type two systems, cas9, is an effector protein of this type that can form a complex with crRNA (CRISPR RNA) in the system to target DNA (20 nt) complementary to the crRNA portion for binding and cleavage. The characteristic of targeting almost all genome sequences specifically and accurately is widely applied to the fields of gene editing, gene therapy and the like. Cas12a (Cpf 1) in the v-type system is also used in the field of gene editing due to its similar properties. Cas13a (C2C 2) in the type VI system demonstrates greater strength in the field of gene detection. However, this property is not fully utilized and the combination of the above-mentioned magnetic bead method has been rarely reported in the field of enrichment, isolation and purification of target nucleic acid DNA from complex biological samples.

Disclosure of Invention

In order to solve the defects in the prior art, the invention aims to provide a method for enriching, separating and purifying nucleic acid based on a CRISPR-Cas system. The invention utilizes the characteristic that a complex formed by Cas effect proteins or other components (such as sgRNA) in a CRISPR-Cas defense system can accurately target and combine nucleic acid, and combines a magnetic bead method to realize enrichment, separation and purification of target nucleic acid in a biological sample to be detected. The specific technical scheme is as follows:

the first aspect of the present invention provides a method for nucleic acid enrichment, separation and purification based on a CRISPR-Cas system, comprising the steps of:

(1) Preparing a complex 1 of nuclease-deficient Cas effector protein and sgRNA, crRNA, or crRNA/tracrRNA, the complex 1 being biotin-labeled or unlabeled;

(2) Adding a nucleic acid sample to be treated into the complex 1, and incubating to form a complex 2 of the complex 1 and the target nucleic acid;

(3) Capturing the complex 2 by using magnetic beads coated with avidin when the complex 1 is labeled with biotin, so as to form a complex 3 of the magnetic beads and the complex 2;

capturing the complex 2 by using magnetic beads modified by antibodies corresponding to the Cas effect proteins when the complex 1 is not labeled by biotin, so as to form a complex 3 of the magnetic beads and the complex 2;

(4) And (3) magnetically separating the compound 3, and eluting magnetic beads by using a buffer solution to realize enrichment, separation and purification of target nucleic acid of the nucleic acid sample to be treated.

Further, the sgrnas are formed by the binding of crrnas and tracrrnas;

the crRNA includes a complementary nucleotide sequence that is immediately 5' to the NGG in the target nucleic acid and a tracRNA recognition binding sequence.

Further, the biotin is labeled on Cas effector protein and/or on sgRNA, crRNA, or crRNA/tracrRNA;

preferably, when the biotin is labeled on the Cas effector protein, the molar ratio of biotin to Cas effector protein is 200:1; at biotin labelling, incubation is carried out overnight at 4 ℃ or room temperature, preferably overnight at 4 ℃;

preferably, the avidin is streptavidin;

preferably, the nucleic acid in the nucleic acid sample to be treated is a linear nucleic acid.

Further, the Cas effector protein is selected from one of Cas9 in a type ii CRISPR-Cas system, cas12a, C2C1, C2C3 in a type v system, cas13a in a type vi system, and effector proteins of other types of systems;

further, the Cas effector protein is derived from archaea or bacteria selected from nuclease-deficient;

preferably, the Cas effect protein is derived from streptococcus pyogenes (Streptococcus pyogenes), a nuclease-deficient Cas 9-derived protein having the amino acid sequence shown in seq id No. 1;

preferably, the Cas effect protein is derived from Mao Luoke bacteria (Lachnospiraceae bacterium), a nuclease-deficient Cpf 1-derived protein having an amino acid sequence as shown in seq id No. 2;

preferably, the Cas effect protein is derived from bacillus acidoterrestris (Alicyclobacillus acidoterrestris), a nuclease-deficient C2C 1-derived protein having the amino acid sequence shown in seq id No. 3.

Further, the method further comprises pre-treating the nucleic acid sample to be treated, the pre-treating comprising disrupting the nucleic acid sample to be treated;

preferably, the pretreatment comprises: sequentially performing ultrasonic treatment, high-temperature treatment and centrifugal treatment on a nucleic acid sample to be treated, and destroying cell walls and cell membranes to release nucleic acid;

preferably, the operation of the ultrasonic treatment is: ultrasonic 100-300W ultrasonic 5 seconds and 5 seconds, total length 15-30 min, preferably 270W ultrasonic 5 seconds and 5 seconds, total length 10min;

preferably, the high temperature treatment is performed by: the treatment is carried out at 90-98 ℃ for 5-10 min, preferably at 95 ℃ for 10min.

Further, the nucleic acid sample to be treated may be disrupted by other means, such as filtration, ultrafiltration, repeated freeze thawing, digestive enzyme hydrolysis, etc., to obtain a purer nucleic acid sample.

Further, the proportion of the nuclease-deficient Cas effector protein to the complex 1 to the nucleic acid sample to be treated is wide, preferably the amount of the complex 1 is nM, and the amount of the target nucleic acid in the nucleic acid sample to be treated is 1/20 to 1/10 of that of the complex 1;

the incubation temperature in step (2) is determined according to the appropriate working temperature of the effector protein, preferably the optimal temperature for Cas effector protein activity, e.g. Cas9 protein working temperature derived from streptococcus pyogenes (Streptococcus pyogenes) is 25 ℃ to 42 ℃, preferably 37 ℃.

The amount of the magnetic beads used in the step (3) is determined according to the amount of biotin supported by the avidin-coated magnetic beads, the amount of the target nucleic acid, and the amount of the complex 1. For a small amount of target nucleic acid, the amount of magnetic beads is preferably 2ul.

Further, the method further comprises identifying the target nucleic acid for the enriched, isolated and purified complex 3;

the target nucleic acid identification method is selected from, but not limited to, polymerase chain reaction, isothermal amplification technology, helicase dependent amplification technology, rolling circle amplification technology, loop mediated amplification technology or recombinase polymerase isothermal amplification technology;

preferably, the method of target nucleic acid identification is polymerase chain reaction.

In the identification operation, the target nucleic acid is not required to be eluted from the magnetic beads, and can be added into an identification amplification reaction system together with the magnetic beads, and the result is not affected.

The second aspect of the present invention provides a CRISPR-Cas system-based nucleic acid enrichment, isolation and purification kit comprising (1) a biotin-labeled Cas effect protein and avidin-coated magnetic beads, and/or (2) a Cas effect protein and magnetic beads modified by Cas effect protein corresponding antibodies, and/or (3) a Cas effect protein, biotin and avidin-coated magnetic beads;

preferably, the avidin is streptavidin.

Further, the kit also comprises a buffer solution;

the buffer solution comprises the following components in percentage by weight: 10mM, naCl:50mM, mgCl ₂ ：10mM、tween20：0.1％、pH7.0～8.0；

The composition of the reaction solution is Tris-HCl:10mM, naCl:50mM, mgCl ₂ ：10mM、tween20：0.1％、pH7.0～8.0。

Further, the Cas effector protein is selected from one of Cas9 in a type ii CRISPR-Cas system, cas12a, C2C1, C2C3 in a type v system, cas13a in a type vi system, and effector proteins of other types of systems.

Further, the Cas effector protein is derived from archaea or bacteria selected from nuclease-deficient;

preferably, the Cas effect protein is derived from streptococcus pyogenes (Streptococcus pyogenes), a nuclease-deficient Cas 9-derived protein having the amino acid sequence shown in seq id No. 1;

preferably, the Cas effect protein is derived from Mao Luoke bacteria (Lachnospiraceae bacterium), a nuclease-deficient Cpf 1-derived protein having an amino acid sequence as shown in seq id No. 2;

preferably, the Cas effect protein is derived from bacillus acidoterrestris (Alicyclobacillus acidoterrestris), a nuclease-deficient C2C 1-derived protein having the amino acid sequence shown in seq id No. 3.

The beneficial effects of the invention are as follows:

1. compared with the existing nucleic acid separation technology, the method is novel, unique, simple and safe, and can accurately target nucleic acid characteristics by utilizing a complex formed by Cas effect protein and sgRNA, crRNA or crRNA/tracrRNA in the CRISPR-Cas system, and perform specific enrichment, separation and purification on target nucleic acid. The method is simple and convenient to operate, and reagents which are harmful to human health or environmental safety are not involved in the process. In addition, this technique is further characterized by focusing on the enrichment and isolation of linear target nucleic acids, rather than the extraction of large genomic fragments. The size of fragmented DNA is between 200 and 1000bp through the biological sample treated by ultrasonic, and the characteristic is combined with the CRISPR-Cas effect, which is enough for the specific detection of various samples.

2. The invention has wide application field, and can enrich and separate target nucleic acids in samples of different sources and further identify the target nucleic acids. The main reason is that when the CRISPR-Cas system is recognizing a target nucleic acid, the complex of Cas effector protein with sgRNA, crRNA or crRNA/tracrRNA can orient the target nucleic acid according to its own characteristics. Such as a complex formed by Cas9 and sgrnas in a type ii system, is capable of recognizing the "-NGG-" sequence (N representing any base) in the 5' to 3' direction in a target nucleic acid, thereby further binding to 20 nucleotides in the 5' direction thereof, along with adjacent sequences for specific separation by the methods of the present invention. The sequences like "-NGG-" are widely distributed throughout the human genome, plant genome, or microbial genome, indicating that the technology can meet different requirements for the isolation of a desired target nucleic acid.

3. The technology of the invention is based on two points, and can efficiently and specifically separate and purify target nucleic acid DNA. The first point is that the effector complex of Cas protein and sgRNA, crRNA, or crRNA/tracrRNA mentioned above can target nucleic acid according to its own characteristics. The second point is that streptavidin or antibodies on the surface of the magnetic beads can specifically recognize other components in the biotin-labeled effector protein or effector protein complex, such as sgrnas, and achieve a high separation effect due to the extremely high binding constant.

Drawings

Fig. 1 is a schematic diagram of the principle of action of enrichment, isolation and purification of target DNA based on CRISPR-Cas system (biotin labeled to Cas9 protein).

FIG. 2 is a schematic representation of the results of PCR validation after enrichment, isolation and purification of trace short fragment DNA containing excess interfering DNA based on CRISPR-Cas system.

FIG. 3 is a result of PCR validation after enrichment isolation of trace short fragment DNA containing excess interfering DNA and cell culture medium based on enrichment, isolation and purification of CRISPR-Cas system.

Detailed Description

For a clearer understanding of the present invention, the present invention will now be further described with reference to the following examples and drawings. The examples are for illustration only and are not intended to limit the invention in any way. In the examples, each of the starting reagent materials is commercially available, and the experimental methods without specifying the specific conditions are conventional methods and conventional conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

The effector proteins Cas9 and sgrnas in a type ii CRISPR-Cas system are used as display in the examples. Schematic of the principle of action of enrichment, isolation and purification of target DNA based on CRISPR-Cas system (biotin labeled to Cas9 protein) is shown in figure 1. sgrnas are formed by the binding of crrnas and tracrnas.

A crRNA is designed for a short piece of DNA. The short piece of DNA sequence is searched for "-NGG-", 20 nucleotides immediately adjacent to the 5' direction are taken as part of the crRNA sequence, and the other part of the crRNA sequence is the tracRNA recognition binding sequence. The sequences of crRNA and tracRNA are as follows:

crRNA：CUUGUAGCUACGCCUGUGAUGUUUUAGAGCUAUGCUGUUUUG

(SEQ.ID.NO:4)

tracRNA：AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG AAAAAGUGGCACCGAGUCGGUGCUUUU(SEQ.ID.NO:5)

primer sequences for validating the target nucleic acid are as follows:

Verification-For：CTGATGTGGGCTGCCTAGAAAGG(SEQ.ID.NO:6)

Verification-Rev：CAAGGATTGACCCAGGCCAGG(SEQ.ID.NO:7)

example 1: enrichment, separation and purification of trace short fragment DNA containing excessive interference DNA based on CRISPR-Cas system

The specific experimental procedure for enrichment, isolation and purification of trace short fragment DNA containing excess interfering DNA based on CRISPR-Cas system is as follows:

1. biotin labeling of Cas9 protein: cas9 is a dual nuclease domain-deficient protein, dCas9, with biotin incubated with dCas9 at a molar ratio of 200:1 overnight at 4 ℃. After the incubation, the mixture is transferred to a desalting column, and centrifuged to remove excessive biotin. After the first centrifugation (5000 rpm,2 min), an appropriate amount of 1 XPBS (pH 7.4) was added and the centrifugation was repeated three times under the same conditions to ensure complete removal of unlabeled biotin.

2. Preparation of sgRNA: annealing the synthesized crRNA and tracRNA according to a molar ratio of 1:2 to form functional sgRNA. The annealing conditions were 98℃for 5min, followed by a gradient of cooling down to 2℃every 20s, up to 25℃at room temperature.

3. Formation of dCAS9-sgRNA Complex: in the reaction solution (Tris-HCl: 10mM, naCl:50mM, mgCl) ₂ :10mM, tween20:0.1%, pH 7-8.0), the biotin-labeled dCAS9 protein and sgRNA are added in a molar ratio of 1:2, wherein the concentration of dCAS9 in a 30ul reaction system is 25 nM-200 nM and the concentration of sgRNA is 50 nM-400 nM, and incubated at 37℃for 10-20 min, preferably 15min in this example, to form a stable dCAS9-sgRNA complex.

4. Formation of dCas9-sgRNA-DNA complex: with preservation solution (Tris-HCl: 10mM, naCl:50mM, mgCl) ₂ :10mM, tween20:0.1%, 0.2mg/ml BSA, 250ng/ul EGFP plasmid, pH 7-8.0) diluted short DNA fragment to a concentration of 25nM2.5fM, 1ul of the reaction solution described in step 2 was added thereto, and 0.5ul of a preservation solution was added thereto to increase the amount of interfering DNA (8 ng/ul), and incubated at 37℃for 15 to 30 minutes, preferably 20 minutes in this example, to form a stable dCAS9-sgRNA-DNA complex.

5. Treatment of magnetic beads: taking 5ul of magnetic beads, adsorbing the beads on the pipe wall through the action force of an external magnetic field, discarding the supernatant, adding 200ul of the reaction solution prepared in the step 3 into the reaction solution, mixing the mixture uniformly by vortex, discarding the supernatant by the magnet adsorption magnetic beads, and repeating the steps for three times. The treated magnetic beads are added into the reaction system of the step 4, and incubated for 10-40 min at normal temperature, preferably 30min in the embodiment, and vortex once every 5min to avoid the deposition of the magnetic beads at the bottom of the tube.

6. Isolation of target nucleic acid DNA: the DNA separated and enriched on the surface of the magnetic beads is adsorbed on the tube wall together by the acting force between the magnet and the magnetic beads, and the supernatant is discarded; adding 200ul of washing buffer (Tris-HCl: 10mM, naCl:50mM, mgCl) ₂ :10mM, tween20:0.1%, pH 7-8.0), then placing the mixture on a magnet to adsorb magnetic beads, standing for 1min, removing supernatant, repeating for three times to purify DNA.

7. Identification of target DNA: in a 50ul system of the polymerase chain reaction, nuclease-free water, 10ul of 5 Xamplification buffer, 1ul of 10mM dNTPs and 0.5ul of polymerase are sequentially added, after uniform mixing, 1.5ul of each of the two 10mM verification primers is added, and then the separated and purified target DNA together with magnetic beads are added to the prepared reaction system. The amplification was performed by repeating the procedure for 30 cycles at 98℃for 10s,55℃for 5s, 72℃for 18 s.

8. To the amplified product, gelgreen fluorescent dye was added at a volume ratio of 100:1, and a 36% aqueous glycerol solution was added at a volume ratio of 6:1. Running gel in 6% non-denaturing gel was identified, buffer at 1 XTBE, running gel at 150V for 20min.

As shown in FIG. 2, the control group (which was not isolated and purified by this technique) had a large amount of nonspecific bands on the amplified product due to the high concentration of interfering DNA and the low concentration of target DNA. The experimental group can amplify the target band more sensitively after enrichment, separation and purification treatment in the embodiment, and the amplified target band can still be observed under the condition of low concentration, which shows the feasibility and high sensitivity of the technology. In addition, the amplification of nonspecific bands was significantly reduced.

Example 2: enrichment, separation and purification of trace short fragment DNA containing excessive interfering DNA and cell culture medium based on CRISPR-Cas system

The specific experimental procedure is as described in example 1, except that in step 3, in addition to the interfering DNA, a final concentration of 10% of the serum-containing cell culture medium is added, and in addition, the DNA concentration is 25fM to 25aM.

The experimental results are shown in fig. 3: because of the complex composition of the cell culture medium, the target band was not amplified for the untreated sample, whereas the experimental group was enriched, isolated and purified in this example to allow amplification of the target band at lower concentrations, although there was a small amount of non-specific band.

Example 3: kit for enriching, separating and purifying nucleic acid based on CRISPR-Cas system

In a specific embodiment, the kit comprises a biotin-labeled Cas effector protein, magnetic beads coated with avidin, a buffer, and a reaction solution, and the avidin is preferably streptavidin.

In a specific embodiment, the kit comprises a Cas effect protein, magnetic beads modified by a Cas effect protein corresponding antibody, a buffer, and a reaction solution.

In a specific embodiment, the kit comprises Cas effect protein, biotin, avidin-coated magnetic beads, buffer and reaction solution, and the avidin is preferably streptavidin.

The buffer solution is used for eluting the magnetic beads, and the components and the concentrations are Tris-HCl:10mM, naCl:50mM, mgCl ₂ ：10mM、tween20：0.1％、pH7～8.0。

The reaction solution is used for the reaction of Cas effect protein and sgRNA, crRNA or complex 1 of crRNA/tracrrRNA, and the components and the concentrations are Tris-HCl:10mM, naCl:50mM, mgCl ₂ ：10mM、tween20：0.1％、pH7～8.0。

In this embodiment, the Cas effector protein is selected from one of Cas9 in a type ii CRISPR-Cas system, cas12a, C2C1, C2C3 in a type v system, cas13a in a type vi system, and effector proteins of other types of systems.

Cas effector proteins are derived from archaea or bacteria and are selected from nuclease-deficient.

As a preferred embodiment, the Cas effector protein is derived from streptococcus pyogenes (streptococcus pyogenes), a nuclease-deficient Cas 9-derived protein having the amino acid sequence shown in seq id No. 1;

as a preferred embodiment, the Cas effector protein is derived from Mao Luoke bacteria (Lachnospiraceae bacterium) ND20061368, a nuclease-deficient Cpf1 derivative protein having the amino acid sequence shown in SEQ ID No. 2;

as a preferred embodiment, the Cas effector protein is derived from Bacillus stearothermophilus (Alicyclobacillus acidoterrestris), a nuclease-deficient C2C 1-derived protein having the amino acid sequence shown in SEQ ID NO. 3.

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.

SEQUENCE LISTING

<110> Shenzhen advanced technology research institute

<120> a method for nucleic acid enrichment, separation and purification based on CRISPR-Cas system

<130> CP121010884C

<160> 7

<170> PatentIn version 3.3

<210> 1

<211> 1368

<212> PRT

<213> Streptococcus pyogenes

<400> 1

Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val

1 5 10 15

Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe

20 25 30

Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile

35 40 45

Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu

50 55 60

Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys

65 70 75 80

Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser

85 90 95

Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys

100 105 110

His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr

115 120 125

His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp

130 135 140

Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His

145 150 155 160

Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro

165 170 175

Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr

180 185 190

Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala

195 200 205

Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn

210 215 220

Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn

225 230 235 240

Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe

245 250 255

Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp

260 265 270

Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp

275 280 285

Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp

290 295 300

Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser

305 310 315 320

Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys

325 330 335

Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe

340 345 350

Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser

355 360 365

Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp

370 375 380

Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg

385 390 395 400

Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu

405 410 415

Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe

420 425 430

Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile

435 440 445

Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp

450 455 460

Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu

465 470 475 480

Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr

485 490 495

Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser

500 505 510

Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys

515 520 525

Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln

530 535 540

Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr

545 550 555 560

Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp

565 570 575

Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly

580 585 590

Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp

595 600 605

Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr

610 615 620

Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala

625 630 635 640

His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr

645 650 655

Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp

660 665 670

Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe

675 680 685

Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe

690 695 700

Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu

705 710 715 720

His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly

725 730 735

Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly

740 745 750

Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln

755 760 765

Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile

770 775 780

Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro

785 790 795 800

Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu

805 810 815

Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg

820 825 830

Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys

835 840 845

Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg

850 855 860

Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys

865 870 875 880

Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys

885 890 895

Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp

900 905 910

Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr

915 920 925

Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp

930 935 940

Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser

945 950 955 960

Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg

965 970 975

Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val

980 985 990

Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe

995 1000 1005

Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala

1010 1015 1020

Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe

1025 1030 1035

Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala

1040 1045 1050

Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu

1055 1060 1065

Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val

1070 1075 1080

Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr

1085 1090 1095

Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys

1100 1105 1110

Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro

1115 1120 1125

Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val

1130 1135 1140

Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys

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Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser

1160 1165 1170

Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys

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Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu

1190 1195 1200

Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly

1205 1210 1215

Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val

1220 1225 1230

Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser

1235 1240 1245

Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys

1250 1255 1260

His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys

1265 1270 1275

Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala

1280 1285 1290

Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn

1295 1300 1305

Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala

1310 1315 1320

Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser

1325 1330 1335

Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr

1340 1345 1350

Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp

1355 1360 1365

<210> 2

<211> 1228

<212> PRT

<213> Lachnospiraceae bacterium

<400> 2

Met Ser Lys Leu Glu Lys Phe Thr Asn Cys Tyr Ser Leu Ser Lys Thr

1 5 10 15

Leu Arg Phe Lys Ala Ile Pro Val Gly Lys Thr Gln Glu Asn Ile Asp

20 25 30

Asn Lys Arg Leu Leu Val Glu Asp Glu Lys Arg Ala Glu Asp Tyr Lys

35 40 45

Gly Val Lys Lys Leu Leu Asp Arg Tyr Tyr Leu Ser Phe Ile Asn Asp

50 55 60

Val Leu His Ser Ile Lys Leu Lys Asn Leu Asn Asn Tyr Ile Ser Leu

65 70 75 80

Phe Arg Lys Lys Thr Arg Thr Glu Lys Glu Asn Lys Glu Leu Glu Asn

85 90 95

Leu Glu Ile Asn Leu Arg Lys Glu Ile Ala Lys Ala Phe Lys Gly Asn

100 105 110

Glu Gly Tyr Lys Ser Leu Phe Lys Lys Asp Ile Ile Glu Thr Ile Leu

115 120 125

Pro Glu Phe Leu Asp Asp Lys Asp Glu Ile Ala Leu Val Asn Ser Phe

130 135 140

Asn Gly Phe Thr Thr Ala Phe Thr Gly Phe Phe Asp Asn Arg Glu Asn

145 150 155 160

Met Phe Ser Glu Glu Ala Lys Ser Thr Ser Ile Ala Phe Arg Cys Ile

165 170 175

Asn Glu Asn Leu Thr Arg Tyr Ile Ser Asn Met Asp Ile Phe Glu Lys

180 185 190

Val Asp Ala Ile Phe Asp Lys His Glu Val Gln Glu Ile Lys Glu Lys

195 200 205

Ile Leu Asn Ser Asp Tyr Asp Val Glu Asp Phe Phe Glu Gly Glu Phe

210 215 220

Phe Asn Phe Val Leu Thr Gln Glu Gly Ile Asp Val Tyr Asn Ala Ile

225 230 235 240

Ile Gly Gly Phe Val Thr Glu Ser Gly Glu Lys Ile Lys Gly Leu Asn

245 250 255

Glu Tyr Ile Asn Leu Tyr Asn Gln Lys Thr Lys Gln Lys Leu Pro Lys

260 265 270

Phe Lys Pro Leu Tyr Lys Gln Val Leu Ser Asp Arg Glu Ser Leu Ser

275 280 285

Phe Tyr Gly Glu Gly Tyr Thr Ser Asp Glu Glu Val Leu Glu Val Phe

290 295 300

Arg Asn Thr Leu Asn Lys Asn Ser Glu Ile Phe Ser Ser Ile Lys Lys

305 310 315 320

Leu Glu Lys Leu Phe Lys Asn Phe Asp Glu Tyr Ser Ser Ala Gly Ile

325 330 335

Phe Val Lys Asn Gly Pro Ala Ile Ser Thr Ile Ser Lys Asp Ile Phe

340 345 350

Gly Glu Trp Asn Val Ile Arg Asp Lys Trp Asn Ala Glu Tyr Asp Asp

355 360 365

Ile His Leu Lys Lys Lys Ala Val Val Thr Glu Lys Tyr Glu Asp Asp

370 375 380

Arg Arg Lys Ser Phe Lys Lys Ile Gly Ser Phe Ser Leu Glu Gln Leu

385 390 395 400

Gln Glu Tyr Ala Asp Ala Asp Leu Ser Val Val Glu Lys Leu Lys Glu

405 410 415

Ile Ile Ile Gln Lys Val Asp Glu Ile Tyr Lys Val Tyr Gly Ser Ser

420 425 430

Glu Lys Leu Phe Asp Ala Asp Phe Val Leu Glu Lys Ser Leu Lys Lys

435 440 445

Asn Asp Ala Val Val Ala Ile Met Lys Asp Leu Leu Asp Ser Val Lys

450 455 460

Ser Phe Glu Asn Tyr Ile Lys Ala Phe Phe Gly Glu Gly Lys Glu Thr

465 470 475 480

Asn Arg Asp Glu Ser Phe Tyr Gly Asp Phe Val Leu Ala Tyr Asp Ile

485 490 495

Leu Leu Lys Val Asp His Ile Tyr Asp Ala Ile Arg Asn Tyr Val Thr

500 505 510

Gln Lys Pro Tyr Ser Lys Asp Lys Phe Lys Leu Tyr Phe Gln Asn Pro

515 520 525

Gln Phe Met Gly Gly Trp Asp Lys Asp Lys Glu Thr Asp Tyr Arg Ala

530 535 540

Thr Ile Leu Arg Tyr Gly Ser Lys Tyr Tyr Leu Ala Ile Met Asp Lys

545 550 555 560

Lys Tyr Ala Lys Cys Leu Gln Lys Ile Asp Lys Asp Asp Val Asn Gly

565 570 575

Asn Tyr Glu Lys Ile Asn Tyr Lys Leu Leu Pro Gly Pro Asn Lys Met

580 585 590

Leu Pro Lys Val Phe Phe Ser Lys Lys Trp Met Ala Tyr Tyr Asn Pro

595 600 605

Ser Glu Asp Ile Gln Lys Ile Tyr Lys Asn Gly Thr Phe Lys Lys Gly

610 615 620

Asp Met Phe Asn Leu Asn Asp Cys His Lys Leu Ile Asp Phe Phe Lys

625 630 635 640

Asp Ser Ile Ser Arg Tyr Pro Lys Trp Ser Asn Ala Tyr Asp Phe Asn

645 650 655

Phe Ser Glu Thr Glu Lys Tyr Lys Asp Ile Ala Gly Phe Tyr Arg Glu

660 665 670

Val Glu Glu Gln Gly Tyr Lys Val Ser Phe Glu Ser Ala Ser Lys Lys

675 680 685

Glu Val Asp Lys Leu Val Glu Glu Gly Lys Leu Tyr Met Phe Gln Ile

690 695 700

Tyr Asn Lys Asp Phe Ser Asp Lys Ser His Gly Thr Pro Asn Leu His

705 710 715 720

Thr Met Tyr Phe Lys Leu Leu Phe Asp Glu Asn Asn His Gly Gln Ile

725 730 735

Arg Leu Ser Gly Gly Ala Glu Leu Phe Met Arg Arg Ala Ser Leu Lys

740 745 750

Lys Glu Glu Leu Val Val His Pro Ala Asn Ser Pro Ile Ala Asn Lys

755 760 765

Asn Pro Asp Asn Pro Lys Lys Thr Thr Thr Leu Ser Tyr Asp Val Tyr

770 775 780

Lys Asp Lys Arg Phe Ser Glu Asp Gln Tyr Glu Leu His Ile Pro Ile

785 790 795 800

Ala Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu Val

805 810 815

Arg Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly Ile Asp

820 825 830

Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly Lys Gly

835 840 845

Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn Asn Phe Asn

850 855 860

Gly Ile Arg Ile Lys Thr Asp Tyr His Ser Leu Leu Asp Lys Lys Glu

865 870 875 880

Lys Glu Arg Phe Glu Ala Arg Gln Asn Trp Thr Ser Ile Glu Asn Ile

885 890 895

Lys Glu Leu Lys Ala Gly Tyr Ile Ser Gln Val Val His Lys Ile Cys

900 905 910

Glu Leu Val Glu Lys Tyr Asp Ala Val Ile Ala Leu Glu Asp Leu Asn

915 920 925

Ser Gly Phe Lys Asn Ser Arg Val Lys Val Glu Lys Gln Val Tyr Gln

930 935 940

Lys Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val Asp Lys

945 950 955 960

Lys Ser Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr Gln Ile

965 970 975

Thr Asn Lys Phe Glu Ser Phe Lys Ser Met Ser Thr Gln Asn Gly Phe

980 985 990

Ile Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser Thr

995 1000 1005

Gly Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala Asp

1010 1015 1020

Ser Lys Lys Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr Val Pro

1025 1030 1035

Glu Glu Asp Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe Ser

1040 1045 1050

Arg Thr Asp Ala Asp Tyr Ile Lys Lys Trp Lys Leu Tyr Ser Tyr

1055 1060 1065

Gly Asn Arg Ile Arg Ile Phe Arg Asn Pro Lys Lys Asn Asn Val

1070 1075 1080

Phe Asp Trp Glu Glu Val Cys Leu Thr Ser Ala Tyr Lys Glu Leu

1085 1090 1095

Phe Asn Lys Tyr Gly Ile Asn Tyr Gln Gln Gly Asp Ile Arg Ala

1100 1105 1110

Leu Leu Cys Glu Gln Ser Asp Lys Ala Phe Tyr Ser Ser Phe Met

1115 1120 1125

Ala Leu Met Ser Leu Met Leu Gln Met Arg Asn Ser Ile Thr Gly

1130 1135 1140

Arg Thr Asp Val Asp Phe Leu Ile Ser Pro Val Lys Asn Ser Asp

1145 1150 1155

Gly Ile Phe Tyr Asp Ser Arg Asn Tyr Glu Ala Gln Glu Asn Ala

1160 1165 1170

Ile Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala

1175 1180 1185

Arg Lys Val Leu Trp Ala Ile Gly Gln Phe Lys Lys Ala Glu Asp

1190 1195 1200

Glu Lys Leu Asp Lys Val Lys Ile Ala Ile Ser Asn Lys Glu Trp

1205 1210 1215

Leu Glu Tyr Ala Gln Thr Ser Val Lys His

1220 1225

<210> 3

<211> 1129

<212> PRT

<213> Alicyclobacillus Acidoterrestris

<400> 3

Met Ala Val Lys Ser Ile Lys Val Lys Leu Arg Leu Asp Asp Met Pro

1 5 10 15

Glu Ile Arg Ala Gly Leu Trp Lys Leu His Lys Glu Val Asn Ala Gly

20 25 30

Val Arg Tyr Tyr Thr Glu Trp Leu Ser Leu Leu Arg Gln Glu Asn Leu

35 40 45

Tyr Arg Arg Ser Pro Asn Gly Asp Gly Glu Gln Glu Cys Asp Lys Thr

50 55 60

Ala Glu Glu Cys Lys Ala Glu Leu Leu Glu Arg Leu Arg Ala Arg Gln

65 70 75 80

Val Glu Asn Gly His Arg Gly Pro Ala Gly Ser Asp Asp Glu Leu Leu

85 90 95

Gln Leu Ala Arg Gln Leu Tyr Glu Leu Leu Val Pro Gln Ala Ile Gly

100 105 110

Ala Lys Gly Asp Ala Gln Gln Ile Ala Arg Lys Phe Leu Ser Pro Leu

115 120 125

Ala Asp Lys Asp Ala Val Gly Gly Leu Gly Ile Ala Lys Ala Gly Asn

130 135 140

Lys Pro Arg Trp Val Arg Met Arg Glu Ala Gly Glu Pro Gly Trp Glu

145 150 155 160

Glu Glu Lys Glu Lys Ala Glu Thr Arg Lys Ser Ala Asp Arg Thr Ala

165 170 175

Asp Val Leu Arg Ala Leu Ala Asp Phe Gly Leu Lys Pro Leu Met Arg

180 185 190

Val Tyr Thr Asp Ser Glu Met Ser Ser Val Glu Trp Lys Pro Leu Arg

195 200 205

Lys Gly Gln Ala Val Arg Thr Trp Asp Arg Asp Met Phe Gln Gln Ala

210 215 220

Ile Glu Arg Met Met Ser Trp Glu Ser Trp Asn Gln Arg Val Gly Gln

225 230 235 240

Glu Tyr Ala Lys Leu Val Glu Gln Lys Asn Arg Phe Glu Gln Lys Asn

245 250 255

Phe Val Gly Gln Glu His Leu Val His Leu Val Asn Gln Leu Gln Gln

260 265 270

Asp Met Lys Glu Ala Ser Pro Gly Leu Glu Ser Lys Glu Gln Thr Ala

275 280 285

His Tyr Val Thr Gly Arg Ala Leu Arg Gly Ser Asp Lys Val Phe Glu

290 295 300

Lys Trp Gly Lys Leu Ala Pro Asp Ala Pro Phe Asp Leu Tyr Asp Ala

305 310 315 320

Glu Ile Lys Asn Val Gln Arg Arg Asn Thr Arg Arg Phe Gly Ser His

325 330 335

Asp Leu Phe Ala Lys Leu Ala Glu Pro Glu Tyr Gln Ala Leu Trp Arg

340 345 350

Glu Asp Ala Ser Phe Leu Thr Arg Tyr Ala Val Tyr Asn Ser Ile Leu

355 360 365

Arg Lys Leu Asn His Ala Lys Met Phe Ala Thr Phe Thr Leu Pro Asp

370 375 380

Ala Thr Ala His Pro Ile Trp Thr Arg Phe Asp Lys Leu Gly Gly Asn

385 390 395 400

Leu His Gln Tyr Thr Phe Leu Phe Asn Glu Phe Gly Glu Arg Arg His

405 410 415

Ala Ile Arg Phe His Lys Leu Leu Lys Val Glu Asn Gly Val Ala Arg

420 425 430

Glu Val Asp Asp Val Thr Val Pro Ile Ser Met Ser Glu Gln Leu Asp

435 440 445

Asn Leu Leu Pro Arg Asp Pro Asn Glu Pro Ile Ala Leu Tyr Phe Arg

450 455 460

Asp Tyr Gly Ala Glu Gln His Phe Thr Gly Glu Phe Gly Gly Ala Lys

465 470 475 480

Ile Gln Cys Arg Arg Asp Gln Leu Ala His Met His Arg Arg Arg Gly

485 490 495

Ala Arg Asp Val Tyr Leu Asn Val Ser Val Arg Val Gln Ser Gln Ser

500 505 510

Glu Ala Arg Gly Glu Arg Arg Pro Pro Tyr Ala Ala Val Phe Arg Leu

515 520 525

Val Gly Asp Asn His Arg Ala Phe Val His Phe Asp Lys Leu Ser Asp

530 535 540

Tyr Leu Ala Glu His Pro Asp Asp Gly Lys Leu Gly Ser Glu Gly Leu

545 550 555 560

Leu Ser Gly Leu Arg Val Met Ser Val Asp Leu Gly Leu Arg Thr Ser

565 570 575

Ala Ser Ile Ser Val Phe Arg Val Ala Arg Lys Asp Glu Leu Lys Pro

580 585 590

Asn Ser Lys Gly Arg Val Pro Phe Phe Phe Pro Ile Lys Gly Asn Asp

595 600 605

Asn Leu Val Ala Val His Glu Arg Ser Gln Leu Leu Lys Leu Pro Gly

610 615 620

Glu Thr Glu Ser Lys Asp Leu Arg Ala Ile Arg Glu Glu Arg Gln Arg

625 630 635 640

Thr Leu Arg Gln Leu Arg Thr Gln Leu Ala Tyr Leu Arg Leu Leu Val

645 650 655

Arg Cys Gly Ser Glu Asp Val Gly Arg Arg Glu Arg Ser Trp Ala Lys

660 665 670

Leu Ile Glu Gln Pro Val Asp Ala Ala Asn His Met Thr Pro Asp Trp

675 680 685

Arg Glu Ala Phe Glu Asn Glu Leu Gln Lys Leu Lys Ser Leu His Gly

690 695 700

Ile Cys Ser Asp Lys Glu Trp Met Asp Ala Val Tyr Glu Ser Val Arg

705 710 715 720

Arg Val Trp Arg His Met Gly Lys Gln Val Arg Asp Trp Arg Lys Asp

725 730 735

Val Arg Ser Gly Glu Arg Pro Lys Ile Arg Gly Tyr Ala Lys Asp Val

740 745 750

Val Gly Gly Asn Ser Ile Glu Gln Ile Glu Tyr Leu Glu Arg Gln Tyr

755 760 765

Lys Phe Leu Lys Ser Trp Ser Phe Phe Gly Lys Val Ser Gly Gln Val

770 775 780

Ile Arg Ala Glu Lys Gly Ser Arg Phe Ala Ile Thr Leu Arg Glu His

785 790 795 800

Ile Asp His Ala Lys Glu Asp Arg Leu Lys Lys Leu Ala Asp Arg Ile

805 810 815

Ile Met Glu Ala Leu Gly Tyr Val Tyr Ala Leu Asp Glu Arg Gly Lys

820 825 830

Gly Lys Trp Val Ala Lys Tyr Pro Pro Cys Gln Leu Ile Leu Leu Glu

835 840 845

Glu Leu Ser Glu Tyr Gln Phe Asn Asn Asp Arg Pro Pro Ser Glu Asn

850 855 860

Asn Gln Leu Met Gln Trp Ser His Arg Gly Val Phe Gln Glu Leu Ile

865 870 875 880

Asn Gln Ala Gln Val His Asp Leu Leu Val Gly Thr Met Tyr Ala Ala

885 890 895

Phe Ser Ser Arg Phe Asp Ala Arg Thr Gly Ala Pro Gly Ile Arg Cys

900 905 910

Arg Arg Val Pro Ala Arg Cys Thr Gln Glu His Asn Pro Glu Pro Phe

915 920 925

Pro Trp Trp Leu Asn Lys Phe Val Val Glu His Thr Leu Asp Ala Cys

930 935 940

Pro Leu Arg Ala Asp Asp Leu Ile Pro Thr Gly Glu Gly Glu Ile Phe

945 950 955 960

Val Ser Pro Phe Ser Ala Glu Glu Gly Asp Phe His Gln Ile His Ala

965 970 975

Asp Leu Asn Ala Ala Gln Asn Leu Gln Gln Arg Leu Trp Ser Asp Phe

980 985 990

Asp Ile Ser Gln Ile Arg Leu Arg Cys Asp Trp Gly Glu Val Asp Gly

995 1000 1005

Glu Leu Val Leu Ile Pro Arg Leu Thr Gly Lys Arg Thr Ala Asp

1010 1015 1020

Ser Tyr Ser Asn Lys Val Phe Tyr Thr Asn Thr Gly Val Thr Tyr

1025 1030 1035

Tyr Glu Arg Glu Arg Gly Lys Lys Arg Arg Lys Val Phe Ala Gln

1040 1045 1050

Glu Lys Leu Ser Glu Glu Glu Ala Glu Leu Leu Val Glu Ala Asp

1055 1060 1065

Glu Ala Arg Glu Lys Ser Val Val Leu Met Arg Asp Pro Ser Gly

1070 1075 1080

Ile Ile Asn Arg Gly Asn Trp Thr Arg Gln Lys Glu Phe Trp Ser

1085 1090 1095

Met Val Asn Gln Arg Ile Glu Gly Tyr Leu Val Lys Gln Ile Arg

1100 1105 1110

Ser Arg Val Pro Leu Gln Asp Ser Ala Cys Glu Asn Thr Gly Asp

1115 1120 1125

Ile

<210> 4

<211> 42

<212> RNA

<213> artificial sequence

<400> 4

cuuguagcua cgccugugau guuuuagagc uaugcuguuu ug 42

<210> 5

<211> 72

<212> RNA

<213> artificial sequence

<400> 5

aaacagcaua gcaaguuaaa auaaggcuag uccguuauca acuugaaaaa guggcaccga 60

gucggugcuu uu 72

<210> 6

<211> 23

<212> DNA

<213> artificial sequence

<400> 6

ctgatgtggg ctgcctagaa agg 23

<210> 7

<211> 21

<212> DNA

<213> artificial sequence

<400> 7

caaggattga cccaggccag g 21

Claims (10)

1. A method for nucleic acid enrichment, separation and purification based on a CRISPR-Cas system, comprising the steps of:

(1) Preparing a complex 1 of nuclease-deficient Cas effector protein and sgRNA, crRNA, or crRNA/tracrRNA, the complex 1 being biotin-labeled or unlabeled;

(2) Adding a nucleic acid sample to be treated into the complex 1, and incubating to form a complex 2 of the complex 1 and the target nucleic acid;

(3) Capturing the complex 2 by using magnetic beads coated with avidin when the complex 1 is labeled with biotin, so as to form a complex 3 of the magnetic beads and the complex 2;

capturing the complex 2 by using magnetic beads modified by antibodies corresponding to the Cas effect proteins when the complex 1 is not labeled by biotin, so as to form a complex 3 of the magnetic beads and the complex 2;

(4) And (3) magnetically separating the compound 3, and eluting magnetic beads by using a buffer solution to realize enrichment, separation and purification of target nucleic acid of the nucleic acid sample to be treated.

2. The method of claim 1, wherein the sgrnas are formed by the binding of crrnas and tracrRNA;

the crRNA includes a complementary nucleotide sequence that is immediately 5' to the NGG in the target nucleic acid and a tracRNA recognition binding sequence.

3. The method of claim 1, wherein the biotin is labeled on Cas effector protein and/or on sgRNA, crRNA or crRNA/tracrRNA;

preferably, when the biotin is labeled on the Cas effector protein, the molar ratio of biotin to Cas effector protein is 200:1;

preferably, the avidin is streptavidin;

preferably, the nucleic acid in the nucleic acid sample to be treated is a linear nucleic acid.

4. The method of claim 1, wherein the Cas effector protein is selected from one of Cas9 in a type ii CRISPR-Cas system, cas12a in a type v system, C2C1, C2C3, cas13a in a type vi system, and effector proteins of other types of systems;

preferably, the Cas effector protein is derived from archaea or bacteria selected from nuclease-deficient;

preferably, the Cas effect protein is derived from streptococcus pyogenes, a nuclease-deficient Cas 9-derived protein having the amino acid sequence shown in seq id No. 1;

preferably, the Cas effect protein is derived from Mao Luoke bacteria, a nuclease-deficient Cpf 1-derived protein having an amino acid sequence as shown in seq id No. 2;

preferably, the Cas effect protein is derived from bacillus stearothermophilus, a nuclease-deficient C2C 1-derived protein having the amino acid sequence shown in seq id No. 3.

5. The method of claim 1, further comprising pre-treating the nucleic acid sample to be treated, the pre-treating comprising disrupting the nucleic acid sample to be treated;

preferably, the pretreatment comprises: sequentially carrying out ultrasonic treatment, high-temperature treatment and centrifugal treatment on a nucleic acid sample to be treated;

preferably, the operation of the ultrasonic treatment is: the ultrasonic power is 100-300W, the ultrasonic power is 5 seconds, the ultrasonic power is stopped for 5 seconds, the total length is 15-30 min, the ultrasonic power is preferably 270W, the ultrasonic power is stopped for 5 seconds, and the total length is 10min;

preferably, the high temperature treatment is performed by: the treatment is carried out at 90-98 ℃ for 5-10 min, preferably at 95 ℃ for 10min.

6. The method of claim 1, wherein the nuclease-deficient Cas effector protein or complex 1 is in nM-order and the amount of target nucleic acid in the nucleic acid sample to be treated is 1/20 to 1/10 of complex 1;

preferably, the incubation temperature in step (2) is the optimal temperature for Cas effector protein activity;

preferably, the amount of the magnetic beads used in step (3) is determined according to the amount of biotin supported by the avidin-coated magnetic beads, the amount of the target nucleic acid, and the amount of the complex 1.

7. The method of claim 1, further comprising identifying the enriched, isolated and purified complex 3 as a target nucleic acid;

the method for identifying the target nucleic acid is selected from the group consisting of polymerase chain reaction, isothermal amplification technology, helicase dependent amplification technology, rolling circle amplification technology, loop-mediated amplification technology, or recombinase polymerase isothermal amplification technology;

preferably, the method of target nucleic acid identification is polymerase chain reaction.

8. A kit for nucleic acid enrichment, isolation and purification based on a CRISPR-Cas system, comprising (1) a biotin-labeled Cas effect protein and an avidin-coated magnetic bead, and/or (2) a Cas effect protein and a magnetic bead modified by a Cas effect protein corresponding antibody, and/or (3) a Cas effect protein, biotin, and an avidin-coated magnetic bead;

preferably, the avidin is streptavidin.

9. The kit of claim 8, further comprising a buffer and a reaction solution;

the buffer solution comprises the following components in percentage by weight: 10mM, naCl:50mM, mgCl ₂ ：10mM、tween20：0.1％、pH7.0～8.0；

The composition of the reaction solution is Tris-HCl:10mM, naCl:50mM, mgCl ₂ ：10mM、tween20：0.1％、pH7.0～8.0。

10. The kit of claim 8, wherein the Cas effector protein is selected from one of Cas9 in a type ii CRISPR-Cas system, cas12a, C2C1, C2C3 in a type v system, cas13a in a type vi system, and effector proteins of other types of systems;

preferably, the Cas effector protein is derived from archaea or bacteria selected from nuclease-deficient;

preferably, the Cas effect protein is derived from streptococcus pyogenes, a nuclease-deficient Cas 9-derived protein having the amino acid sequence shown in seq id No. 1;

preferably, the Cas effect protein is derived from Mao Luoke bacteria, a nuclease-deficient Cpf 1-derived protein having an amino acid sequence as shown in seq id No. 2;

preferably, the Cas effect protein is derived from bacillus stearothermophilus, a nuclease-deficient C2C 1-derived protein having the amino acid sequence shown in seq id No. 3.

Images