CN113278646A - Method for constructing rice polygene editing mutant library and application - Google Patents
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Abstract
The invention discloses a method for constructing a rice polygene editing mutant library and application thereof, wherein the method comprises the following steps: s1, designing an sgRNA library; s2, preparing a CRISPR/Cas9 vector library; s3, genetic transformation; s4, identifying the sgRNA; s5, detecting the gene editing seedlings; according to the invention, the CRISPR/Cas9 technology is adopted, the whole rice polygene editing mutant library is obtained at one time, compared with the existing one-by-one gene knockout, the efficiency is improved, and the workload is obviously reduced; meanwhile, the invention designs a complete, systematic and efficient plant multi-genome editing and detecting method, can accurately edit a large number of pathway genes or gene families with high efficiency and low cost, and can detect and analyze edited seedlings, thereby bringing help and reference significance to the CRISPR/Cas9 technology in plant signal pathway research or gene family function research and screening.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for constructing a rice polygene editing mutant library and application thereof.
Background
The CRISPR/Cas9 system is an acquired immune system that is present in most bacteria and archaea, and consists of a series of short highly conserved direct repeats with spacers of similar length. Cas genes are a gene family, encoded proteins have the function of combining with nucleic acid and the activity of combining with nuclease, polymerase, helicase and the like, a CRISPR/Cas9 system has a series of Cas genes, the genes play different roles when a host responds to the invasion of exogenous genetic materials, and in different species, the types of the Cas genes are different and have abundant diversity, and a plurality of homologous but functionally unrelated Cas proteins can be expressed. The CRISPR/Cas9 technology, as a third-generation artificial endonuclease technology, is the simplest in a Cas system and mainly comprises a gene encoding a Cas protein, a short leader and a CRISPR locus consisting of a spacer sequence and a repeat sequence. The leader region is composed of a non-coding sequence rich in AT base and plays a role as CRISPR promoter. Although the mechanism of action is not yet fully understood, the process of action can be understood in three stages, first the acquisition of the CRISPR highly variable spacer, second the expression of the CRISPR locus, and third the exertion of the CRISPR/Cas9 system activity or interference with foreign genetic material.
Creating mutant libraries of different animals and plants is very important for high-throughput research of gene functions and creation of germplasm resources. At present, high-throughput large-scale genome editing is realized in animals and plants based on a CRISPR/Cas9 technology, site-directed knockout of tens of thousands or even hundreds of thousands of gene loci can be realized, and gene functions can be efficiently and rapidly researched. However, in the process of creating a plant mutation library, when different mutant individuals are identified, a generation sequencing technology (Sanger sequencing) is still used at present, the cost is very high, and the precise mutation types of the mutants cannot be obtained. On the other hand, in order to study a certain gene family or related pathway genes, it is necessary to knock out the gene family and related pathway genes to establish a small mutant library, but at present, there are few detailed technical data related thereto.
Therefore, the invention aims to develop a complete system method for constructing the rice polygene mutant library, and the method can efficiently obtain a large batch of gene editing seedlings and detect the mutation types of the gene editing seedlings at low cost.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for constructing a rice polygene editing mutant library and application thereof. The method for constructing the rice polygene mutant library based on the complete system realizes the purposes of efficiently obtaining large batches of gene editing seedlings and detecting the mutation types of the gene editing seedlings at low cost.
The invention aims to provide a method for constructing a rice polygene editing mutant library.
The method for constructing the rice multi-gene editing mutant library comprises the following steps:
s1, sgRNA library design: designing specific sgRNA sequences aiming at different functional genes of rice according to a rice genome sequence, and then adding a sequence complementary with the adhesive tail end of a CRISPR/Cas9 vector to prepare a sgRNA library;
s2, preparing CRISPR/Cas9 vector library: integrating the sgRNA prepared in the step S1 into a CRISPR/Cas9 vector, and forming a CRISPR/Cas9 vector library after the construction is correct by high-throughput sequencing verification;
s3, genetic transformation: transforming agrobacterium with the CRISPR/Cas9 vector prepared in the step S2, obtaining bacterial suspension after screening, identifying and culturing, transforming the bacterial suspension into rice callus, and obtaining transgenic positive plants through eukaryotic resistance screening;
s4, sgRNA identification: sequencing and analyzing the vector framework fragment of the positive seedlings obtained in the step S3, identifying sgRNA, and determining the type of the sgRNA corresponding to each seedling;
s5, detecting a gene editing seedling: based on the sgRNA sequence information of the positive seedlings determined in step S4, detection primers are designed in batches, PCR amplification and sequencing analysis are performed on the positive seedlings, and finally, detailed gene editing types and results are obtained.
Further, in step S1, the sgRNA sequence has a GC content of 40-80%, the target is located on the first exon as much as possible, and is at least 3 bases different from the potential off-target site, and 4 consecutive T bases are not allowed; adding a sequence complementary to the cohesive end of 4 bases formed by digestion of a CRISPR/Cas9 vector by Bsa I enzyme at both ends of the sgRNA sequence, adding a complementary sequence,
the forward primer sequence is: 5 '-TGCA-NNNNNNNNNNNNNNNNNNNN-3',
reverse primer sequence: 5 '-AAAC-NNNNNNNNNNNNNNNNNNN-3', N represents any base and is the designed specific sgRNA sequence.
Further, in step S2, in the CRISPR/Cas9 vector library construction process, the adopted gene editing vector is a binary vector, and the binary vector comprises a sgRNA expression cassette and a Cas9 expression cassette; and each plasmid in the CRISPR/Cas9 vector library expresses only 1 sgRNA.
Further, in step S2, the CRISPR/Cas9 vector is first digested with Bsa i enzyme, then mixed with the sgRNA library in step S1, and T4 ligase is added for enzymatic ligation, and finally escherichia coli is transformed, and a positive clone is obtained through resistance screening and identification, and the positive clone is subjected to mass culture, plasmid extraction, and the like to obtain a plasmid library.
Further, designing detection primers for plasmids in the plasmid library according to a vector framework sequence, wherein the sequences of the detection primers are shown as SEQ ID NO.1 and SEQ ID NO.2, amplifying a sequence containing an sgRNA interval by using the detection primers, and controlling the size of an amplification product to be 180-260 bp; and (3) sending the amplification product to high-throughput sequencing, and detecting the CRISPR/Cas9 vector library coverage.
Further, in step S3, the method for transforming agrobacterium with the plasmid vector is chemical transformation or electric shock transformation, and the concentration of the agrobacterium suspension is OD600：0.1～0.5。
Further, in step S4, the positive seedling genome obtained in step S3 is extracted, sequences shown as SEQ ID No.1 and SEQ ID No.2 are used to amplify the sequence containing the sgRNA region, and the size of the amplified product is controlled between 180-260 bp; and (3) sending the amplification product to a high-throughput sequencing system, and detecting the sgRNA coverage condition of each seedling.
Further, in step S5, based on the Primer design software Primer3, using the PREL language, the detection primers of each sgRNA are designed in batch, the size of the amplified target fragment is between 180 and 260bp, and the TM value range of the detection primers is: 50-62 ℃, the most preferable TM value is 58 ℃, and the length range of the detection primer is as follows: 18-25 bp, most preferably 20bp, wherein the GC content range of the detection primer is as follows: 40-68%, and the most preferable GC content of the primer is 50%; then, a barcode sequence and a sequencing joint are added to the target fragment by utilizing a multiplex PCR amplification technology, high-throughput sequencing is carried out, and the gene editing mutation condition of each positive seedling is detected.
The invention also aims to provide application of the method for constructing the rice polygene editing mutant library in constructing the Nippon polygene editing mutant library.
Compared with the prior art, the invention has the following advantages:
1) according to the invention, the CRISPR/Cas9 technology is adopted, the whole rice polygene editing mutant library is obtained at one time, compared with the existing one-by-one gene knockout, the efficiency is improved, and the workload is obviously reduced;
2) the invention utilizes a high-throughput detection method to detect positive Nipponbare plants, when the mixed number of sgRNAs is 8, 7 knockout genes are detected, and the editing efficiency is 87.5%; when the mixed number of the sgrnas is 100, 67 knockout genes are detected, and the editing efficiency is 67%; when the mixed number of the sgrnas is 200, 121 knockout genes are detected, and the editing efficiency is 60.5%;
3) the invention designs a complete, systematic and efficient plant multi-genome editing and detecting method, can accurately edit a large number of pathway genes or gene families with high efficiency and low cost, and can detect and analyze edited seedlings, thereby bringing help and reference significance to the CRISPR/Cas9 technology in plant signal pathway research or gene family function research and screening.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a schematic view of the experimental operation of the present invention;
FIG. 2 is a schematic diagram of the design of a CRISPR/Cas9 vector library coverage primer for vector construction and detection.
Detailed Description
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 it is obvious that the described embodiments are only a part of the embodiments of the present invention, and 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.
It should be noted that the reagents and equipment used in the present invention are generally commercially available unless otherwise specified.
Example 1sgRNA library design
According to the invention, Nipponbare is selected as an experimental material, and an Ensembl website is used for downloading a Nipponbare rice genome sequence (http:// plants. ensemblel. org/Oryza _ sativa/Info/Index) as a reference genome for designing sgRNA; then 8, 100 and 200 genes with different functions of the japonica fine rice are respectively selected, 8, 100 and 200 genes sgRNA with different functions of the japonica fine rice are respectively designed by using a compiled Perl script (also an sgRNA design tool published at home and abroad can be used), each gene selects 1sgRNA sequence, and the design principle of the sgRNA is shown in fig. 1, specifically: the GC content is between 40% and 80%, the target is located on the first exon as much as possible, at least 3 bases of difference exists between the potential off-target sites, and 4 continuous T bases are not allowed.
In addition, if the sgRNA sequence exceeds 1 ten thousand, the prior art synthesizes all single-stranded oligonucleotides in vitro using a chip synthesis method, which is advantageous in that the cost is reduced. However, small-scale library construction is carried out, only hundreds of sgRNAs are designed, so that a primer synthesis method is used, a sequence which is complementary to a sticky end of 4 bases formed by the CRISPR/Cas9 vector after being cut by Bsa I enzyme is added to a synthesized sgRNA fragment, and after the complementary sequence is added,
the forward primer sequence is: 5 '-TGCA-NNNNNNNNNNNNNNNNNNNN-3',
reverse primer sequence: 5 '-AAAC-NNNNNNNNNNNNNNNNNNN-3', N represents any base and is a designed specific sgRNA sequence; and synthesizing double-stranded sgRNA based on a primer annealing process, and then mixing the double-stranded sgRNA in an equimolar ratio to form a sgRNA library. The primer reaction system and annealing procedure are shown in Table 1 below.
TABLE 1 primer reaction System and annealing procedure
Example 2 preparation of CRISPR/Cas9 vector library
The CRISPR/Cas9 vector was cleaved enzymatically using Bsa i enzyme, and then the sgRNA library of example 1 was ligated with the linearized CRISPR/Cas9 vector. mu.L of the ligation product was transformed into E.coli competent cells (50. mu.L), which were then plated on a kanamycin-resistant plate, and after 12 hours of culture at 37 ℃, all E.coli clones were directly resuspended in PBS buffer (pH 7.2), and plasmids were extracted to obtain a CRISPR/Cas9 vector library. Utilizing vector framework primers (shown in SEQ ID NO.1 and SEQ ID NO. 2) to amplify a sequence containing a sgRNA interval, wherein the amplification size is between 180 and 260bp, sending the sequence to second-generation sequencing, wherein the specific flow is shown in FIG. 2, the coverage of the sgRNA is counted based on the second-generation sequencing result, and the result is shown in the following table 2:
table 2 sgRNA coverage
| Mixed number/number of sgRNAs | High throughput sequencing analysis detects number/number | Coverage degree |
| 8 | 8 | 100％ |
| 100 | 90 | 90％ |
| 200 | 192 | 96％ |
The results in Table 2 show that coverage can be substantially over 90%, and that coverage can completely satisfy the requirements for knockout of small libraries.
Example 3 genetic transformation
Adopting one of a chemical transformation method or an electric shock transformation method, wherein the chemical transformation method or the electric shock transformation method is an existing conventional transformation method, and details are not repeated herein, transforming the CRISPR/Cas9 vector library in the embodiment 2 into agrobacterium, and then coating the agrobacterium on a solid culture medium of kanamycin; after culturing for 2-3 days, scraping agrobacterium tumefaciens colonies, resuspending all agrobacterium tumefaciens bacterial liquid, infecting the rice callus, and diluting the bacterial liquid concentration of the infection liquid to OD600Is between 0.1 and 0.5, and the experiment is carried outThe concentration is 0.5, and a large amount of regeneration positive plants of the Nipponbare are obtained after tissue culture.
Example 4sgRNA identification
Identifying the sgRNA species of the transformed seedlings obtained in example 3, wherein the operation method is the same as that in example 2, a vector framework design primer (shown in SEQ ID No.1 and SEQ ID No. 2) is used to amplify a fragment containing the sgRNA interval, and the fragment is subjected to second-generation sequencing, wherein the specific flow is shown in fig. 2, and based on the second-generation sequencing result, the sgRNA species contained in each transformed seedling is identified and obtained, and the results are shown in the following table 3:
TABLE 3 proportion of sgRNA species contained in transformed seedlings
| Mixed number/number of sgRNAs | High throughput sequencing analysis detects number/number | Coverage degree |
| 8 | 8 | 100％ |
| 100 | 92 | 92％ |
| 200 | 187 | 94％ |
As is clear from the results in Table 3, the coverage can be substantially 90% or more, and the coverage obtained increases as the number of transformed seedlings detected increases.
Example 5 high-throughput design of detection primers and detection of Gene-edited seedlings
According to the method of the embodiment 4, after the sgRNA species contained in each positive seedling are identified, the positive seedling mutant is identified by adopting a second-generation high-throughput sequencing method; firstly, primers are designed by batch production using PERL programming language according to the position of each sgRNA on the genome, and the designed primer sequences are shown in the following table 4, wherein id represents the number of each sgRNA, LEFT represents a forward primer, and RIGHT represents a reverse primer. Respectively extracting the genome of each seedling according to the designed detection primer, carrying out PCR amplification, adding a barcode sequence and a high-throughput sequencing joint by utilizing a multiple PCR amplification technology, and sending to next-generation sequencing. The sequencing gene editing for each seedling was obtained as shown in table 5 below, where in the mutation types, M: represents a perfect match, D: represents a deletion, I: indicating an insertion.
TABLE 4 detection of knockout efficiency detection primers (partial display)
TABLE 5 high throughput sequencing test editing Miao results (partial display)
As can be seen from the results in Table 5, the editing condition of each mutant strain can be accurately obtained based on the method of the present invention, which provides great convenience for gene function research and germplasm resource creation.
Further, the edit efficiency statistics for the different blending gradients are shown in table 6 below:
TABLE 6 detection of editing efficiency of knockout genes
From the results in table 6, 3 positive seedlings of each sgRNA were selected for sequencing, the editing efficiency was detected, the editing efficiency was up to 60% or more, and most of the gene edited seedlings could be obtained by one transformation event, which greatly saved the cost.
Compared with the existing independent editing, the editing method of the invention has the following advantages:
1) vector construction: the existing method is constructed one by one, and has low working efficiency and large workload; the invention can obtain the whole library at one time, the efficiency is improved, and the workload is greatly reduced;
2) genetic transformation: the existing method is one-by-one conversion, and the cost is very high (calculated by 8 sgrnas, 3000 yuan is charged for each conversion (by the current commercial charge standard), and 2.4 ten thousand yuan is needed); the method has the advantages that the method can be realized by one-time conversion, the cost is 3000 yuan, and the cost is greatly reduced;
3) detection and analysis of gene editing seedlings: in the existing method, each sgRNA is detected independently and is detected and sequenced respectively, so that the personnel management cost is increased and the efficiency is reduced; according to the invention, positive seedlings with different sgRNAs can be distinguished by one-time high-throughput sequencing, and the efficiency is greatly improved.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
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<213> Artificial Sequence (Artificial Sequence)
<400> 38
acctccatcg ttttgaccct 20
<210> 39
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
tcatcaccct tagccttgca 20
<210> 40
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
actcctcttc ttgacccctt 20
<210> 41
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
ttgaggaagt agaggacgag 20
<210> 42
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
tgttctctgt ggaaggagct 20
<210> 43
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
tcaccaccac caccaactaa 20
<210> 44
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
tcctcgacgc agaagaagta 20
Claims (9)
1. A method for constructing a rice multi-gene editing mutant library is characterized by comprising the following steps:
s1, sgRNA library design: designing specific sgRNA sequences aiming at different functional genes of rice according to a rice genome sequence, and then adding a sequence complementary with the adhesive tail end of a CRISPR/Cas9 vector to prepare a sgRNA library;
s2, preparing CRISPR/Cas9 vector library: integrating the sgRNA prepared in the step S1 into a CRISPR/Cas9 vector, and forming a CRISPR/Cas9 vector library after the construction is correct by high-throughput sequencing verification;
s3, genetic transformation: transforming agrobacterium with the CRISPR/Cas9 vector prepared in the step S2, obtaining bacterial suspension after screening, identifying and culturing, transforming the bacterial suspension into rice callus, and obtaining transgenic positive plants through eukaryotic resistance screening;
s4, sgRNA identification: sequencing and analyzing the vector framework fragment of the positive seedlings obtained in the step S3, identifying sgRNA, and determining the type of the sgRNA corresponding to each seedling;
s5, detecting a gene editing seedling: based on the sgRNA sequence information of the positive seedlings determined in step S4, detection primers are designed in batches, PCR amplification and sequencing analysis are performed on the positive seedlings, and finally, detailed gene editing types and results are obtained.
2. The method for constructing a pool of rice polygene-editing mutants according to claim 1, wherein in step S1, the sgRNA sequence has a GC content of 40-80%, the target is located as far as possible on the first exon, and is at least 3 bases different from the potential off-target site, and 4 consecutive T bases are not allowed; adding a sequence complementary to the cohesive end of 4 bases formed by digestion of a CRISPR/Cas9 vector by Bsa I enzyme at both ends of the sgRNA sequence, adding a complementary sequence,
the forward primer sequence is: 5 '-TGCA-NNNNNNNNNNNNNNNNNNNN-3',
reverse primer sequence: 5 '-AAAC-NNNNNNNNNNNNNNNNNNN-3', N represents any base and is the designed specific sgRNA sequence.
3. The method for constructing a rice polygene editing mutant library as claimed in claim 1, wherein in step S2, the gene editing vector used in the construction of the CRISPR/Cas9 vector library is a binary vector, and the binary vector comprises a sgRNA expression cassette and a Cas9 expression cassette; and each plasmid in the CRISPR/Cas9 vector library expresses only 1 sgRNA.
4. The method for constructing a polygene editing mutant library of rice as claimed in claim 1, wherein in step S2, the CRISPR/Cas9 vector is first digested with Bsa i enzyme, then mixed with the sgRNA library in step S1, T4 ligase is added for enzymatic ligation, finally escherichia coli is transformed, positive clones are obtained through resistance screening and identification, and the plasmid library is obtained through the steps of mass culture, plasmid extraction and the like of the positive clones.
5. The method for constructing the rice polygene editing mutant library as claimed in claim 4, wherein the detection primers are designed according to the vector framework sequences of the plasmids in the plasmid library, the sequences of the detection primers are shown as SEQ ID No.1 and SEQ ID No.2, then the detection primers are used for amplifying the sequences containing the sgRNA intervals, and the size of the amplification products is controlled between 180-260 bp; and (3) sending the amplification product to high-throughput sequencing, and detecting the CRISPR/Cas9 vector library coverage.
6. The method for constructing a pool of rice multi-gene editing mutants according to claim 1, wherein in step S3The method for transforming agrobacterium by the plasmid vector is a chemical transformation method or an electric shock transformation method, and the concentration of the agrobacterium suspension is OD600：0.1～0.5。
7. The method for constructing a rice polygene editing mutant library as claimed in claim 1, wherein in step S4, the positive seedling genome obtained in step S3 is extracted first, the sequences comprising sgRNA intervals are amplified using the sequences shown as SEQ ID No.1 and SEQ ID No.2, and the size of the amplified product is controlled to be 180-260 bp; and (3) sending the amplification product to a high-throughput sequencing system, and detecting the sgRNA coverage condition of each seedling.
8. The method for constructing a pool of rice polygene editing mutants according to claim 1, wherein in step S5, the detection primers for each sgRNA are designed in batch based on Primer design software Primer3 using PREL language, the amplified target fragment size is 180-260bp, and the TM value of the detection primers is in the range: and the detection primer length ranges from 50 ℃ to 62 ℃: 18-25 bp, and the GC content range of the detection primer is as follows: 40-68%; then, a barcode sequence and a sequencing joint are added to the target fragment by utilizing a multiplex PCR amplification technology, high-throughput sequencing is carried out, and the gene editing mutation condition of each positive seedling is detected.
9. The method for constructing a rice polygene editing mutant library as claimed in any one of claims 1 to 8, wherein the method is applied to the construction of a Nipponbare polygene editing mutant library.
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