CN117568343A - sgRNA for targeted editing of PRLR gene and application thereof - Google Patents

sgRNA for targeted editing of PRLR gene and application thereof Download PDF

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CN117568343A
CN117568343A CN202311459090.6A CN202311459090A CN117568343A CN 117568343 A CN117568343 A CN 117568343A CN 202311459090 A CN202311459090 A CN 202311459090A CN 117568343 A CN117568343 A CN 117568343A
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prlr
sgrna
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高元鹏
经佳懿
张景程
张涌
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Northwest A&F University
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Abstract

The invention discloses sgRNA for targeted editing of PRLR genes and application thereof, and belongs to the technical field of animal genetic engineering. The invention discloses a sgRNA for targeted editing of PRLR genes, which comprises PRLR sgRNA139, PRLR sgRNA188 and PRLR sgRNA189. The sgRNA for targeted editing of the PRLR genes provided by the invention lays a foundation for subsequent determination of a cytosine base editing system and a targeting site with better PRLR gene editing efficiency, obtaining somatic cell nuclear transfer donor cells cultivated by PRLR gene editing heat-resistant stress cows and bovine in-vitro fertilization microinjection embryos, and finally provides technical support for breeding of heat-resistant bovine varieties in China.

Description

sgRNA for targeted editing of PRLR gene and application thereof
Technical Field
The invention relates to the technical field of animal genetic engineering, in particular to a targeting editing PRLR gene sgRNA and application thereof, and in particular relates to a targeting single base gene editing bovine prolactin receptor gene sgRNA.
Background
The natural mutation of the prolactin receptor (prolactin receptor, PRLR) gene in a certain sequence region of the genome leads to the truncation of the C-terminal region of the prolactin receptor protein (PRLR protein) and does not affect the SLICK phenotype generated by other normal physiological functions, and the SLICK phenotype dairy cows can show excellent temperature regulation capability when facing heat stress, and are not easy to produce the decline of milk production and other production performance, immunity and reproductive capacity under the heat stress environment.
Therefore, it is a urgent need for a person skilled in the art to provide a sgRNA for targeted editing of PRLR gene and its application.
Disclosure of Invention
In view of the above, the invention provides a sgRNA for targeted editing of PRLR genes and application thereof, provides a gene editing target locus sgRNA which can be obtained and has SLICK character genotypes, and provides a gene editing locus for breeding of heat-resistant cattle.
Ext> theext> inventionext> providesext> aext> targetingext> geneext> forext> editingext> sgRNAext> ofext> PRLRext> geneext>,ext> whichext> realizesext> singleext> baseext> Cext> -ext> Text> orext> Gext> -ext> Aext> substitutionext> (ext> Cext>:ext> Gext>:ext> Text>:ext> Aext> orext> Gext>:ext> Cext>:ext> Aext>:ext> Text> transitionext>)ext> ofext> theext> PRLRext> geneext> byext> usingext> aext> cytosineext> baseext> editorext>.ext>
In order to achieve the above purpose, the present invention adopts the following technical scheme:
an sgRNA for targeted editing of PRLR gene,
the sgrnas include PRLR sgRNA139, PRLR sgRNA188, PRLR sgRNA189;
the nucleotide sequence of the PRLR sgRNA139 is shown as SEQ ID NO.1;
the nucleotide sequence of the PRLR sgRNA188 is shown as SEQ ID NO.2;
the nucleotide sequence of the PRLR sgRNA189 is shown in SEQ ID NO. 3.
Further, an expression vector for targeted editing of sgrnas of PRLR genes, comprising a nucleotide sequence encoding the sgrnas.
Further, the sgRNA or the expression vector is applied to breeding heat-resistant cattle.
Compared with the prior art, the invention discloses the sgRNA for targeted editing of the PRLR gene and the application thereof, which lays a foundation for subsequent determination of a cytosine base editing system and a targeting site with better PRLR gene editing efficiency, obtaining somatic cell nuclear transfer donor cells cultured by PRLR gene editing heat-resistant stress cows and bovine in-vitro fertilization microinjection embryos, and finally provides technical support for breeding of heat-resistant varieties of cattle in China.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 shows 3 sgRNA sequences corresponding to the present invention;
FIG. 2 shows SSA test results according to the invention;
NC was used as a negative control (transfection of recombinant SSA reporter SSA-1-3-sgRNA and 0.025. Mu.g Renilla luciferase reporter PRL-TK alone), and each of the remaining experimental groups was subjected to T-test with NC group, error bars represent SEM; * P <0.01, p <0.001, p <0.0001;
FIG. 3 shows the result of agarose gel electrophoresis for identifying the cleavage activity of sgRNA by T7E1 cleavage according to the present invention;
wherein, the sizes of two bands generated by the enzyme digestion of the 139 lanes are 520bp+270bp respectively; lanes 188 and 189, the size of the two bands produced by the digestion is 570bp+220bp; m: trans DNA Marker II; NC: transfection of Cas9 expression vector without insertion of sgrnas;
FIG. 4 shows the editing efficiency of the target base gene of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
1) PRLR gene editing sgRNA site prediction design
According to the http:// www.rgenome.net/be-designer/, a 240bp sequence (SEQ ID NO. 4) of a ninth exon part of the PRLR gene is used as a screening area of a PRLR gene editing site, a common primordial spacer sequence adjacent motif (Protospacer Adjacent Motif, PAM) NGG is selected, and three sgRNA sequences meeting the editing purpose are designed and obtained, wherein the three sgRNA sequences comprise PRLR sgRNA139, PRLR sgRNA188 and PRLR sgRNA189 (figure 1); the nucleotide sequence of the PRLR sgRNA139 is shown as SEQ ID NO.1, the nucleotide sequence of the PRLR sgRNA188 is shown as SEQ ID NO.2, and the nucleotide sequence of the PRLR sgRNA189 is shown as SEQ ID NO. 3.
PRLR sgRNA139:5'-ACCAAGCAGAGGGAGTCAGA-3'; SEQ ID NO.1; wherein the 3' end is provided with AGG as PAM sequence;
PRLR sgRNA188:5'-GACCAAGACACGGTGTGGCC-3'; SEQ ID NO.2; wherein, the 5' end is provided with CCT as PAM sequence;
PRLR sgRNA189:5'-AGACACGGTGTGGCCACGAC-3'; SEQ ID NO.3; wherein the 5' end carries CCA as PAM sequence.
TACCACAACATTGCTGACGTGTGTGAGCTGGCCCTGGGCATGGCCGGCACCACAGCCACTTCGCTGGACCAAACAGACCAACATGCTTTAAAAGCCTCAAAAACCATTGAAACTGGCAGGGAAGGAAAGGCAACCAAGCAGAGGGAGTCAGAAGGCTGCAGTTCCAAGCCTGACCAAGACACGGTGTGGCCACGACCCCAAGACAAAACCCCCTTGATCTCTGCTAAACCCTTGGAATAC；SEQ ID NO.4。
2) CRISPR/Cas9 eukaryotic expression vector construction
The experiment selects pSpCas9 (BB) -2A-Puro (PX 459, plasmid # 48139) vector and pSpCas9 (BB) -2A-GFP (PX 458, plasmid # 48138) vector as targeting vector. For the three sgRNA sequences designed, complementary target sequences with linkers were designed, see table 1. Then, after an annealing procedure (95 ℃ C., 10 min), the mixture was taken out and cooled to room temperature to form a double strand, and the reaction system was shown in Table 2. The linearized vector pSpCas9 (BB) -2A-Puro/GFP, after cleavage by BbsI (37 ℃,2 h), was recovered (cleavage reaction system, see Table 3), and ligated with annealed double-stranded sgRNA139, sgRNA188, sgRNA-189, respectively, using T4 DNA ligase (overnight ligation at 4 ℃ C. Or 16 ℃ C., 2 h), the ligation system was shown in Table 4. The ligation products were transformed into DH 5. Alpha. Competent cells, then monoclonal colonies were picked for Sanger sequencing, and after verification of correct sequences 6 positive plasmids (pSpCas 9 (BB) -2A-Puro-sgRNA, pSpCas9 (BB) -2A-GFP-sgRNA) were extracted and stored at-20℃for subsequent experiments.
TABLE 1 sgRNA cloning primer sequences
TABLE 2 sgRNA annealing System
sgRNA Volume (mu L)
sgRNA Top Guide Oligo 5μL(100μM)
sgRNA Bottom Guide Oligo 5μL(100μM)
TABLE 3 pSpCas9 (BB) -2A-Puro/GFP cleavage reaction System
Reagent name Amount of reagent
pSpCas9(BB)-2A-Puro/GFP 2μg
BbsⅠ-HF 1μL
10×rCutSmart Buffer 5μL
ddH 2 O To50μL
Table 4 connection system
3) Construction of SSA reporter vectors
pGL3-Control was used as a template, and a portion of the Luciferase fragment having EcoRI cleavage site and targeting site sequences was amplified using PCR primers RvP-f and SSA-1-3-sgRNA-r reverse primer (see Table 5 for PCR reaction system) to replace the sequence between BglII and EcoRI double cleavage sites of pSSA-1-3 Plasmid (Plasmid # 35091), and transformed into DH 5. Alpha. Competent cells after cleavage ligation. After the plasmid is successfully constructed, the primers LucRep-f and LucRep-r are used for PCR amplification (the PCR reaction system is shown in Table 6), and the SSA report carrier SSA-1-3-sgRNA required to be constructed is obtained after the sequencing and identification of the products are correct.
RvP3-f and SSA-1-3-sgRNA-r reverse primer sequences were as follows:
RvP3-f：5’-CTAGCAAAATAGGCTGTCCC-3’；SEQ ID NO.11；
SSA-1-3-sgRNA-139-r：
5’-CTCGAATTCCCCCTTCTGACTCCCTCTGCTTGGTctcacataggacctctca cacacag-3’；SEQ ID NO.12；
SSA-1-3-sgRNA-188-r：
5’-CTCGAATTCCGGCCACACCGTGTCTTGGTCAGGctcacataggacctctca cacacag-3’；SEQ ID NO.13；
SSA-1-3-sgRNA-189-r：
5’-CTCGAATTCCGTCGTGGCCACACCGTGTCTTGGGctcacataggacctct cacacacag-3’；SEQ ID NO.14。
the primer sequences for LucRep-f and LucRep-r are as follows:
LucRep-f：5’-CGAAGGTTGTGGATCTGGATACC-3’；SEQ ID NO.15；
LucRep-r：5’-TAGCTGATGTAGTCTCAGTGAGC-3’；SEQ ID NO.16。
TABLE 5 PCR reaction System for amplifying partial Luciferase fragment with target site sequence
PCR reaction conditions of Table 5: 98 ℃ for 5min;98 ℃ for 10s,60 ℃ for 10s,72 ℃ for 30s,33 cycles; 5-10min at 72 ℃; preserving at 4 ℃.
Table 6 identification of PCR reaction System
Reagent name Amount of reagent
SSA reporter vector plasmid 1μL(30ng/μL)
LucRep-f 1μLof10μM
LucRep-r 1μLof10μM
2×Taq polymerase mix 25μL
ddH 2 O 22μL
The PCR conditions of Table 6 were as follows: 3min at 95 ℃;95℃30s,58℃30s,72℃1min,33 cycles; 5-10min at 72 ℃; preserving at 12 ℃.
4) SSA (Single Strand Annealing) -Dual fluorescence reporter assay System for detecting sgRNA Activity
0.75 μg of the recombinant CRISPR/Cas9 eukaryotic expression vector pSpCas9 (BB) -2A-Puro-sgRNA,0.225 μg of the recombinant SSA reporter SSA-1-3-sgRNA and 0.025 μg of the renilla luciferase reporter PRL-TK were co-transfected into 293T cells with a confluency of about 60% -70% in 24 well plates using PEI transfection reagents;
after 48h of transfection, a double fluorescence reporting system kit (TransDouble-Luciferase Reporter Assay Kit, FR 201-01-V2) detection, firefly luciferase reporter to Renilla luciferase reporter ratio indicates that sgRNA directs the cleavage activity of Cas9, and the results are shown in FIG. 2.
Results: the ratio of firefly luciferase reporter gene to Renilla luciferase reporter gene was the greatest in the transfected with sgRNA-139 test group, with sgRNA189 centered and sgRNA188 lowest, but the ratios were all significantly higher in the three test groups than in the control group.
SSA results showed that the relative activity of all three sgRNA treated groups was significantly higher than that of the negative control group, the three experimental groups: the result of sgRNA-139 > sgRNA-189 > sgRNA-188 indicates that the cleavage activity of the sgRNA-139 for Cas9 is highest, followed by the sgRNA-189, and the sgRNA-188 is lowest.
5) Mismatch endonuclease (T7 endonucleolytic i, T7EN 1) assay to detect the cleavage activity of sgRNA-guided Cas9
By means of3000Reagent transfection Reagent 8.4. Mu.g of CRISPR/Cas9 targeting vector pSpCas9 (BB) -2A-GFP-sgRNA was transfected into bovine mammary epithelial cells (MAC-T) with cell confluency of over 80% in 6cm dishes, GFP positive cells were flow sorted and recovered 72h after transfection, and clone lysates (1 mL of preparation: 40mM TrisHCl,0.4mg/mL,0.9% Triton X-100,0.9% NP-40, solvent ddH) were used 2 0) Cell lysis is carried out to obtain a whole genome of the cell serving as a PCR template, and the clone lysis reaction conditions are as follows: 15min at 65 ℃,10min at 95 ℃ and hold at 16 ℃; cell lysates were used as PCR templates.
The primers T7EN1-PRLR-amplicon-F and T7EN1-PRLR-amplicon-R are used for amplifying sequences containing target sites, agarose gel nucleic acid electrophoresis is carried out after the PCR products are digested by the T7EN1, and the proportion of the contained indels heterozygous double-stranded DNA can be calculated by analyzing the gray value, so that the cleavage activity of Cas9 nuclease guided by three sgRNAs is semi-quantitatively determined, and the result is shown in figure 3.
The T7EN1-PRLR-amplicon-F and T7EN1-PRLR-amplicon-R primer sequences were as follows:
T7EN1-PRLR-amplicon-F：5’-GACTGCGAGGACTTGCTGAT-3’；SEQ ID NO.17；
T7EN1-PRLR-amplicon-R：5’-ACAGAGTCAGGTTTTGCGCT-3’；SEQ ID NO.18。
the results of fig. 3 show that: indels rate and cleavage efficiency were both sgRNA139 > sgRNA188 > sgRNA189.
The results indicated that the cleavage activity of Cas9 guided by sgRNA-139 was highest, consistent with the SSA results, but the results of the T7EN1 detection showed that the cleavage activity of sgRNA-188, which was higher than that of sgRNA-189, was opposite to that of sgRNA-188 and sgRNA-189. This difference may be due to differences in transfected cells, which require further verification.
Example 2 cytosine base editor edit efficiency detection
1) Construction of sgRNA expression vector
In the test, pGL-U6-sgRNA-PGK-puromycin (Plasmid # 51133) vector with puromycin screening marker is selected as an sgRNA expression vector, an sgRNA primer sequence with BsI restriction enzyme site is designed for the sgRNA sequence (see table 7), and double-stranded sgRNA with BsI restriction enzyme site synthesized after annealing procedure is connected with linearized Plasmid after BsI endonuclease. And then the connection product is transformed into DH5 alpha competent cells, and positive plasmids are extracted after the correct sequence is verified.
TABLE 7sgRNA primer sequences of the sgRNA expression vectors
2) Amplicon sequencing to detect CBE editing efficiency on different locus sgRNAs
Using a program available from Invitrogen corporation of America3000Reagent transfection kit 700ng of sgRNA expression vector and 1.4. Mu.g of Cytosine Base Editor (CBE) - - - -pCMV-AncBE4max were transfected into MAC-T cells with a cell confluency of about 60% in 12-well plates. After 48h of transfection, medium DF12 (12% FBS) of puromycin at a concentration of 2. Mu.g/mL was added, and cells were recovered after 24h of drug screening. And obtaining the whole genome of the cell through cloning lysate.
Target sites are amplified by using PRLR-amplicon-F2/R2 primers with barcode sequences (see Table 8), PCR products are recovered and sent to Beijing-NodeB biogenetic biotechnology Co., ltd for amplicon sequencing, and the data are analyzed by using an http:// crispresso.
The PRLR-amplicon-F2/R2 primer sequences were as follows:
PRLR-amplicon-F2：5’-CCACAACATTGCTGACGTGT-3’；SEQ ID NO.25；
PRLR-amplicon-R2：5’-TACTCCTTGCTGGCTTCAGG-3’；SEQ ID NO.26。
TABLE 8 PRLR-amplicon-F2/R2 primer sequences with barcode sequences
Results: after amplicon sequencing, the editing efficiency of the sgRNA-139 target site is 3.09%, the editing efficiency of the sgRNA-188 target site is 2.54% and the editing efficiency of the sgRNA-189 target site is 0.31% under the same CBE-pCMV-AncBE 4max editing. The highest gene editing efficiency of the sgRNA-139 target site is consistent with the SSA result and the T7EN1 result, and the editing efficiency of the sgRNA-188 target site is higher than that of the sgRNA-189 site and is consistent with the T7EN1 result.
Conclusion(s)
Thus, the highest editing efficiency of the sgRNA139 site was demonstrated in different cells and assays, and the comparison of the results of the sgRNA189 and the sgRNA189 sites was not consistent in the different assays, i.e., the endogenous gene cleavage activity (T7 EN1 assay) and the exogenous DNA cleavage activity (SSA assay). The editing efficiency of the sgRNA188 site is higher than that of the sgRNA189 site in bovine mammary epithelial cells, whereas in human 293-T cells, the sgRNA 188-directed Cas9 cleavage activity is lower than that of the sgRNA 189-directed Cas9 cleavage activity.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (3)

1. A sgRNA for targeted editing of PRLR gene, which is characterized in that,
the sgrnas include PRLR sgRNA139, PRLR sgRNA188, PRLR sgRNA189;
the nucleotide sequence of the PRLR sgRNA139 is shown as SEQ ID NO.1;
the nucleotide sequence of the PRLR sgRNA188 is shown as SEQ ID NO.2;
the nucleotide sequence of the PRLR sgRNA189 is shown in SEQ ID NO. 3.
2. An expression vector for targeted editing of sgrnas of PRLR genes, comprising a nucleotide sequence encoding the sgrnas of claim 1.
3. Use of the sgRNA of claim 1 or the expression vector of claim 2 for breeding heat-resistant cattle.
CN202311459090.6A 2023-11-03 2023-11-03 sgRNA for targeted editing of PRLR gene and application thereof Pending CN117568343A (en)

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