CN108384812B - A yeast genome editing vector and its construction method and application - Google Patents

Abstract

The invention discloses a kind of Yeast genome editor carrier and its construction method and applications, it is the carrier that sets out with gRNA encoding plasmids P, the LoxP sequence of 34bp in the same direction is introduced respectively at the both ends replication origin Ori of the carrier, and Cre recombinase expression casette is introduced in plasmid P frame position, obtain Yeast genome editor carrier.Application of the carrier in the gene editing using CRISPR/Cas9 as medium is able to achieve efficient, more wheels editor to saccharomyces cerevisiae genome.

Description

A yeast genome editing vector and its construction method and application

technical field

The present invention relates to a method for genome editing of Saccharomyces cerevisiae and its application. This editing method utilizes a universal vector that can achieve self-loss, combined with the CRISPR/Cas9 system, to achieve efficient and convenient editing of the Saccharomyces cerevisiae genome.

Background technique

With the rapid development of molecular biology, a variety of genome editing technologies that can achieve precise editing of the genome of organisms have emerged one after another. This technology mainly uses a sequence-specific endonuclease (SSN) composed of a sequence-specific DNA-binding domain and a non-specific DNA-modifying domain to perform directional cleavage of double-stranded DNA at a specific location in the genome. And then activate the cell's own repair function to achieve targeted transformation such as gene knockout, site-directed insertion or replacement.

At present, gene editing technologies mainly include: zinc finger nucleases (zincfinger nucleases, ZFNs), transcription activator-like effector nucleases (transcription activator-like effector nucleases, TALEN) systems, and regularly clustered short palindromic repeats (clustered repeats). regularly interspaced short palindromicrepeats, CRISPR)/CRISPR-associated proteins (Cas) system, Argonaute protein (Natronobacterium gregoryi Argonaute, NgAgo) nuclease and structure-guided endonuclease (structure-guided) Nuclease, SGN) system, etc. Among them, the most widely used in recent years is the CRISPR/Cas9 system.

The CRISPR-Cas system is an adaptive immune defense mechanism formed during the long-term evolution of bacteria, which enables bacteria to resist the invasion of foreign DNA through the RNA-mediated DNA splicing mechanism. Currently, the CRISPR/Cas system can be divided into type I, type II and type III according to the different gene sequences of Cas proteins. Compared with type I and type III, the mechanism of action of the type II CRISPR system is simpler. In addition to crRNA and tracrRNA, the action process requires only one Cas9 protein, which is easy to design in vitro. The artificial nuclease system—CRISPR/Cas9 system.

In 2013, FengZhang's laboratory applied the CRISPR/Cas9 system to perform site-directed mutagenesis in eukaryotic cells for the first time, and formally developed the system as a new gene editing technology: that is, under the guidance of gRNA, the Cas9 protein recognizes target gene loci and It is specifically cut to form a double-strand break (DSB) gap, thereby triggering the cell's own repair mechanism to perform non-homologous end joining (NHEJ) or homologous recombination at the DSB site. Repair (Homology-directed repair, HR), to achieve the purpose of efficient genome editing (Gilbert, L.A., Larson, M.H., Morsut, L. et al, CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell, 2013, 154, 442- 451.). However, the current CRISPR/Cas9 system still has certain defects. For example, when the genome sequence of more than 5 sites is edited simultaneously, the positive clone rate is less than 60%. And because the gRNA encoding vector is transferred into the edited cell, the cell cannot perform the second genome editing due to the presence of the selectable marker.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a yeast genome editing method using CRISPR/Cas9 as the core, the genome editing technology solves the problem that the general CRISPR/Cas9 technology has low efficiency of simultaneous editing at multiple sites, and strains whose genomes have been modified cannot undergo secondary editing. editing and applying this method to the editing modification of the Saccharomyces cerevisiae genome.

The purpose of the present invention is to achieve the recovery of the selection marker by artificially controllable loss of the gRNA encoding vector, thereby facilitating a new round of gRNA encoding vector introduction and efficient genome editing. This method effectively solves the bottleneck problem of low positive clone rate of multi-site genome editing by decomposing multi-site genome editing into multiple rounds of genome editing. Specifically, the present invention is achieved through the following technical solutions:

A method for constructing a yeast genome editing vector, using gRNA encoding plasmid P as a starting vector, introducing 34bp LoxP sequences in the same direction at both ends of Ori, the origin of replication of the vector, and introducing Cre recombination at the position of the plasmid P backbone (backbone) Enzyme (Cyclization Recombination Enzyme) gene expression cassette to obtain yeast genome editing vector (P-CL vector).

The LoxP site is a gene sequence of 34 bp in size, including two 13 bp inverted repeats separated by 8 bp. Preferably, the LoxP sequence is shown in SEQ ID NO:1.

Preferably, the Cre recombinase gene has the nucleotide sequence shown in SEQ ID NO:2.

Preferably, the starting vector is P426 (commercialized plasmid p426-SNR52p-gRNA.CAN1.Y-SUP4t of Addgene Company), and the Cre recombinase expression cassette is inserted into the KpnI site of P426.

Preferably, the promoter in the expression cassette is a galactose-induced GAL1 promoter, and the terminator is a CYC1 transcription terminator.

The application of the P-CL vector constructed by the above method in gene editing mediated by CRISPR/Cas9, under the mediation of the gRNA encoded by the P-CL vector, after the yeast genome editing is completed, the expression of Cre recombinase is induced, and Cre recognizes Ori two. flanking the LoxP sequence, delete the Ori site on the P-CL vector. The vector that has lost the origin of replication cannot replicate normally and is eventually lost during yeast cell division, which means that the P-CL selectable marker is lost, and efficient recovery of the selectable marker is achieved. Specifically include the following steps:

1) Starting the vector with the constructed yeast genome editing vector, constructing an expression vector containing the targeting gRNA gene sequence;

2) introducing the gRNA expression vector obtained in step 1) into Saccharomyces cerevisiae containing the corresponding Cas9 system, obtaining a recombinant Saccharomyces cerevisiae, and realizing genome editing under the guidance of the CRISPR/Cas9 system;

3) After the genome editing is completed, the recombinant Saccharomyces cerevisiae is placed in a galactose medium to induce culture, block the self-replication of the vector, and realize the loss of the passaged plasmid;

4) Screen the recombinant Saccharomyces cerevisiae with lost plasmid, and introduce a new gRNA expression vector for the next round of genome editing.

Preferably, steps 3) and 4) are repeated to achieve multiple rounds of genome editing.

Preferably, in step 3), the conditions for the induction and culture of the recombinant Saccharomyces cerevisiae in galactose medium are: temperature 25-32° C., time 36-42 h, and rotating speed 150-300 rpm.

Preferably, the Saccharomyces cerevisiae is S. cerevisiae BJ5464 strain.

The Cas9 system is preferably the commercial plasmid p414-TEF1p-Cas9-CYC1t of Addgene Company.

Compared with the prior art, the present invention has the following beneficial effects:

In the present invention, the Cre/LoxP system is introduced into the gRNA expression vector, so that the plasmid expresses Cre recombinase under the induction of galactose and cuts off the Ori sequence after completing the knockout work of the gene, so as to realize self-loss (the Ori sequence is the starting point of the replication of the plasmid). Initiation sites, including genes that express RNAs and proteins necessary for replication encoded by plasmids, such as the lack of Ori sequences, cannot replicate in cells) to achieve the recovery of genetic markers. The results showed that the homologous LoxP sequences were introduced at both ends of Ori, the origin of replication of plasmid P426. Under the action of recombinase Cre, the plasmid loss rate exceeded 99.9%, thereby completing the recovery of the selection marker of P426 vector. Further, another gRNA expression vector can be introduced into the genome-edited engineered bacterial cells for genome editing again, thereby achieving high-efficiency (positive rate close to 100%) and multiple rounds (unrestricted) genome editing.

Description of drawings

Figure 1 is the P426-CL plasmid map.

Detailed ways

The methods used in the following examples are conventional molecular cloning methods unless otherwise specified.

Example 1 Construction of universal vector for yeast genome editing

Take the commercialized plasmid p426-SNR52p-gRNA.CAN1.Y-SUP4t (referred to as P426) of Addgene Company as the starting vector, and introduce the same direction LoxP sequence (SEQ ID NO: 1) at both ends of the Ori site of the vector, and in P426 The KpnI site was integrated into the complete recombinase Cre expression cassette. The specific implementation is as follows:

1. Introduction of LoxP sequences at both ends of Ori at the origin of replication

Using the P426 vector as a template, the LoxP1 fragment was obtained by amplifying the primer pair P1(5'-TCGTATAGCATACATTATACGAAGTTATACTTATATGCGTCTATTTATGT AG-3')/P2(5'-GTATAGCATACATTATACGAAGTTATTCCCCGAAAAGTGCCACCTGA-3'). II recombinant cloning kit (Nanjing Novizan Biotechnology Co., Ltd.), the plasmid P426-LoxP1 was obtained by circularizing LoxP1 by self-recombination and splicing.

Further, using P426-LoxP1 as a template, using P3(5'-TCGTATAATGTATGCTATACGAAGTTATAATGTGCGCGGAACCCCTAT-3')/P4(5'-GTATAATGTATGCTATACGAAGTTATACGCATTTAAGCATAAACAC-3') primer pair to amplify to obtain LoxP2 fragment. II Recombination Cloning Kit The self-recombination splicing of LoxP2 is circularized to obtain the vector P426-LoxP that introduces the same direction LoxP sequence at both ends of the origin of replication Ori.

2. Construction of Recombinase Cre Expression Cassette

The recombinase Cre gene (SEQ ID NO: 2) was synthesized in TaKaRa company, inserted into the pUC57 vector, marked as pUC57-Cre, and SacI and SmaI restriction sites were respectively introduced upstream and downstream of the enzyme encoding gene. Saccharomyces cerevisiae expression vector pYES2 containing promoter GAL1 and terminator CYC1 was purchased from Invitrogen.

Using pYES2 as the template, the primer pair P5(5'-GTTACCGAATTCTTTACCGTCGACAGTACGGATTAGAAGCCGCC-3')/P6(5'-GGTAACGAGCTCGGTACCAAG CTTAATATTC-3') was used to amplify the promoter GAL1 fragment, and EcoRI and SacI restriction sites were introduced upstream and downstream of the GAL1 fragment, respectively. , YEp352 and promoter GAL1 fragments were digested with EcoRI and SacI respectively, and YEp352-P _GAL1 containing promoter GAL1 was obtained by conventional digestion, ligation and transformation.

Using pYES2 as the template, the primer pair P7(5′-TCGCTCTCTAGAGGGCCGCATCATGTAATTAGTTA-3′)/P8(5′-GTTACCCTGCAGTTTACCCTCGAGGCAAATTAAAGCCTTCGAGCGT-3′) was used to amplify the terminator CYC1 fragment, and XbaI and PstI restriction sites were introduced upstream and downstream of the CYC1 fragment, respectively. The YEp352-P _GAL1 and terminator CYC1 fragments were digested with XbaI and PstI respectively, and YEp352-P _GAL1 -T _CYC1 containing the terminator CYC1 was obtained by conventional digestion, ligation and transformation.

Using pUC57-Cre as template, P9(5′-GTTACCGAGCTCGTTACCGGATCCATGAGTAACCTGCTGACAGTG-3′)/P10(5′-GAGCGATCTAGAGAGCGACCCGGGTTAATCCCCATCTTCTAATAACC-3′) primer pair was used to amplify the recombinase Cre fragment, and pUC57-Cre and YEp352 were digested with SacI and SmaI, respectively. -P _GAL1 -T _CYC1 vector, through conventional digestion, ligation and transformation, to obtain YEp352-P _GAL1 -Cre-T _CYC1 containing the recombinase Cre expression cassette. The expression of recombinase Cre is regulated by the promoter GAL1.

3. Construction of yeast genome editing universal vector p426-CL

Using YEp352-P _GAL1 -Cre-T _CYC1 as a template, the P11(5'-CTTTAATTTGCGGCCGGTACCAGTACGGATTAGAAGCC-3')/P12(5'-CTATAGGGCGAATTGGGTACCGCAAATTAAAGCCTTCGAG-3') primer pair was used to amplify the Cre expression cassette fragment P _GAL1 -Cre-T _CYC1 .

use II Recombination Cloning Kit The PCR product gene fragment P _GAL1 -Cre-T _CYC1 and KpnI linearized P426-LoxP homologous recombination to obtain yeast genome editing universal vector p426-CL. Example 2 Genome of Saccharomyces cerevisiae Geranylgeranyl pyrophosphate synthase (BTS1) and Heme-dependent repressor of hypoxic genes (ROX1) gene knockout

The vector P426-CL constructed in Example 1 was used as the gRNA expression vector to construct the backbone to achieve the knockout of the BTS1 and ROX1 genes in the yeast genome.

1. PCR amplification of bts1-gRNA and rox1-gRNA expression vector fragments

Using P426-CL as template, P13(5′-TAATAATGGTTTCTTAGTATGA-3′)/P14(5′-AACAGGATCATTATTGATCAGATCATTTATCTTTCACTGC-3′) and P15(5′-TGATCAATAATGATCCTGTTGTTTTAGAGCTAGAAAATA-3′)/P16(5′-ACTAAGAAACCATTATTATCAT-3′) ') is a primer pair, respectively amplified to obtain BTS1-1 and BTS1-2 fragments.

Using P426-CL as the template and P13/P17 (5′-TTCGTCTATTAAGATCCTGTGATCATTTATCTTTCACTG-3′) and P16/P18 (5′-ACAGGATCTTAATAGACGAAGTTTTTAGAGCTAGAAATA-3′)/P16 as primer pairs, ROX1-1 and ROX1- 2 Fragments.

2. Construction of recombinant vector containing BTS1 guide RNA sequence

use II Recombination cloning kit, recombination of BTS1-1 and BTS1-2 fragments to obtain the recombinant vector P426-CL-BTS1 carrying the BTS1 guide RNA sequence.

3. Construction of recombinant vector containing ROX1 guide RNA sequence

use II Recombination Cloning Kit, the two fragments of ROX1-1 and ROX1-2 were recombined to obtain the recombinant vector P426-CL-ROX1 carrying the ROX1 guide RNA sequence.

4. PCR amplification of bts1-rox1-gRNA expression vector fragment

Using P426-CL-BTS1 as a template and P13/P19 (5'-CTAATTACATGACTCGA-3') as a primer pair, a BTS1 fragment containing the bts1 target sequence was obtained by amplification. Using P426-CL-ROX1 as a template and P16/P20 (5′-TCGAGTCATGTAATTAGTCTTTGAAAAGATAATGTATGA-3′)/P16 as a primer pair, the ROX1 fragment containing the rox1 targeting sequence was amplified.

5. Construction of recombinant vector containing both BTS1 guide RNA sequence and ROX1 guide RNA sequence

use II Recombination cloning kit, recombination of BTS1 and ROX1 fragments to obtain the recombinant vector P426-CL-BTS1-ROX1 carrying the BTS1 guide RNA sequence and the ROX1 guide RNA sequence.

6. Yeast genome BTS1 and ROX1 editing validation

The P426-CL-BTS1-ROX1 and p414-TEF1p-Cas9-CYC1t (Addgene) plasmids were simultaneously transferred into the host S. cerevisiae BJ5464 competent cells. With the assistance of Cas9 protein, the BTS1 gene gRNA and ROX1 gene gRNA sequence The BTS1 gene and the ROX1 gene in the yeast genome were knocked out respectively, and the mutant strain S.cerevisiae BJ5464ΔBTS1ΔROX1/P426-CL-BTS1-ROX1 was obtained.

10 Saccharomyces cerevisiae transformants were randomly selected and treated with P21(5'-ATGGAGGCCAAGATAGATGAGC-3')/P22(5'-ATGAAAGGAACCAACGAATGGC-3') and P23(5'-TCCTAAATCCTCTACACCTAAG-3')/P24(5'-ATCATGCGTCAAAGGTAGTCCA- 3') The primer pair is used for colony PCR amplification, and the PCR product is sent for testing. The sequencing results showed that the BTS1 and ROX1 sites of the target genes of 10 transformants were mutated, and the positive mutation rate was 10/10. The results showed that the introduction of the LoxP sequences at both ends of the replication origin and the Cre expression cassette did not affect the editing ability of the yeast genome by the CRISPR/Cas9 system.

Example 3 Saccharomyces cerevisiae S. cerevisiae BJ5464ΔBTS1ΔROX1 genome editing of ERG9 gene, ypl062w gene and yjl064w gene

Using the P426-CL constructed in Example 1 as a template as a gRNA expression vector to construct a backbone, the editing of the yeast genome ERG9 gene, ypl062w gene and yj1064w gene was realized.

1. Loss of S. cerevisiae BJ5464ΔBTS1ΔROX1/P426-CL-BTS1-ROX1 intracellular vector P426-CL-BTS1-ROX1

Pick a single colony of S.cerevisiae BJ5464ΔBTS1ΔROX1/P426-CL-BTS1-ROX1 and inoculate it in SG/ΔTrp liquid medium (galactose 20.0g/L, DO Supplement 0.62g/L, amino-free yeast nitrogen source YNB 6.7g/L , Ura 0.02g/L, Leu 0.06g/L), cultured at 30°C and 220rpm for 36h. The bacterial solution was diluted ¹⁰ times and spread on SD/ΔTrp solid plate containing 1g/L 5-FOA (glucose 20.0g/L, agar 20.0g/L, DO Supplement 0.62g/L, no amino yeast nitrogen source YNB 6.7g/L, Ura 0.02g/L, Leu 0.06g/L), cultured at 30°C for 36h, through 5-FOA negative screening, the colonies grown on the plate are those without the carrier P426-CL-BTS1 - S. cerevisiae BJ5464ΔBTS1ΔROX1 of ROX1.

2. PCR amplification of erg9-gRNA, ypl062w-gRNA and yjl064w-gRNA expression vector fragments using P426-CL as the template, P13/P25 (5′-ATGCAAAGTGCAGTGGAAAAGATCATTTATCTTTCACTG-3′) and P16/P26 (5′-TTTTCCACTGCACTTTGCATGTTTTAGAGCTAGAAATAGCA- 3') is a primer pair, respectively amplified to obtain ERG9-1 and ERG9-2 fragments.

Using P426-CL as the template, P13/P27 (5′-CATCAGCCACGGCGACGTGCGATCATTTATCTTTCACTGC-3′) and P16/P28 (5′-GCACGTCGCCGTGGCTGATGGTTTTAGAGCTAGAAATA-3′) were used as primer pairs to amplify the ypl062w-1 and ypl062w-2 fragments respectively. .

Using P426-CL as a template, P13/P29 (5′-AGATGAACTCACGCTGTCGTGATCATTTATCTTTCACTGC-3′) and P16/P30 (5′-ACGACAGCGTGAGTTCATCTGTTTTAGAGCTAGAAATA-3′) were used as primer pairs to amplify and obtain yj1064w-1 and yj1064w-2 fragments respectively. .

3. Construction of recombinant vector containing ERG9 guide RNA sequence

use II recombination cloning kit, the two fragments of ERG9-1 and ERG9-2 were recombined to obtain the recombinant vector P426-CL-ERG9 carrying the ERG9 guide RNA sequence.

4. Construction of recombinant vector containing ypl062w guide RNA sequence

use II recombination cloning kit, recombination of two fragments ypl062w-1 and ypl062w-2, to obtain the recombinant vector P426-CL-ypl062w carrying the ypl062w guide RNA sequence.

5. Construction of recombinant vector containing yjl064w guide RNA sequence

use II recombination cloning kit, recombination of two fragments yjl064w-1 and yjl064w-2, to obtain the recombinant vector P426-CL-yjl064w carrying the yjl064w guide RNA sequence.

6. PCR amplification of ERG9 and ypl062w fragments

Using P426-CL-ERG9 as the template and P13/P19 as the primer pair, the ERG9 fragment containing the erg9 target sequence was obtained by amplification. Using P426-CL-ypl062w as a template and P20/P16 as a primer pair, a ypl062w fragment containing the ypl062w targeting sequence was obtained by amplification.

7. Construction of recombinant vector containing ERG9 guide RNA sequence and ypl062w guide RNA sequence

use II recombination cloning kit, the two fragments of ERG9 and ypl062w were recombined to obtain the recombinant vector P426-CL-ERG9-ypl062w carrying the ERG9 guide RNA sequence and the ypl062w guide RNA sequence.

8. PCR amplification of ERG9-ypl062w and ypj064w fragments

Using P426-CL-ERG9-ypl062w as the template and P31(5′-GAACAAAAGCTGGAGCT-3′)/P32(5′-CCTTTAGTGAGGGTTAA-3′) as the primer pair, amplify the erg9 target sequence and the ypl062w target sequence. ERG9-ypl062w fragment. Using P426-CL-ypj064w as the template and P33(5'-TTAACCCTCACTAAAGGTCTTTGAAAAGATAATGTATGA-3')/P34(5'-AGCTCCAGCTTTTTTCAAGACATAAAAAACAAAAAAAG-3') as the primer pair, the ypj064w fragment containing the ypj064w targeting sequence was amplified.

9. Construction of the recombinant vector containing the three gene guide RNA sequences of ERG9, ypl062w and ypj064w at the same time

use II Recombination Cloning Kit, the two fragments of ERG9-ypl062w and ypj064w were recombined to obtain the recombinant vector P426-CL-ERG9-ypl062w-ypj064w carrying the BTS1 guide RNA sequence, the ROX1 guide RNA sequence and the ypj064w guide RNA sequence.

10. Yeast genome ROX1 site, ypl062w site and ypj064w site editing verification

The P426-CL-ERG9-ypl062w-ypj064w and p414-TEF1p-Cas9-CYC1t plasmids were simultaneously transferred into the host S. cerevisiae BJ5464ΔBTS1-ΔROX1 competent cells, with the assistance of Cas9 protein, the ROX1 gene gRNA sequence, YPL062W gene sequence and ypj064w gene sequences were located respectively to achieve the knockout of the yeast genome ROX1 gene, ypl062w gene and ypj064w gene, and the mutant strain S.cerevisiae BJ5464ΔBTS1ΔROX1ΔERG9Δypl062wΔypj064w/P426-CL-ERG9-ypl062w-ypj064w was obtained.

10 Saccharomyces cerevisiae transformants were randomly selected and treated with P35(5′-CAACAACAATACCGACTTACCAT-3′)/P36(5′-CCGTCGAAACTCCATTTAGTTA-3′), P37(ATGATAGAATTGGATTATGTAAAAGG)/P38(GTTCCCAAGAGGAGATCAATGT) and P39(ATGACACTTGTAGTATATCT)/P40( GACTCTCCCGTGATGTCCGA) primer pair was used for colony PCR amplification, and the PCR products were sent for testing. The sequencing results showed that the target genes ERG9, ypl062w and ypj064w of 10 transformants were mutated at the same time, and the positive mutation rate was 10/10. The results show that the introduction of the LoxP sequences at both ends of the replication origin and the Cre expression cassette (1) does not affect the editing ability of the CRISPR/Cas9 system for yeast genomes; (2) can efficiently realize multi-gene step-by-step editing operations.

Example 4 Evaluation of Saccharomyces cerevisiae genome editing vector loss rate

Taking S. cerevisiae BJ5464ΔBTS1/P426-CL-BTS1 as the research object, single colonies were picked, inoculated into SG/ΔTrp liquid medium, and cultured at 30°C and 220rpm for 36 hours. The bacterial solution was counted with a hemocytometer, and the cell concentration A ₁ of the bacterial solution was 6.7×10 ⁸ CFU. After diluting to an appropriate multiple, take 200 μL of the diluted bacterial solution and spread it on SD/△Trp-△Ura (glucose 20.0g/L, agar 20.0g/L, DO Supplement 0.62g/L, amino-free yeast nitrogen source YNB 6.7g/L , Leu 0.06g/L) on a solid plate, cultured at 30°C for 48 hours. The colonies grown at this time are recombinant bacteria that still contain the plasmid P426-CL-BTS1. The number of colonies grown on the plate was counted, and the plasmid loss rate was calculated. Do 3 parallels for each sample.

Among them, A ₁ represents the cell density of yeast liquid, CFU (unit/mL)

A ₂ represents the number of colonies grown on the plate (units)

D is the dilution factor

The statistical results are shown in Table 1

Table 1 Statistics and calculation of plasmid loss rate*

*The cell density of yeast broth is 6.2×10 ⁸ cells/mL.

The results showed that the loss rate of plasmid was over 99.9% under the action of Cre recombinase by introducing the directional LoxP sequences at both ends of Ori, the origin of replication of plasmid P426. This completes the recovery of the P426 vector selection marker, which provides technical support for the smooth implementation of the next round of CRISPR/Cas9-mediated yeast genome editing.

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Claims (6)

1. a kind of construction method of Yeast genome editor carrier, which is characterized in that it with gRNA encoding plasmids P is to set out carrier, The LoxP sequence of 34 bp in the same direction is introduced respectively at the both ends replication origin Ori of the carrier, and is introduced in plasmid P frame position Cre recombinase expression casette obtains Yeast genome editor carrier；

The LoxP sequence is as shown in SEQ ID NO: 1；

The Cre recombinates enzyme gene nucleotide sequence as shown in SEQ ID NO: 2；

The carrier that sets out is P426, and is inserted into Cre recombinase expression cassette in the site KpnI of P426；

Promoter in the expression cassette is the GAL1 promoter of galactolipin induction, and terminator is CYC1 transcription terminator.

2. Yeast genome editor's carrier of claim 1 the method building.

3. application of the carrier described in claim 2 in the gene editing using CRISPR/Cas9 as medium, which is characterized in that packet Include following steps:

It 1) is the carrier that sets out, expression vector of the building containing targeting gRNA gene order with the carrier of claim 2；

2) step 1) is obtained into gRNA expression vector and imports the saccharomyces cerevisiae for containing corresponding Cas9 system, obtain recombination wine brewing ferment Mother, and genome editor is realized under CRISPR/Cas9 System guides；

3) after the completion of genome editor, recombinant Saccharomyces cerevisiae is placed in Fiber differentiation in gala sugar culture-medium, blocks carrier oneself I replicates, and realizes passage plasmid loss；

4) recombinant Saccharomyces cerevisiae for screening plasmid loss imports new gRNA expression vector and carries out next round genome editor.

4. application according to claim 3, which is characterized in that repeat step 3), 4) realize more wheel genome editors.

5. application according to claim 3 or 4, which is characterized in that recombinant Saccharomyces cerevisiae described in step 3) is trained in galactolipin Support the condition of Fiber differentiation in base: temperature 25-32^oC, time 36-42h, revolving speed 150-300rpm.

6. application according to claim 3 or 4, which is characterized in that the saccharomyces cerevisiae isS.cerevisiaeBJ5464； The Cas9 system is plasmid p414-TEF1p-Cas9-CYC1t.