CRISPR / Cpf1-based hansenula polymorpha efficient gene editing system and application thereof

Abstract

The invention discloses a hansenula polymorpha efficient gene editing system based on CRISPR (clustered regularly interspaced short palindromic repeats) / Cpf1 and application of the hansenula polymorpha efficient gene editing system. The hansenula polymorpha gene editing system comprises: (1) a Cpf1 integration plasmid, wherein the Cpf1 integration plasmid is expressed by an endogenous constitutive promoter, and the Cpf1 is integrated into a hansenula polymorpha genome; (2) a crRNA expression plasmid, wherein the crRNA expression plasmid comprises a crRNA expression cassette; wherein the crRNA expression cassette is composed of an RNA pol III type promoter and a terminator.

Description

Technical Field

[0001] The present invention relates to a CRISPR / Cpf1-based efficient gene editing system for Hansenula polymorpha and application thereof, belonging to the technical field of microbial genetic engineering. Background Art

[0002] Hansenula polymorpha is a non-traditional methylotrophic yeast that can grow with methanol as the sole carbon source. Hansenula polymorpha can utilize a variety of carbon sources such as glucose, sucrose, glycerol, cellobiose and xylose, and has a wide substrate spectrum, making it an ideal host for microbial cell factories. In addition, Hansenula polymorpha is resistant to high temperatures, with an optimal growth temperature of 37-43°C. It remains active at temperatures above 50°C, which can greatly reduce industrial production costs. Hansenula polymorpha has the advantages of high secretory protein expression and low glycosylation, and is used in the expression of a variety of proteins. It is an ideal protein secretion expression system. As an unconventional yeast, Hansenula has many excellent properties, but it lacks a stable expression vector, has low homologous recombination efficiency, and is difficult to manipulate genes, which greatly limits the application of Hansenula in industrial production.

[0003] The CRISPR / Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) system can perform specific targeted editing of genes and has become a conventional tool for genome editing. Currently, various types of CRISPR / Cas systems have been developed and widely used in gene editing in various biological systems. Among them, the most widely used are the CRISPR / Cas9 system and the CRISPR / Cpf1 system. The gene editing function of the CRISPR / Cas9 system depends on the protein complex formed by the Cas9 protein and the guide RNA (sgRNA). The Cas9 protein is an RNA-mediated nuclease whose HNH functional domain and RuvC functional domain can cut double-stranded DNA molecules. At the same time, the Cas9 protein requires a nuclear localization signal (NLS) to enable it to enter the cell nucleus and thus function. The sgRNA consists of a 20bp targeting RNA (crRNA) and a reverse activating RNA (tracrRNA). The crRNA is derived from the targeting sequence, and the 3' end of the target contains a G-rich PAM sequence (NGG). sgRNA can form a specific secondary structure and bind to the Cas9 protein, guiding the Cas9 protein to target a specific sequence and complete the break of double-stranded DNA to form a blunt-end incision. Compared with the CRISPR / Cas9 system, CRISPR / Cpf1 has better characteristics. The CRISPR / Cpf1 system consists of Cpf1 protein and smaller crRNA, and does not require the participation of tracrRNA. The Cpf1 protein does not contain the HNH domain and relies on the RuvC domain to complete the break of double-stranded DNA. At the same time, the Cpf1 protein also has RNA endonuclease activity, which can autonomously recognize crRNA and complete the maturation of crRNA, independent of other RNA endonucleases. The CRISPR / Cpf1 system specifically recognizes the T-rich PAM region (TTTN) at the 5' end of the target sequence, which can break double-stranded DNA and form sticky ends. Compared with blunt ends, sticky ends are more conducive to subsequent genetic manipulation.

[0004] At present, the CRISPR / Cas9 system has been successfully applied to Hansen yeast, and can achieve site-specific knockout and homologous integration of genes. Juergens et al. used a free plasmid containing a panARS replicon to express Cas9 protein and sgRNA. Both Cas9 and sgRNA were expressed using a type II promoter and ribozyme recognition sites were introduced at both ends of the sgRNA to ensure the expression of sgRNA (Juergens et al. Genome editing in Kluyveromyces and Ogataea yeasts using abroad-host-range Cas9 / gRNA co-expression plasmid. FEMS Yeast Res. 2018; 18(3):foy012.). Although gene editing was successfully achieved, the editing efficiency was only about 9%. Numamoto et al. used a type III promoter tRNA derived from Hansen yeast itself. CUG To express sgRNA, the construction process was simplified, and the mutation rate of different genes reached 17-71% (Numamoto et al. Efficient genome editing by CRISPR / Cas9 with atRNA-sgRNA fusion in the methylotrophic yeast Ogataea polymorpha. J Biosci Bioeng. 2017; 124 (5): 487-492.). Wang et al. integrated both Cas9 protein and sgRNA into the genome for expression, and achieved a gene knockout efficiency of more than 50%, and was able to achieve multi-gene editing (Wang et al. Efficient CRISPR–Cas9 mediated multiplex genome editing in yeasts. Biotechnol Biofuels. 2018; 11 (1): 277). Gao et al. integrated Cas9 into the genome, used type II endogenous promoters for sgRNA, and introduced ribozyme recognition sites at both ends, and achieved efficient gene editing by free plasmid expression. At the same time, the homology efficiency of Hansen yeast was improved by overexpressing homologous recombination-related genes, which greatly improved the gene integration efficiency (Gao et al. Recombination machinery engineering for precise genome editing in methylotrophic yeast Ogataea polymorpha. iScience. 2021; 24(3): 102168.).

[0005] Although the CRISPR / Cas9 system can be used to edit the Hansen yeast genome, the efficiency is relatively low, especially in multi-gene editing. The low efficiency of multi-gene editing and the cumbersome operation process are far from meeting the needs of metabolic engineering transformation. Summary of the invention

[0006] The present invention aims to construct a complete CRISPR / Cpf1 gene editing system, provide efficient gene editing efficiency and a simpler and faster operation method. At the same time, it improves the homologous recombination efficiency of the host and provides a good molecular basis for more accurate genetic engineering operations.

[0007] The present invention provides a CRISPR / Cpf1 system with high genome editing efficiency in Hansenula polymorpha. The system is composed of Hansenula polymorpha expressing Cpf1 and a free plasmid expressing crRNA.

[0008] One aspect of the present invention provides a plasmid combination for gene editing of Hansenula polymorpha based on CRISPR / Cpf1, comprising:

[0009] (1) Cpf1 integration plasmid: The Cpf1 integration plasmid is expressed by an endogenous constitutive promoter, an NLS nuclear localization sequence derived from SV40 is added to the C-terminus of the Cpf1 gene, and the fusion gene is integrated into the genome of Hansenula polymorpha;

[0010] (2) crRNA expression plasmid: The crRNA expression plasmid includes a crRNA expression frame; preferably, the promoter of crRNA is an RNA pol III type promoter.

[0011] Specifically, the Cpf1 gene utilizes the endogenous pol II type promoter P PGD , P ADH2-1 , P MAL or P GAP Express.

[0012] In a specific embodiment, crRNA is expressed using an endogenous pol III type promoter selected from 5s rRNA, tRNA AUG , tRNA UGG , tRNA AAC , tRNA CAC , tRNA AGA , tRNA GAC , tRNA GAG or tRNA UCU .

[0013] The present invention provides a gene editing method for Hansenula polymorpha based on CRISPR / Cpf1, which utilizes the plasmid combination for CRISPR / Cpf1 gene editing of Hansenula polymorpha to perform targeted gene editing.

[0014] In a specific embodiment, the Cpf1 protein is integrated into the genome of Hansenula polymorpha via a Cpf1 integration plasmid, and the crRNA expression plasmid and a donor DNA with homology arms are co-transformed into the Hansenula polymorpha into which Cpf1 has been integrated, thereby achieving knockout or insertion of the target gene;

[0015] Preferably, the homology arm size of the donor DNA is 500-1000 bp.

[0016] In some embodiments, a crRNA expression vector containing multiple crRNAs is co-transformed with corresponding donor DNAs with different homology arms (in tandem) to achieve simultaneous editing of multiple genes.

[0017] Preferably, the genome of the Hansenula polymorpha has knocked out the KU70 gene and / or the KU80 gene. The CRISPR / cpf1 gene editing system of the Hansenula polymorpha includes deleting the endogenous non-homologous end joining key gene KU70 / KU80, which can significantly improve the homologous recombination efficiency of Hansenula.

[0018] Specifically, the crRNA expression vector and donor DNA are co-transformed to achieve site-directed deletion of large fragments of the Hansenula genome.

[0019] The present invention also provides a plasmid combination for regulating gene expression of Hansenula polymorpha based on CRISPR / dCpf1, wherein the plasmid combination comprises:

[0020] (1) dCpf1 or dCpf1-VP64 integration plasmid: The dCpf1 or dCpf1-VP64 integration plasmid is expressed by an endogenous constitutive promoter and is used to integrate dCpf1 or dCpf1-VP64 into the genome of Hansenula polymorpha;

[0021] (2) crRNA expression plasmid: The crRNA expression plasmid includes a crRNA expression cassette; preferably, the promoter of the crRNA expression cassette is an RNA pol III type promoter.

[0022] The present invention provides a method for regulating gene expression of Hansenula polymorpha, which utilizes the plasmid combination to regulate gene expression of Hansenula polymorpha; the regulation is to inhibit or activate the expression of the corresponding gene;

[0023] Specifically, when inhibiting the expression of a gene, the crRNA expression plasmid is transformed into Hansenula in which the dCpf1 protein is integrated;

[0024] When activating the expression of the gene, the crRNA expression vector is transformed into Hansenula polymorpha in which the dCpf1-VP64 protein is integrated;

[0025] Further preferably, the simultaneous inhibition or activation of multiple genes is achieved by a crRNA expression vector containing multiple crRNAs.

[0026] Compared with the Hansenula CRISPR / Cas9 system, the present invention has the following characteristics:

[0027] (1) The CRISPR / Cpf1 gene editing system of the present invention has a simpler and faster operation process and extremely high gene editing efficiency;

[0028] (2) The CRISPR / Cpf1 gene editing system described in the present invention has extremely high multi-gene editing efficiency;

[0029] (3) The CRISPR / Cpf1 gene editing system described in the present invention has extremely high gene integration efficiency;

[0030] (4) The CRISPR / Cpf1 gene editing system described in the present invention has extremely high efficiency in knocking out large genome fragments;

[0031] (5) The gene regulation system mediated by dCpf1 constructed based on the CRISPR / Cpf1 gene editing system described in the present invention can achieve effective inhibition of gene expression;

[0032] (6) The gene regulation system mediated by dCpf1-VP64 constructed based on the CRISPR / Cpf1 gene editing system described in the present invention can achieve effective activation of gene expression;

[0033] The CRISPR / Cpf1 gene editing system constructed in the present invention makes the genetic manipulation of Hansenula more concise, rapid, accurate and efficient, and expands the application prospects of Hansenula in chassis cell construction and as a cell factory. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of Cpf1 integration plasmid, including FnCpf1 expression cassette, resistance selection gene and homology arms.

[0035] Figure 2 The Cpf1 proteins with different promoters were integrated into the genome, and the expression intensity of Cpf1 was displayed by detecting the intensity of GFP.

[0036] Figure 3 Schematic diagram of expressing crRNA using type II promoter. Among them, a is the crRNA sequence including DR and targeting sequence; b is the introduction of ribozyme recognition sites at both ends of crRNA; c is the introduction of ribozyme recognition sites only at the 3' end of crRNA; d is the introduction of DR sequence at the 3' end of crRNA.

[0037] Figure 4 Editing efficiency of different crRNA compositions when expressed using type II promoters and terminators.

[0038] Figure 5 Schematic diagram of the construction of crRNA expression using type III promoter and terminator.

[0039] Figure 6 Editing efficiency when expressing crRNA using different type III promoters.

[0040] Figure 7 The effect of the upstream region of tRNA on editing efficiency when it acts as a promoter.

[0041] Figure 8 To utilize the optimized Hansenula CRISPR / Cpf1 gene editing system to construct crRNA matrix and improve the editing efficiency of multiple genes.

[0042] Fig. 9 To verify the transformants using nutrient-deficient medium, the efficiency of triple gene editing by the CRISPR / Cpf1 gene editing system was tested.

[0043] Fig.10 Figure 2 shows the three DNA repair modes in Hansenula in the presence of donor DNA after double-stranded DNA breaks were induced using the CRISPR / Cpf1 system.

[0044] Fig.11 To improve the homologous recombination efficiency of Hansenula by overexpressing homologous recombination-related proteins or deleting key proteins for non-homologous end joining.

[0045] Fig.12 To select large fragments in the genome for deletion.

[0046] Fig.13 To improve the efficiency of large genome fragment deletion on the basis of improving the efficiency of homologous recombination.

[0047] Figure 14(a) shows crRNA located at different positions of the promoter and gene.

[0048] Figure 14(b) shows the inhibitory effect of dCpf1 on gene expression when crRNA is located at different positions of the promoter and gene.

[0049] Figure 15(a) shows crRNA located at different positions of the promoter.

[0050] Figure 15(b) shows the activation effect of dCpf1-VP64 on gene expression when crRNA is located at different positions in the promoter. DETAILED DESCRIPTION

[0051] The present invention will be further described below in conjunction with examples, but the examples are not intended to limit the present invention.

[0052] Example 1: Construction of CRISPR / Cpf1 gene editing system

[0053] (1) Construction of Cpf1 integration plasmid

[0054] The NLS nuclear localization sequence (QRU95054.1) derived from SV40 was added to the C-terminus of the Cpf1 gene, and it was connected to GFP using the P2A peptide. The recombinant sequence was codon-optimized and synthesized by Jin Weizhi according to Pichia pastoris. First, the Cpf1 integration vector framework was constructed. The promoter fragments were obtained by PCR from the genomes of Pichia pastoris (PpGAP) and Hansenula (HpGAP, HpADH2-1, HpPGD) using designed primers, and the homologous arm sequences were obtained by PCR from the Hansenula genome using designed primers. The NAT resistance gene expression frame was added as a screening gene, and the ori and Amp resistance genes were obtained from the Escherichia coli expression vector by PCR. The different fragments were connected using the seamless cloning method to construct the cpf1 integration plasmid framework (such as Figure 1 As shown). Secondly, the target gene and the vector backbone containing different promoters were double-digested with EcoRI and SalI endonucleases, and then connected with T4 ligase. The ligation product was transformed into DH5α competent cells, and the plasmid was extracted after the transformant grew, and the plasmid was sequenced to verify the plasmid.

[0055] (2) Integration of Cpf1 gene

[0056] The Cpf1 integration plasmid was digested with restriction endonuclease SacII and digested at 37°C for 4 hours to obtain a linearized vector. After the linearized vector was purified, it was added to Hansenula competent cells and transformed into Hansenula using electroporation. After electroporation, it was quickly placed in a recovery medium and incubated at 37°C for 1 hour. It was then spread on a YPD plate containing NAT (30 μg / mL) for culture and integrated into the Hansenula genome by double exchange. The obtained transformants were fermented and cultured, and the expression intensity of Cpf1 under different promoters was compared by detecting GFP intensity. Among them, the endogenous GAP promoter of Hansenula has the highest Cpf1 expression intensity (such as Figure 2), named F06 and used as the strain for subsequent experiments.

[0057] (3) Construction of crRNA expression vector

[0058] crRNA is expressed using a free expression vector. In previous reports, when using a pol III promoter to express sgRNA, there is a problem of no editing activity or low editing activity. Therefore, in order to ensure the functionality of the system, crRNA is temporarily expressed with a pol II promoter. Primers were designed to obtain the HpTEF1 promoter from the Hansenula genome by PCR, the AOX1 terminator from the PPIC9K plasmid, the panARS replicon from the Kluyveromyces lactis genome PCR, and the ori and Amp resistance genes from the Escherichia coli expression vector. At the same time, the URA3 gene was obtained by PCR amplification from the Hansenula genome as a screening gene. The connection was performed by seamless cloning, and the connection product was transformed into a DH5α competent cell, and the correct plasmid was extracted and verified. The crRNA was further optimized, and the ribozyme recognition site HDV or DR sequence (such as ) was introduced at the 3' end of the crRNA targeting sequence by the long primer method. Figure 3 Different types of crRNA expression vectors were transformed into F06.

[0059] The results showed that there was no editing activity when there were no DR or ribozyme recognition sequences at both ends of crRNA. When DR or ribozyme recognition sequences were added at both ends of crRNA, the editing efficiency was >90% (e.g. Figure 4 The results show that pol II promoters and terminators may leave some redundant sequences in crRNA, which can seriously affect the editing efficiency. However, the DR of crRNA in the CRISPR-Cpf1 system can be recognized and cut by Cpf1 protein, which can replace the ribozyme recognition site.

[0060] (4) Optimization of crRNA expression vector

[0061] To further simplify the construction process of crRNA plasmid, available pol III type promoters were mined from Hansenula. 5S rRNA and different tRNA sequences were mined from the Hansenula genome. It has been reported that the upstream sequence of 5S rRNA may have an important influence on the transcription of 5S rRNA. Therefore, when using 5S rRNA as a promoter, the upstream 300bp is also used as a promoter (such as Figure 5 The mined tRNA sequences were also used as promoters together with their upstream 300 bp, including tRNA AUG , tRNA UGG , tRNA AAC , tRNA CAC , tRNAAGA , tRNA GAC , tRNA GAG , tRNA UCU The type III terminator SUP4 from Saccharomyces cerevisiae was used as the terminator. Different crRNA expression vectors were transformed into F06 to verify the editing efficiency.

[0062] Table 1. Type III promoter sequences used.

[0063]

[0064]

[0065] The results showed that the editing efficiency of 5S rRNA alone as a promoter was only 35.6%, while the editing efficiency of 5S rRNA together with the upstream 300bp as a promoter reached 90.1% (e.g. Figure 6 ). This indicates that in Hansenula, there are unknown regulatory elements upstream of 5S rRNA, which play an important regulatory role in the transcription of 5S rRNA. Compared with 5S rRNA, tRNA has a higher editing efficiency when used as a promoter, and the editing efficiency of all types of tRNA is above 96.5%. In order to explore whether the upstream sequence of tRNA has an important regulatory function on the transcription of tRNA, the upstream of tRNA was truncated to different degrees to detect its effect on the editing efficiency. The results showed that the presence of the upstream region of tRNA had no significant effect on the editing efficiency (as shown in Figure 2). Figure 7 This indicates that there are no related elements regulating tRNA transcription upstream of tRNA, and tRNA can be used directly as a promoter.

[0066] Example 2: Application of CRISPR / Cpf1 gene editing system in multi-gene editing

[0067] After optimizing the expression of crRNA, the most concise crRNA expression cassette so far was obtained: tRNA-crRNA-SUP4, and the editing efficiency of the gene was greater than 96.5%. One of the characteristics of the CRISPR / Cpf1 system is that Cpf1 can recognize the DR region in crRNA and cut it, promoting the maturation of crRNA, which also illustrates the natural advantage of CRISPR-Cpf1 in multi-gene editing. In order to verify the efficiency of the CRISPR-Cpf1 system in multi-gene editing, a crRNA matrix containing 3 crRNAs was constructed using the long primer PCR method: tRNA-crRNA1-crRNA2-crRNA3-SUP4. The target genes of the three crRNAs are ADE2, HIS4 and GUT1. Using the crRNA expression plasmid as a vector, PCR was performed using long primers. After the PCR product was recovered, it was digested with DpnI enzyme and treated at 37°C for 1h to fully digest the template. After DpnI treatment, the PCR product was transformed into DH5α competent cells, spread on LB plates containing ampicillin (100 μg / mL) and cultured overnight at 37°C. The transformants were picked for expansion and plasmid extraction, and sequencing verification was performed to obtain the correct plasmid. The free vector expressing the correct crRNA matrix was electroporated into F06 competent cells, and after recovery, it was spread on MD medium plates supplemented with leucine, adenine and histidine for culture. After culturing at 37°C for 2 to 3 days, the transformants were picked and spread on MD medium plates supplemented with leucine, adenine and histidine as the control group, and at the same time spread on the corresponding nutritional deficiency plates as the experimental group. The gene editing efficiency was calculated by calculating the proportion of bacteria that could not grow on the nutritional deficiency plates. The results are shown in Figure 8 , Fig. 9 As shown, the editing efficiency of single genes is ≥94.0%, the editing efficiency of double genes is ≥92.7%, and the editing efficiency of triple genes is 92.7%. This is the highest triple gene editing efficiency obtained so far without the use of other sequence assistance and only using the natural crRNA matrix.

[0068] Example 3: Improving the efficiency of homologous recombination in Hansenula

[0069] The essence of the CRISPR / Cpf1 gene editing system is to rely on the Cpf1-crRNA complex to cause double-stranded DNA breaks, which are then repaired through the host's own DNA repair system to complete gene deletion, insertion, etc. Among them, the main repair methods are homologous recombination and non-homologous end joining. Previous studies have reported that non-homologous end joining plays a dominant role in DNA repair in Hansenula, which also affects the efficiency of gene integration using the CRISPR / Cpf1 system. In exploring the integration efficiency in Hansenula, the co-transformation method of crRNA and donor DNA was used, and the positive transformants were sequenced to detect whether the gene integration was completed. The results showed that there are three repair methods in the positive clones: homologous recombination, one-end homologous recombination and one-end non-homologous end joining, and complete non-homologous end joining (such as Fig.10 As shown). In order to further improve the efficiency of homologous recombination, we tried to overexpress homologous recombination-related proteins RAD51 and RAD52 and delete non-homologous end joining-related protein KU70. Rad51 and Rad52 genes were obtained by PCR in the Hansenula genome and Pichia pastoris genome, and expressed using the endogenous promoter and terminator of Hansenula. Rad51 and Rad52 genes from different sources were integrated into the Hansenula genome for expression. At the same time, the non-homologous end joining-related gene Ku70 was mined in the Hansenula genome, and the Ku70 sequence was completely deleted using the CRISPR-Cpf1 system. The crRNA expression vector and donor DNA were transformed into Hansenula strains that overexpressed Rad51 and Rad52 genes or deleted the Ku70 gene, and colony PCR verified the probability of the donor DNA in the transformant being integrated into the genome through homologous recombination. The results are shown in Fig.11 As shown, overexpression of homologous recombination-related genes did not significantly improve the host's homologous recombination efficiency, while mining and deleting the non-homologous end joining-related gene Ku70 made the host's homologous recombination efficiency reach 100%, that is, the host's non-homologous end joining mode was completely blocked.

[0070] Example 4: Application of CRISPR / Cpf1 system in large genome fragment deletion

[0071] Using CRISPR / Cpf1 to achieve site-directed deletion of large genomic DNA fragments is another application of the CRISPR / Cpf1 system. In order to verify the efficiency of the constructed Hansenula polymorpha CRISPR / Cpf1 system in deleting large genomic fragments, non-essential gene regions were selected from the Hansenula genome. One end target was fixed, and the deletion of DNA fragments of different lengths was achieved by designing targeting sequences at different lengths. The deletion of genomic DNA fragments was achieved by co-transformation of a crRNA matrix expression vector containing two crRNAs and the corresponding donor DNA. The Ira3 gene was selected to design the downstream crRNA, and the upstream crRNA was designed at 10k, 15k and 20k upstream of the crRNA. Different double crRNA expression matrices were formed using unused upstream crRNA and downstream crRNA, and corresponding upstream homologous arms were designed according to different upstream crRNAs, and the downstream homologous arms corresponding to the downstream crRNA formed the donor DNA. Among them, the homologous arm length of the donor DNA was set to 1000bp. The lengths of the DNA fragments to be deleted are 10kb, 15kb and 20k, respectively (such as Fig.12 As shown). By designing verification primers at both ends of the crRNA target site, the transformants were verified by PCR. That is, the PCR product size of the transformant after deletion was 2000 bp, and the transformant that was not successfully deleted could not obtain a PCR product due to its large length. The results are shown in Fig.13 As shown, the deletion efficiency of the 10kb fragment reached 95.8%, and the deletion efficiency of the 20kb fragment reached 91.7%. This is also the most efficient and longest large DNA fragment deletion achieved in Hansenula so far.

[0072] Example 5: Application of the CRISPR / dCpf1 gene expression regulation system constructed by expanding the CRISPR / Cpf1 system

[0073] (1) Application of CRISPR / dCpf1 gene expression regulation system in inhibiting gene expression

[0074] In order to further expand the application of CRISPR / Cpf1 system in Hansenula, a dCpf1-mediated gene expression regulation system was constructed based on the constructed CRISPR / Cpf1 system. The dCpf1 protein is an inactive mutant of the Cpf1 protein, that is, it loses the activity of cutting double-stranded DNA, but retains the ability to target and bind to the target DNA. The aspartic acid at position 917 of the Cpf1 protein was mutated to alanine to form dCpf1. The dCpf1 expression cassette and the mRFP expression cassette were simultaneously constructed into the integration plasmid, and dCpf1 and mRFP were integrated into the Hansenula genome using the same method as the Cpf1 protein integration to obtain strain F29. Different crRNA target sites were selected in the promoter region and the mRFP gene region (as shown in Figure 14 (a)), and different crRNA expression vectors were constructed. These crRNAs were transformed into F29 respectively, and the expression intensity of mRFP in the transformants was verified. As shown in Figure 14(b), in the experimental groups containing different crRNA targets, the expression intensity of mRFP decreased by about 20-40%, and the expression of mRFP was inhibited to varying degrees, indicating that dCpf1 can play a role in inhibiting gene expression. Furthermore, the crRNA matrix can also be used to simultaneously inhibit the expression of multiple genes.

[0075] (2) Application of CRISPR / dCpf1 gene expression regulation system in activating gene expression

[0076] In order to achieve the activation of gene expression, it is necessary to fuse dCpf1 with the transcription activator VP64, and use dCpf1-VP64 fusion protein to complete. The dCpf1-VP64 fusion protein and the mRFP reporter gene are integrated into the Hansen yeast genome using the same method as the dCpf1 integration to obtain strain F30. Design crRNA targets in different regions of the promoter to compare whether different targets have differences in the degree of activation of gene expression (as shown in Figure 15 (a)). Different crRNA expression vectors are transformed into F30 competent cells respectively to verify the expression intensity of mRFP of the transformants. As shown in Figure 15 (b), the mRFP expression intensity of all transformants containing different crRNAs is improved, indicating that the activation of gene expression can be effectively achieved by using the dCpf1-VP64 fusion method. Further, the expression activation of multiple genes can also be achieved synchronously by using the crRNA matrix method.

[0077] The above are only several embodiments of the present invention, and do not limit the present invention in any form. Although the present invention is disclosed as above in the preferred embodiment, it is not used to limit the present application. Any technician familiar with this profession, without departing from the scope of the technical solution of the present invention, using the above disclosed technical content to make slight modifications or changes are equivalent to equivalent implementation cases and belong to this technical scope.

Claims

1. A plasmid combination for gene editing of Hansenula polymorpha based on CRISPR / Cpf1, characterized in that: It includes: (1) Cpf1 integration plasmid: The Cpf1 integration plasmid is expressed by an endogenous constitutive promoter, an NLS nuclear localization sequence derived from SV40 is added to the C-terminus of the Cpf1 gene, and the fusion gene is integrated into the genome of Hansenula polymorpha; (2) crRNA expression plasmid: The crRNA expression plasmid comprises a crRNA expression cassette; preferably, the promoter of crRNA is an RNA pol III type promoter.

2. The plasmid combination of CRISPR / Cpf1 gene of Hansenula polymorpha according to claim 1, characterized in that The Cpf1 gene is expressed using the endogenous pol II promoter of Hansenula polymorpha PGD , P ADH2-1 , P MAL or P GAP Express.

3. The plasmid combination for CRISPR / Cpf1 gene editing of Hansenula polymorpha according to claim 1, characterized in that: crRNA is expressed using an endogenous pol III type promoter and is selected from 5s rRNA, tRNA AUG , tRNA UGG , tRNA AAC , tRNA CAC , tRNA AGA , tRNA GAC , tRNA GAG or tRNA UCU .

4. A gene editing method for Hansenula polymorpha based on CRISPR / Cpf1, characterized in that: Targeted gene editing is performed using the CRISPR / Cpf1 gene editing plasmid combination of Hansenula polymorpha according to any one of claims 1 to 3.

5. The method according to claim 4, characterized in that The Cpf1 protein is integrated into the genome of Hansenula polymorpha through the Cpf1 integration plasmid, and the crRNA expression plasmid and the donor DNA with homology arms are co-transformed into the Hansenula polymorpha in which Cpf1 is integrated to achieve knockout or insertion of the target gene; Preferably, the homology arm size of the donor DNA is 500-1000 bp.

6. The method according to claim 4 or 5, characterized in that: Simultaneous editing of multiple genes can be achieved by co-transforming a crRNA expression vector containing multiple crRNAs with corresponding donor DNAs with different homology arms.

7. The method according to claim 6, characterized in that The genome of the Hansenula polymorpha has KU70 gene knocked out and / or KU80 Gene.

8. The method according to claim 7, characterized in that Co-transformation of crRNA expression vector and donor DNA was used to achieve site-directed deletion of large fragments of the Hansenula genome.

9. A plasmid combination for regulating gene expression in Hansenula polymorpha based on CRISPR / dCpf1, characterized in that: The plasmid combination includes: (1) dCpf1 or dCpf1-VP64 integration plasmid: The dCpf1 or dCpf1-VP64 integration plasmid is expressed by an endogenous constitutive promoter and is used to integrate dCpf1 or dCpf1-VP64 into the genome of Hansenula polymorpha; (2) crRNA expression plasmid: The crRNA expression plasmid comprises a crRNA expression cassette; preferably, the promoter of the crRNA expression cassette is an RNA pol III type promoter.

10. A method for regulating gene expression in Hansenula polymorpha, characterized in that: Using the plasmid combination of claim 9 to regulate gene expression of Hansenula polymorpha; the regulation is to inhibit or activate the expression of the corresponding gene; Specifically, when inhibiting the expression of a gene, the crRNA expression plasmid is transformed into Hansenula in which the dCpf1 protein is integrated; When activating the expression of the gene, the crRNA expression vector is transformed into Hansenula polymorpha in which the dCpf1-VP64 protein is integrated; Further preferably, the simultaneous inhibition or activation of multiple genes is achieved by a crRNA expression vector containing multiple crRNAs.

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