Plasmid-free gene editing system based on T7 RNA polymerase and application thereof

Abstract

The invention relates to a plasmid-free gene editing system based on T7RNA polymerase and application of the plasmid-free gene editing system, and belongs to the technical field of gene editing. The plasmid-free gene editing system constructed by the invention comprises a T7 RNA polymerase, a Cas12a nuclease, a selection marker gene and a short double-stranded DNA expression cassette, wherein the short double-stranded DNA expression cassette sequentially comprises a T7 promoter, a first forward repetitive sequence, a spacer sequence and a second forward repetitive sequence. According to the plasmid-free gene editing system, T7RNA polymerase is expressed in host bacteria, short double-stranded DNA is transcribed to generate guide RNA, Cas12a nuclease is driven to achieve rapid and efficient gene editing, the problems that in the prior art, linear fragment transformation is complex and tedious, and part of strains lack genetic plasmids are solved, iterative editing and multi-site editing are supported at the same time, and the plasmid-free gene editing system is suitable for large-scale popularization and application. And a brand-new convenient tool is provided for gene editing of non-model fungi.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of gene editing, in particular to a plasmid-free gene editing system based on T7 RNA polymerase and application thereof. BACKGROUND

[0002] Genetic manipulation based on CRISPR / Cas has been widely used in various fields of life science, which uses RNA-guided nuclease variants to target and cleave nucleic acid sequences. Nuclease effector proteins (such as Cas9 and Cas12a / Cpf1) in type II CRISPR / Cas system are widely used. Although bacteria and archaea have endogenous CRISPR / Cas system, it can be used for genome editing and other related purposes. However, for most organisms other than bacteria and archaea, CRISPR-Cas components (at least including guide RNA and Cas nuclease) must be delivered into cells to perform editing. These components can be delivered in different forms, such as DNA, RNA or protein. Among them, episomal plasmid encoding Cas protein and / or guide RNA is the most common delivery reagent. However, many organisms, especially eukaryotes, lack native plasmids, and it is often difficult to construct artificial inheritable plasmids. Therefore, it is urgent to develop plasmid-independent gene editing tools.

[0003] Several plasmid-free gene editing strategies are currently available. For example, by in vitro transcription and protein purification, a ribonucleoprotein (RNP) complex composed of a guide RNA and a Cas protein is delivered (Kim, S., Kim, D., Cho, S. W., Kim, J., & Kim, J. S. (2014). Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Research, 24(6), 1012-1019). This method has been widely used for gene editing in animals, plants, filamentous fungi, yeast, and bacteria. Rapid degradation of RNP complexes within cells can reduce off-target effects, however, the preparation of RNP complexes is time-consuming and expensive, and lower editing efficiency has been observed in certain organisms, for example, the editing efficiency of Cas9 RNP complexes transformed into Saccharomyces cerevisiae is very low (Nishimura, A., Tanahashi, R., Oi, T., Kan, K., & Takagi, H. (2023). Plasmid-free CRISPR / Cas9 genome editing in Saccharomyces cerevisiae. Bioscience Biotechnology Biochemistry, 87(4), 458-462). Other methods also include Agrobacterium-mediated single-stranded transport DNA (T-DNA) transformation, delivery of DNA fragments encoding Cas proteins and / or guide RNAs, transient expression or integrated expression. For example, Agrobacterium-mediated transformation can achieve transient expression of Cas and guide RNA in plants, inducing target double-strand breaks, thereby avoiding stable insertion of T-DNA into the chromosome (Krenek, P., Samajova, O., Luptovciak, I., Doskocilova, A., Komis, G., & Samaj, J. (2015). Transient plant transformation mediated by Agrobacterium tumefaciens: Principles, methods and applications. Biotechnology Advances, 33(6 Pt 2), 1024-1042).However, in fungi, transient transformation has not been fully explored, and the Cas protein and guide RNA expression cassette provided by T-DNA are usually randomly integrated into the fungal genome, so multiple rounds of transformation require different selection marker genes or marker gene recovery, and the cumulative integration or multiple copy tandem integration of the guide RNA cassette can have unpredictable harmful effects on the host cell. In addition, the integration of Cas nuclease and guide RNA expression cassettes into the chromosome by homologous recombination is also a commonly used method, but depends on whether there is a suitable selection marker gene, and iterative editing is usually limited.

[0004] Previous studies have attempted to transform linear DNA fragments to transiently express CRISPR / Cas components, such as PCR fragments expressing U6 promoter guide RNA used for transformation of mammalian cells. In yeast, guide RNA expression cassettes were delivered as linear DNA fragments to Cas9-expressing strains, achieving efficient genome editing. The recently developed LINEAR platform uses a single linear DNA fragment to encode Cas9 variants, guide RNAs, homologous recombination repair donor DNA, and selection marker genes to mediate efficient editing through homology-directed repair (HDR). However, the linear fragments transformed by these methods are complex and difficult to prepare, limiting rapid and efficient gene editing. SUMMARY

[0005] To solve the above technical problems, the present application provides a plasmid-free gene editing system, comprising elements T7 RNA polymerase, Cas12a nuclease, selection marker gene and short double-stranded DNA expression cassette, wherein the short double-stranded DNA expression cassette comprises T7 promoter, first forward repeat sequence, spacer sequence and second forward repeat sequence in turn. The plasmid-free gene editing system realizes the transcription of guide RNA from short double-stranded DNA by expressing T7 RNA polymerase in host bacteria, and drives Cas12a to achieve gene editing.

[0006] The first object of the present application is to provide a T7 RNA polymerase-based plasmid-free gene editing system, which comprises the following elements:

[0007] (1) T7 RNA polymerase;

[0008] (2) Cas12a nuclease;

[0009] (3) selection marker gene;

[0010] (4) short double-stranded DNA expression cassette, which comprises T7 promoter, first forward repeat sequence, spacer sequence and second forward repeat sequence in turn.

[0011] In an embodiment of the present application, the amino acid sequence of the T7 RNA polymerase is as shown in WP_001092355.

[0012] In an embodiment of the present application, the T7 RNA polymerase is expressed using the host cell’s own TDH3 promoter (upstream 667 bp sequence of the TDH3 gene NCBI Reference Sequence: NM_001181321.3 as the promoter) and TEF2 terminator (AgTEF2 terminator derived from the yeast expression vector pUD628).

[0013] In an embodiment of the present application, the T7 RNA polymerase is expressed using the host cell’s own ENO1 promoter (ENO1 gene encoding enolase, accession number: MG719760, upstream 1494 bp sequence of the gene as the promoter) and Tsynth7 terminator.

[0014] In an embodiment of the present application, the Cas12a nuclease is derived from Francisella novicida, and the amino acid sequence is as shown in ATB19155.

[0015] In an embodiment of the present application, the Cas12a nuclease is expressed using the TEF1 promoter and CYC1 terminator (both derived from pUDC175).

[0016] In an embodiment of the present application, the Cas12a nuclease is expressed using the host cell’s own TEF1 promoter and TEF1 terminator (TEF1 gene encoding transcription elongation factor EF-1a, accession number: MG719756, upstream 1479 bp sequence of the gene as the promoter, and downstream 200 bp sequence of the gene as the terminator).

[0017] Further, the target position protospacer adjacent motif (PAM) is downstream of a sequence of 19-25 bp in length.

[0018] Preferably, the length is 19 bp.

[0019] Further, the selection marker gene is a hygromycin resistance gene, a G418 resistance gene, a uracil auxotrophic gene, or a leucine auxotrophic gene.

[0020] Further, the T7 RNA polymerase and the Cas12a nuclease are terminally modified with a nuclear localization sequence.

[0021] Further, the number of the nuclear localization sequences is 1-3.

[0022] Further, the T7 RNA polymerase and the Cas12a nuclease are modified with an epitope tag sequence for detecting the expression of the T7 RNA polymerase and the Cas12a nuclease.

[0023] In an embodiment of the present application, the epitope tag sequence is a hemagglutinin tag.

[0024] In an embodiment of the present application, three segments of hemagglutinin HA tag are fused to the C-terminus of the Cas12a nuclease, and the amino acid sequence of the HA tag is as shown in SEQ ID NO. 3.

[0025] Further, the plasmid-free gene editing system further comprises a RAD52 protein.

[0026] Overexpression of Rad52 protein in the host strain expressing Cas12a nuclease and T7 RNA polymerase can improve the homologous recombination activity.

[0027] A second object of the present application is to provide the use of the above-mentioned plasmid-free gene editing system in fungal gene editing.

[0028] Further, the gene editing includes iterative gene editing and multi-site gene editing.

[0029] A third object of the present application is to provide a method for gene editing, which comprises transforming the above-mentioned plasmid-free gene editing system and an expression cassette comprising a donor DNA into a host strain, and the host strain is a fungus.

[0030] Further, the host strain is selected from Kluyveromyces, Saccharomyces, Schizosaccharomyces, Zygosaccharomyces, Pichia, Candida, Hansenula, Debaryomyces, Yarrowia, Lipomyces, Aspergillus, Mortierella, Penicillium, Trichoderma, Schizochytrium, Starmerella, Wickerhamiella.

[0031] Further, the transformation method comprises the step of electroporating the host strain.

[0032] In an embodiment of the present application, the overnight culture is inoculated into 50 mL YPD medium with an initial OD 600 of 0.15-0.2, and then cultured at 30℃ with shaking at 250 rpm until the OD 600The pH value was set at 0.4-2.0. Each 400 μL electroporation buffer used for transformation contained approximately 5 × 10⁻⁶ mg / L. 7 Cells were used. 50 μg of single-stranded vector DNA was added to the electroporation buffer for co-transformation with the linear DNA fragment. After transformation, cells were incubated at 30°C for 1–2 hours in a 1:1 mixture of 1 mL of 1 M sorbitol and YPD medium, and then plated onto selective medium plates. This improved protocol increased transformation efficiency by more than 10-fold.

[0033] Furthermore, the T7 RNA polymerase and the Cas12a nuclease are integrated and expressed in the host bacteria.

[0034] Furthermore, it also includes the step of splicing the short double-stranded DNA expression cassette and the expression cassette containing donor DNA.

[0035] Furthermore, the splicing includes any of the following:

[0036] (1) Link the donor DNA expression cassette downstream of the second positive repeat sequence of the short double-stranded DNA expression cassette;

[0037] (2) Link the donor DNA expression cassette downstream of the spacer sequence of the short double-stranded DNA expression cassette;

[0038] (3) Link the donor DNA expression cassette upstream of the first positive repeat sequence of the short double-stranded DNA expression cassette.

[0039] Preferably, the donor DNA expression cassette is linked downstream of the spacer sequence of the short double-stranded DNA expression cassette.

[0040] Further, the molar ratio of the donor DNA expression cassette, the short double-stranded DNA expression cassette or splice fragment to the screening marker gene is (1-1000):1.

[0041] Furthermore, the amount of the screening marker gene added is 0.25-3 μg.

[0042] Furthermore, the amount of the screening marker gene added is 0.25-1 μg.

[0043] Furthermore, it also includes a step of thiomodifying the ends of the donor DNA or the short double-stranded DNA expression cassette.

[0044] The beneficial effects of this invention are:

[0045] The application constructs and obtains a plasmid-free gene editing system suitable for fungi, which uses T7 RNA polymerase to transcribe guide RNA from short double-stranded DNA for CRISPR / Cas12a gene editing in the host cell, eliminates the cumbersome in vitro transcription or plasmid construction step, shortens the editing time, integrates the screening marker gene at the same site in the genome by turns, does not need plasmid loss and marker gene recovery, supports fast iterative editing, supports efficient simultaneous editing of multiple genes by simultaneously transforming multiple pairs of crDNA and homologous recombination repair donor DNA, is suitable for gene editing of non-model strains lacking heritable plasmids, and provides a new efficient and convenient tool for fungal gene editing. BRIEF DESCRIPTION OF DRAWINGS

[0046] In order to make the content of the application more easily and clearly understood, the application will be further described in detail below according to specific embodiments of the application and in conjunction with the drawings, in which:

[0047] Figure 1 The principle schematic diagram of the plasmid-free gene editing system provided by the application;

[0048] Figure 2 The construction of the plasmid-free gene editing system in Saccharomyces cerevisiae in Example 1 of the application, wherein A is a workflow schematic diagram of the plasmid-free gene editing system, and B is the editing efficiency of each step of the plasmid-free gene editing;

[0049] Figure 3 The construction schematic diagram of the recombinant strain NCZQX01 of Starmerella bombicola in Example 2 of the application;

[0050] Figure 4 The identification PCR electrophoresis result schematic diagram of the recombinant strain NCZQX01 of Starmerella bombicola in Example 2 of the application, wherein lane M is a molecular weight marker, lane C is a negative control strain, and the arrow indicates the expected fragment size of the edited colony;

[0051] Figure 5 The destruction of SbURA3 for constructing the recombinant strain NCZQX03 of Starmerella bombicola in Example 3 of the application, wherein A is the colony PCR result, and B is a construction principle schematic diagram;

[0052] Figure 6 The replacement of SbURA3 with HygR screening marker gene in Example 4 of the application, wherein A is the targeted editing of HygR by crDNA and / or homologous recombination repair donor DNA, and B is the construction schematic diagram of the recombinant strain NCZQX05 of Starmerella bombicola;

[0053] Figure 7 The growth of the recombinant strain of Starmerella bombicola in the YPD medium in Example 5 of the application;

[0054] Figure 8 For the editing system of Ogataea parasp. in Example 5 of the present application, wherein A is the selection marker gene for integrating HygR at the SbLEU2 locus, and the crDNA and homologous recombination repair donor DNA for editing SbADE2, B is the transformation efficiency of two different electroporation schemes, C is the efficiency of editing SbADE2 by transforming the indicated three linear fragments, D is the effect of the amount of selection marker gene fragment pmol on editing efficiency;

[0055] Figure 9 For plasmid-free genome editing of Ogataea parasp. in Example 6 of the present application, wherein A is the Ogataea parasp. strain designed for efficient plasmid-free CRISPR / FnCas12a genome editing, B is plasmid-free and iterative genome editing in strain NCZQX05;

[0056] Figure 10 For multiple genome editing of Ogataea parasp. in Example 7 of the present application, wherein A is a schematic overview of multiple genome editing, B is the editing efficiency of multiple targets;

[0057] Figure 11 For editing efficiency of different lengths of homologous recombination repair donor DNA (120 bp and 600 bp) and different amounts of homologous recombination repair donor DNA in Ogataea parasp. recombinant strain NCZQX05 in Example 8 of the present application;

[0058] Figure 12 For editing efficiency of SbADE2 gene deletion using short (120 bp) or long (600 bp) homologous recombination repair donor DNA in different Ogataea parasp. recombinant strains in Example 8 of the present application;

[0059] Figure 13 For comparison of editing efficiency of SbADE2 deletion in Ogataea parasp. recombinant strain NCZQX10 in Example 8 of the present application, wherein lane M is a molecular weight standard, and lane C is a negative control strain;

[0060] Figure 14 For optimizing the amount of selection marker gene fragment added to improve the gene editing ability using short homologous recombination repair donor DNA based on Ogataea parasp. recombinant strain NCZQX10 in Example 9 of the present application;

[0061] Figure 15 For three different crDNA and homologous recombination repair donor DNA fusions in Ogataea parasp. in Example 10 of the present application, wherein A is the three fusion schemes, B is the gene editing efficiency of different fusion schemes, and C is the comparison of gene editing efficiency after using sulfur modification of DNA fragments. DETAILED DESCRIPTION

[0062] The present application is further described in connection with the accompanying drawings and specific embodiments so that it can be better understood and carried out by persons skilled in the art. The embodiments are not intended to limit the present application.

[0063] The culture medium and reagents involved in the embodiments are as follows:

[0064] YPD culture medium: 20 g / L glucose, 20 g / L peptone, 10 g / L yeast powder.

[0065] SD-URA: 1.7 g / L YNB (yeast nitrogen base, without ammonium sulfate, other nitrogen source, from Shenguo Bioengineering Co., Ltd.), 5 g / L ammonium sulfate ((NH4)2SO4), 20 g / L glucose, 0.77 g / L-Ura DO supplement, 2% agar was added to the solid culture medium.

[0066] SD-LEU: 1.7 g / L YNB (yeast nitrogen base, without ammonium sulfate, other nitrogen source, from Shenguo Bioengineering Co., Ltd.), 5 g / L ammonium sulfate ((NH4)2SO4), 20 g / L glucose, 0.69 g / L Complete Supplement Mixture without leucine (CSM-LEU), 2% agar was added to the solid culture medium.

[0067] SD+5FOA: 1.7 g / L YNB (yeast nitrogen base, without ammonium sulfate, other nitrogen source), 5 g / L ammonium sulfate, 20 g / L glucose, 0.77 g / L-Ura DO supplement (Takara), 1 mg / L 5-FOA (Shenguo Bioengineering Co., Ltd.), 2 g / L Uracial, 2% agar was added to the solid culture medium.

[0068] LB culture medium: 10 g / L Tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 2% agar was added to the solid culture medium.

[0069] SOB medium: 20 g / L Bacto Tryptone, 5 g / L Bacto Yeast Extract, 0.5 g NaCl (final concentration 8.6 mM), 2.5 mL 1 M KCl (final concentration 2.5 mM), adjust pH to 7.0, autoclave. 5 mL 2 M MgCl2(final concentration 10 mM) and 5 mL 2 M MgSO4(final concentration 10 mM) are added after autoclaving.

[0070] TB solution: final concentrations 10 mM PIPES, 250 mM KCl, 15 mM CaCl2, 55 mM MnCl2, pH adjusted to 6.7 with 5 M KOH. Filter sterilized with 0.45 μm filter.

[0071] Ampicillin (AmpR) concentration: 100 μg / mL.

[0072] G418 concentration: 200 mg / L.

[0073] Hygromycin B: 500 mg / L.

[0074] Delft+His medium: (NH4)2SO4 7.5 g / L, KH2PO4 14.4 g / L, MgSO4-6H2O 0.5 g / L, trace elements solution 2 mL / L, vitamin solution 1 mL / L, glucose 20 g / L, histidine (pH=6) 100 mg / L.

[0075] Vitamin solution (L -1 ): 50 mg biotin, 200 mg p-aminobenzoic acid, 1 g nicotinic acid, 1 g calcium pantothenate, 1 g pyridoxal hydrochloride, 1 g thiamine hydrochloride, 25 g myo-inositol. Dissolve biotin in 20 mL 0.1 M NaOH solution, then add 900 mL water. Adjust pH to 6.5 with hydrochloric acid, then add the remaining vitamins. Adjust pH to 6.5 before and after adding myo-inositol. Adjust the final volume to 1 L and sterile filter, then store at 4°C.

[0076] Trace element solution: 4.5 g CaCl2·2H2O, 4.5 g ZnSO4·7H2O, 3 g FeSO4·7H2O, 1 g H3BO3, 1 g MnCl2·4H2O, 0.4 g Na2MoO4·2H2O, 0.3 g CoCl2·6H2O, 0.1 g CuSO4·5H2O, 0.1 g KI, 15 g EDTA. The trace metal solution was prepared by dissolving all components except EDTA in 900 mL ultrapure water, pH 6. The solution was then gently heated and EDTA was added. Finally, the pH was adjusted to 4, the volume was adjusted to 1 L, and the solution was autoclaved (121 °C, 20 min).

[0077] CSM: Adenine 10 mg / L, L-Arginine 50 mg / L, L-Aspartic acid 80 mg / L, L-Histidine hydrochloride 20 mg / L, L-Isoleucine 50 mg / L, L-Leucine 100 mg / L, L-Lysine hydrochloride 50 mg / L, L-Methionine 20 mg / L, L-Phenylalanine 50 mg / L, L-Threonine 100 mg / L, L-Tryptophan 50 mg / L, L-Tyrosine 50 mg / L, Uracil 20 mg / L, Valine 140 mg / L.

[0078] 1x LiAc-TE solution: 100 mM LiAc, 10 mM Tris-HCl, 1 mM EDTA.

[0079] 10x TE buffer: 100 mM Tris-HCl, 10 mM EDTA.

[0080] The culture method involved in the examples is as follows:

[0081] Deep well plate culture: The high throughput microbial growth curve analysis system of Jingling Instrument Manufacturing (Tianjin) Co., Ltd. was used with a matching 48-well deep well plate. The culture volume was 1 mL, the temperature was 30 °C or 37 °C, and the shaking speed was 800 rpm. The growth data of the strain was collected every 1 h.

[0082] The strains involved in the examples are shown in Table 1:

[0083] Table 1 Strain information

[0084]

[0085]

[0086] The plasmids involved in the examples are shown in Table 2:

[0087] Table 2 Plasmid information

[0088]

[0089] The primers involved in the examples are shown in Table 3:

[0090] Table 3 Primer information

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097] Example 1: Construction of plasmid-free gene editing system in Saccharomyces cerevisiae

[0098] (1) Preparation of crDNA and short homologous recombination repair donor DNA

[0099] crDNA was prepared by annealing two 87 nt reverse complementary oligonucleotides. When the oligonucleotides were annealed, the mixture containing 50 μΜ oligonucleotides in 1 × TE buffer was heated to 95 °C for 5 min, and then gradually cooled to 30 °C in 150 min. The volume of the annealing product was 1.5 μL (75 pmol or ~ 4 μg), which was directly used for transformation.

[0100] Short homologous recombination repair donor DNA was prepared by overlap extension of two oligonucleotides. The concentration of two oligonucleotides in 25 μL PCR mixture was 32 μΜ, which was amplified for 18 cycles, and then 5 μL (160 pmol) of homologous recombination repair donor DNA was directly used for transformation.

[0101] (2) Construction of plasmid pQX-KlURA3-T7RNAP

[0102] The pUD628 plasmid expressing crRNA targeting the ADE2 gene was purchased from Addgene. To express T7 RNA polymerase, the T7 RNA polymerase gene (amino acid sequence see WP_001092355) was amplified from the genomic DNA of E. coli BL21(DE3) using primer pair P23 / P24. The TDH3 promoter was amplified from the genomic DNA of S. cerevisiae S.cerevisiae SY03 (Reference: Yang, S., Cao, X., Yu, W., Li, S., & Zhou, Y.J. (2020). Efficient targeted mutation of genomic essential genes in yeast Saccharomyces cerevisiae. Applied Microbiology and Biotechnology, 104(7), 3037-3047) using primer pair P21 / P22, and the AgTEF2 terminator was amplified from pUD628 using primer pair P25 / P26. These three fragments were assembled into a vector backbone amplified from pECAS9-gRNA-KlURA3-tHFD1 (Zhu, Z., Zhou, Y.J., Kang, M.-K., Krivoruchko, A., Buijs, N.A., & Nielsen, J. (2017). Enabling the synthesis of medium chain alkanes and 1-alkenes in yeast. Metabolic Engineering, 44, 81-88) using primer pair P5 / P6, resulting in plasmid pQX-KlURA3-T7RNAP for expression of wild-type T7 RNA polymerase with an N-terminal nuclear localization signal.

[0103] (3) Construction of plasmids pECAS9-KlURA3-gRNA.X-2 and pECAS9-KlURA3-gRNA.ATF1

[0104] The 20 bp guide DNA sequence in pECAS9-gRNA-KlURA3-tHFD1 was replaced with sequences targeting the upstream region of the X-2 and ATF1 genes to obtain plasmids pECAS9-KlURA3-gRNA.X-2 and pECAS9-KlURA3-gRNA.ATF1. Specifically, four DNA fragments were amplified from plasmid pECAS9-gRNA-KlURA3-tHFD1 using primer pairs P1-P8, and T5 exonuclease-dependent assembly (Xia, Y., Li, K., Li, J., Wang, T., Gu, L., & Xun, L. (2018). T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis. Nucleic Acids Research, 47(3), e15) was used to obtain plasmid pECAS9-KlURA3-gRNA.X-2 targeting the X2 site. Similarly, four DNA fragments were amplified using primer pairs P2 / P3, P5-P8, gRNA-ATF1-F, and gRNA-ATF1-R, and T5 exonuclease-dependent assembly was used to obtain plasmid pECAS9-KlURA3-gRNA.ATF1 targeting the upstream of the ATF1 gene.

[0105] (4) Construction of crRNA expression plasmids

[0106] To construct the crRNA expression plasmids, the crRNA expression cassette (T7p-DR-crADE2-DR-T7t, where T7p is the T7 promoter, DR is the direct repeat sequence, T7t is the T7 terminator, and crADE2 is the crRNA for editing ADE2) was prepared by overlap extension of primer pairs P49 / P50, and the vector fragment was amplified from pECAS9-gRNA-kanMX-tHFD1 (derived from the same literature as pECAS9-gRNA-KlURA3-tHFD1) or pECAS9-gRNA-KlURA3-tHFD1 using primer pairs P5 / P6. The crRNA expression cassette and the vector fragment were assembled to obtain plasmids pKanMX-crADE2 or pKlURA3-crADE2.

[0107] (5) Construction of plasmids pIKanMX-crADE2 and pIScURA3-crADE2

[0108] The kanMX expression cassette in pKanMX-crADE2 (nucleotide sequence as shown in SEQ ID NO. 1, composed of AgTEF2p-kanMX-AgTEF2t) was replaced by a new kanMX and URA3 expression cassette (nucleotide sequence as shown in SEQ ID NO. 2), both of which were added with homologous arm sequences of about 70bp targeting the ura3-52 or URA3 allele upstream and downstream of the selection marker gene, allowing the selection marker gene to be integrated into the ura3-52 / URA3 locus. The kanMX cassette open reading frame plus overhang was amplified from pKanMX-crADE2 using primer pair P109 / P110, the URA3 cassette open reading frame plus overhang was amplified from pRS316 using primer pair P113 / P114, and the two fragments were assembled with the vector backbone amplified from pKanMX-crADE2 using primer pair P111 / P112, to generate plasmids pIKanMX-crADE2 and pIScURA3-crADE2.

[0109] (6) Construction of plasmids pIScURA3-crKanMX-2 and pIKanMX-crScURA3-2

[0110] The spacer sequence of the crRNA targeting ADE2 in pIKanMX-crADE2 was replaced by the spacer sequence targeting ura3-52 or URA3 to construct pIKanMX-crScURA3-2, and the spacer sequence of crADE2 in pIScURA3-crADE2 was replaced by the spacer sequence targeting kanMX to construct pIScURA3-crKanMX-2. The two DNA fragments amplified from pIKanMX-crADE2 were assembled using primer pair P119 / P116 and P120 / P118 to obtain pIKanMX-crScURA3-2. The two DNA fragments amplified from pIScURA3-crADE2 were assembled using primer pair P119 / P116 and P120 / P118 to obtain pIScURA3-crKanMX-2. The selection marker gene fragments without crDNA were amplified from pIKanMX-crScURA3-2 and pIScURA3-crKanMX-2 using primer pair P126 / P128, and the selection marker gene fragments with crDNA were amplified from pIKanMX-crScURA3-2 and pIScURA3-crKanMX-2 using primer pair P126 / P127; the amplified selection marker gene fragments were purified and used for yeast transformation.

[0111] (7) Construction of plasmids pUCIKanMX-crScURA3-2 and pUCIScURA3-crKanMX-2

[0112] To avoid false positive transformants caused by the contamination of intact plasmid DNA when purifying the screening marker gene fragments, the fragments amplified from pIKanMX-crScURA3-2 and pIScURA3-crKanMX-2 using primer pair P129 / P130 were circularized to obtain pUCIKanMX-crScURA3-2 and pUCIScURA3-crKanMX-2, respectively. These two plasmids do not have 2μ replication element to prevent replication in S. cerevisiae. These two plasmids were then used as templates to amplify the screening marker gene fragments using the following primer pairs: P126 / P128 (for screening marker gene fragments without crDNA) and P126 / P127 (for screening marker gene fragments with crDNA).

[0113] (8) Construction of strain ZQX01

[0114] FnCas12a expression cassette was integrated into the chromosome by CRISPR / Cas9-mediated genome editing (Zhu, Z., Zhou, Y. J., Kang, M.-K., Krivoruchko, A., Buijs, N. A., & Nielsen, J. (2017). Enabling the synthesis of medium chain alkanes and 1-alkenes in yeast. Metabolic Engineering, 44, 81-88). The 20 bp spacer sequence in pECAS9-gRNA-KlURA3-tHFD1 was replaced by the sequence targeting the upstream region of X-2 gene to construct the plasmid. FnCas12a expression cassette was amplified from pUDC175 using primer pair P9 / P10 and co-transformed with pECAS9-KlURA3-gRNA.X-2 into strain SY03 to integrate FnCas12a expression cassette into the X-2 site of SY03 strain to obtain ZQX01. The pUDC175 plasmid expressing Francisella novicida Cpf1 nuclease (FnCpf1) with a C-terminal nuclear localization signal and a 3xHA tag was purchased from Addgene.

[0115] (9) Construction of strain ZQX02

[0116] T7 RNA polymerase expression cassette was amplified from plasmid pQX-KlURA3-T7RNAP using primer pair P41 / P42, and 1 μg of pECAS9-KanMX-gRNA.ATF1 plasmid and 1 μg of T7 RNA polymerase expression cassette fragment were co-transformed into ZQX01 strain to integrate T7 RNA polymerase expression cassette upstream of ATF1 gene to obtain strain ZQX02.

[0117] (10) Removal of resistance plasmid

[0118] After colony PCR, streak the liquid culture with a loop onto YPD solid plates and place in a 30 °C incubator. After 36 h of growth, pick single colonies and inoculate into YPD liquid medium. After 12 h of growth, streak the liquid culture with a loop onto YPD solid plates and place in a 30 °C incubator. After 36 h of growth, pick single colonies and streak onto YPD plates with 5-FOA and 10 mM uridine. + The antibiotic plates are used to verify the loss of plasmid. If no lawn grows, it indicates that the plasmid has been lost.

[0119] (11) DNA fragment transformation

[0120] The linear PCR-amplified selection marker gene is integrated into the genome, and the crDNA targeting the gene of interest and the homologous recombination repair donor DNA fragment are transformed at the same time, which can achieve the editing of the gene of interest. Since there is no circular plasmid DNA transformation, there is no tedious plasmid loss step after successful editing. By alternately integrating two different selection marker gene expression cassettes at the fixed site of the genome, multiple rounds of editing can be conveniently completed. After each round of editing is completed, the selection marker gene does not need to be recovered, and the next round of editing can be performed, so that the time of each round of editing is shortened to 3 days. The process is shown in Figure 2 A.

[0121] Chemical transformation of S. cerevisiae is used to co-transform crDNA targeting ADE2, PDR12 or TPO1 and the corresponding homologous recombination repair donor DNA, as well as a selection marker gene fragment, to achieve the iterative replacement of the gene of interest and the selection marker gene. The specific transformation components are shown in Table 4.

[0122] Table 4 Transformation components

[0123]

[0124] (12) Result analysis

[0125] Two versions of the selection marker gene were constructed and compared for editing effect. One contains crDNA targeting the integration site of the selection marker gene, and the other does not contain crDNA. The results are shown in Figure 2 B.

[0126] Firstly, the ADE2 gene was edited using a kanMX selection marker gene fragment integrated into the ura3-52 locus, a crDNA targeting the ADE2 gene (crDNA.ADE2), and a 120 bp ADE2 knockout homologous recombination repair donor DNA (donor.ADE2). Both versions of the kanMX selection marker gene were 100% integrated, while the ADE2 gene knockout efficiency was over 30%. The addition of crDNA to the selection marker gene increased the number of transformants by more than 10 times, indicating that CRISPR / Cas12a-induced double-strand breaks significantly enhanced homologous recombination repair-mediated integration of the selection marker gene. Without the need for marker gene recovery, editing of PDR12 was immediately performed using a URA3 selection marker gene fragment integrated into the same locus, a crDNA targeting the PDR12 gene (crDNA.PDR12), and a 120 bp ADE2 knockout homologous recombination repair donor DNA (donor.PDR12). Both versions of the URA3 selection marker gene were 100% integrated, while the PDR12 gene knockout efficiency was over 50%. Next, editing of TPO1 was performed using the same kanMX selection marker gene fragment as in the first round of editing, a crDNA targeting the TPO1 gene (crDNA.TPO1), and a 120 bp ADE2 knockout homologous recombination repair donor DNA (donor.TPO1). Both versions of the kanMX selection marker gene were 100% integrated, while the PDR12 gene knockout efficiency was over 18%. The above experimental results demonstrate the feasibility of iterative gene editing independent of plasmid transformation.

[0127] Example 2: Integration of expression of T7 RNA polymerase and Cas12a nuclease in Starmerella bombicola

[0128] Using the hygromycin resistance gene as a selection marker gene, FnCas12a and T7 RNA polymerase were integrated for expression in the intergenic region of the chromosome of Starmerella bombicola NBRC 10243 strain to obtain NCZQX01. Starmerella bombicola NBRC 10243 was purchased from the Japan NBRC Strain Collection, which is equivalent to ATCC 22214, and is a strain that produces high levels of sophorolipid surfactants (Saerens, K. M. J., Saey, L., & Soetaert, W. (2011). One-step production of unacetylated sophorolipids by an acetyltransferase negative Candida bombicola. Biotechnology and Bioengineering, 108(12), 2923-2931.).

[0129] To integrate T7 RNA polymerase with 3xNLS sequence at N terminus, FnCas12a with 3xNLS sequence at C terminus and Hygromycin resistance gene (HygR), four DNA fragments (Fig. 1) were prepared: Figure 3

[0130] (1) Primer pair P155 / P156 was used to amplify 1 kb homologous sequence adjacent to SbFET3 gene from genomic DNA of S. bombicola (NBRC 10243). Primer pair P139 / P140 was used to amplify T7 RNA polymerase gene from plasmid pQX-KlURA3-T7RNAP, which also introduced a terminator T synth7 (Curran, K. A., Morse, N. J., Markham, K. A., Wagman, A. M., Gupta, A., & Alper, H. S. (2015). Short Synthetic Terminators for Improved Heterologous Gene Expression in Yeast. ACS Synthetic Biology, 4(7), 824-832). These two fragments were assembled into the first fragment by overlap extension PCR;

[0131] (2) Primer pair P37 / P140 was used to amplify 5’ end of T7 RNA polymerase gene from pQX-KlURA3-T7RNAP. Primer pair P141 / P142 and P143 / P144 were used to amplify SbENO1 promoter and SbTEF1 promoter from genomic DNA of S. bombicola, respectively. These three fragments were assembled into the second fragment;

[0132] (3) Primer pair P145 / P146 was used to amplify FnCas12a gene from pUDC175. Primer pair P143 / P144, P147-P150 was used to amplify SbTEF1 promoter, SbTEF1 terminator and SbGPD1 terminator from genomic DNA of S. bombicola. These four fragments were assembled into the third fragment;

[0133] (4) Primer pair P151 / P152 was used to amplify HygR gene from pUDP082. Primer pair P147-P150, P153 / P154, P157 / P158 was used to amplify SbTEF1 terminator, SbGPD1 terminator, SbGPD1 promoter and 1 kb homologous sequence adjacent to SbGWT1 from genomic DNA of S. bombicola. These five fragments were assembled into the fourth fragment.​

[0134] Four fragments were equimolar amounts (2.97 μg in total) transformed into S. bombicola NBRC10243 strain. Transformants were selected on YPD+Hyg medium. PCR detection was performed using the following primers to confirm the correct integration of the expression cassette: P159 / P38, P141 / P152, P161 / P162, P160 / P13. Eight transformants were colony PCR verified, of which five carried the expected bands, the expression cassette was successfully integrated, the resulting strains were named NCZQX01 (Strain identification PCR results are shown in Figure 4

[0135] Example 3: Construction of uracil auxotrophic strain of S. bombicola

[0136] The 300 bp homologous sequences flanking SbURA3 were amplified from the genomic DNA of S. bombicola S. bombicola using primers P170-P173, which were used to replace the selection marker gene in plasmid pUCIKanMX-crScURA3-2, i.e. the spacer sequence of crRNA in the plasmid was replaced with the target sequence of SbURA3 gene and the vector backbone of the plasmid was obtained using primer pairs P166 / P175 and P174 / P169, the above four DNA fragments were obtained by T5 exonuclease-dependent assembly T5 cloning to obtain plasmid pUCIura3-crSbURA3-1.

[0137] The 300 bp homologous sequences flanking SbURA3 were amplified from the genomic DNA of S. bombicola S. bombicola using primers P170-P173, which were used to replace the selection marker gene in plasmid pUCIKanMX-crScURA3-2, i.e. the spacer sequence of crRNA in the plasmid was replaced with the target sequence of SbURA3 gene and the vector backbone of the plasmid was obtained using primer pairs P166 / P167 and P168 / P169, the above four DNA fragments were obtained by T5 exonuclease-dependent assembly to obtain plasmid pUCIura3-crSbURA3-2.

[0138] ​The SbURA3 knockout cassettes were amplified from plasmids pUCIura3-crSbURA3-1 and pUCIura3-crSbURA3-2 using primer pair P176 / P127, which contained the same homologous arm sequences and crDNA targeting different positions in the coding region of SbURA3. Two μg of each of the two knockout cassette DNA fragments were co-transformed into the NCZQX01 strain, and the transformation components are shown in Table 5. Transformants were selected on YPD+5FOA plates. Colony PCR was performed using primer pair P164 / P165 to verify the presence of a 423 bp deletion in the SbURA3 coding sequence. Out of six randomly picked colonies, one colony had the desired 423 bp deletion in SbURA3, and this uracil auxotrophic strain was named NCZQX03 Figure 5

[0139] Table 5 Transformation components

[0140]

[0141] Example 4: Plasmid-free gene editing to knock out the hygromycin resistance gene

[0142] To verify that crDNA can facilitate gene editing, the hygromycin resistance gene integrated in Example 2 was chosen as the target, and crDNA was prepared. The crDNA was prepared by annealing two 87 nt reverse complementary oligonucleotides. To anneal the oligonucleotides, a mixture containing 50 μΜ oligonucleotides in lx TE buffer was heated to 95 °C for 5 min, and then gradually cooled to 30 °C over 150 min. The volume of the annealed product was 1.860 μί (93 pmol or ~5 μg), which was used directly for transformation (Method same as Example 1).

[0143] A homologous recombination repair donor DNA to knock out the hygromycin resistance gene was constructed using the SbURA3 gene as a selection marker. Two DNA fragments flanking the HygR gene were amplified from the genomic DNA of strain NCZQX01 using primer pairs P184 / P185 and P187 / P188, and fused with the SbURA3 fragment amplified from the genomic DNA using primer pair P164 / P186 to generate a homologous recombination repair donor DNA cassette HygR::SbURA3 containing SbURA3 flanked by 300 bp homologous arms.

[0144] Five μg of the homologous recombination repair donor DNA and 5 μg (1.860 μί, ~93 pmol) of the crDNA targeting HygR (crDNA.HygR-2) were co-transformed into strain NCZQX03 Figure 6 A), and the transformation components are shown in Table 6. The number of transformants was more than 10 3 ​, colony PCR verification showed that all colonies successfully replaced the selection marker gene. In the control experiment, only the homologous recombination repair donor DNA or crDNA was transformed into strain NCZQX03. Transformants were selected on SD-URA medium, and 10 transformant colonies were picked for PCR identification. Colony PCR identification used primer pair P14 / P186, and strain NCZQX03 was used as a control. Only the transformation of the homologous recombination repair donor DNA produced 29 colonies, and only 90% (9 / 10) of the colonies completed the selection marker gene replacement. Only the transformation of crDNA did not produce transformants due to the inability to obtain the SbURA3 gene. The above results show that in the T7 RNA polymerase and FnCas12a-expressing Y. lipolytica strain, crDNA can form functional crRNA in vivo, and guide Cas12a to produce double-strand breaks at the target site, thereby enhancing homologous recombination. SbURA3 successfully replaced the HygR gene, and the resulting strain was named NCZQX05 Figure 6 B), which is a uracil prototrophic strain and has lost the hygromycin resistance.

[0145] Table 6 Transformation components

[0146]

[0147]

[0148] Example 5: LEU2 gene editing in Y. lipolytica

[0149] 1. Plasmid construction

[0150] The HygR open reading frame in pUDP082 was amplified using primer pair P151 / P152 and the 300bp flanking sequences of the SbLEU2 gene were amplified from the Y. bombicola genome using primer pair P189-P192. The selection marker fragment without crDNA (leu2::HygR) was obtained by fusion through overlap PCR. The fusion fragment and two DNA fragments containing the vector backbone (P193 / P194, P195 / P196) amplified from pUCIScURA3-crKanMX-2 were used to construct the plasmid pUCIHygR-crSbLEU2-1 through T5 cloning.

[0151] 2. DNA fragment preparation

[0152] The selection marker gene fragments amplified from pUCIHygR-crSbLEU2-1 with primer pairs P189 / P192 (for selection marker DNA fragment leu2::HygR without crDNA) and P189 / P127 (for selection marker DNA fragment leu2::HygR-crDNA.LEU2) with the crDNA were purified.

[0153] The fusion product of the two 300 bp homologous sequences flanking the target editing site was used as the long homology recombination repair donor DNA (about 600 bp). The long homology recombination repair donor DNA fragments (e.g. 600 bp) were generated by overlap extension PCR and then purified before transformation. For example, long homology recombination repair donor DNA.SbADE2 was generated by overlap extension PCR using primer pairs P217-P220 to amplify two 300 bp flanking sequences from S. bombicola genome.

[0154] The crDNA was prepared by annealing two 87 nt reverse complementary oligonucleotides. The mixture containing 50 μΜ oligonucleotides in lx TE buffer was heated to 95 °C for 5 min and then gradually cooled to 30 °C over 150 min. The volume of the annealed product was 1.860 μί (93 pmol or ~5 μg) and was used directly for transformation (same as Example 1).

[0155] 3. Transformation method optimization

[0156] The electroporation conditions were optimized to improve transformation efficiency. The optimized electroporation protocol was performed with slight modifications from the previously described protocol (Benatuil, L., Perez, J. M., Belk, J., & Hsieh, C.-M. (2010). An improved yeast transformation method for the generation of very large human antibody libraries. Protein Engineering, Design and Selection, 23(4), 155-159). Briefly, the overnight culture was inoculated into 50 mL YPD medium with initial OD 600 at 0.15-0.2 and then incubated at 30 °C with 250 rpm shaking until OD 600 at 0.4-2.0. The 400 μί electroporation buffer used for each transformation contained about 5 x 10 750 μg of single-stranded carrier DNA was also added to the electroporation buffer for co-transformation with the linear DNA fragments. After transformation, the cells were incubated in 1 mL of 1 M sorbitol and YPD medium at 1 : 1 mixture at 30°C for 1-2 hours, and then plated on the selection medium plates. This modified protocol increased the transformation efficiency by more than 10 times.

[0157] The optimized electroporation method is shown in the following steps:

[0158] (1) Pick a single colony of yeast cells into a test tube, and culture in YPD medium to an OD 600 value that can be transferred.

[0159] (2) Transfer the yeast cells to 50 mL of YPD medium (250 mL sterile flask), with an initial concentration of OD 600 = 0.10-0.2.

[0160] (3) Continue to culture in a constant temperature shaker at 30°C and 250 rpm until the OD 600 is about 0.4-2.0.

[0161] (4) Centrifuge the yeast cells at 3000 g for 5 minutes, and then remove the culture medium.

[0162] (5) Wash the cells twice with 50 mL of ice-cold sterile water, and once with 20-50 mL of ice-cold electroporation buffer (1 M sorbitol / 1 mM CaCl2).

[0163] (6) After centrifugation, resuspend the cell pellet in 20 mL of 0.1 M LiAc / 10 mM DTT, and shake in a flask at 250 rpm and 30°C for 30 minutes to adjust the yeast cells.

[0164] (7) Centrifuge to collect the matured cells, wash once with 20-50 mL of ice-cold electroporation buffer, and resuspend the cell pellet in an appropriate amount of electroporation buffer (an appropriate amount for adjusting the number of cells for electroporation).

[0165] (8) Use 5 x 10 7 cells / 400 μL for electroporation transformation (the total volume of DNA is less than 50 μL).

[0166] (9) Gently mix 400 μL of electroporation competent cells and DNA (including crDNA, homologous recombination repair donor DNA, selection marker DNA fragments, and 50 μg of denatured salmon sperm DNA), and transfer to a pre-cooled electroporation cup (0.2 cm electrode gap), and keep on ice for 5 minutes until electroporation transformation.

[0167] (10) Electroporate cells at 2.5 kV (typical time constant range 3.0 to 4.5 ms) and immediately add 1 mL of sorbitol:YPD = 1:1 medium to the cuvette.

[0168] (11) Transfer the electroporated cells and medium from each cuvette to a 2 mL sterile centrifuge tube. After incubation at 250 rpm and 30 °C for 1-2 hours on a constant temperature shaker, plate an appropriate amount of cells on selection medium.

[0169] 4. DNA fragment transformation

[0170] Based on the above optimization configuration, this embodiment uses an improved electroporation method to achieve the replacement of the target gene and the selection marker DNA by transforming crDNA targeting SbADE2 and the corresponding homologous recombination repair donor DNA, and screening the selection marker DNA fragment. The transformation components are shown in Table 7.

[0171] Table 7 Transformation components

[0172]

[0173]

[0174] 5. Result analysis

[0175] Although SbURA3 can be used as a selection marker gene, Ura + Transformants need to be cultured for a long time (about 9 days) on SD-URA plates to form. In addition, the growth of Ura-parental strains (such as NCZQX03) is very poor Figure 7 , and it takes more than 13 days to form colonies on YPD+5FOA plates. Therefore, SbLEU2 is used as an alternative selection marker gene, and Leu + colonies are usually formed within 5 days, which can significantly shorten the gene editing time of S. bombicola.

[0176] NCZQX05 was transformed with 5 μg of the selection marker gene leu2::HygR-crSbLEU2 or an equimolar amount of leu2::HygR (4.4 μg) using the reported transformation method (Saerens KMJ, Saey L, Soetaert W. One-step production of unacetylated sophorolipids by an acetyltransferase negative Candida bombicola[J]. Biotechnology and Bioengineering, 2011, 108(12):2923-2931.). The number of transformants was 162 and 2, respectively, and the integration efficiencies of the selection marker gene were 88% (14 / 16) and 50% (1 / 2), respectively.

[0177] Due to the small number of transformants, the transformation method was optimized, and the integration rate of the selection marker gene fragments transformed by the two methods was compared. Using the previously reported electroporation protocol (Saerens, KMJ, Saey, L., & Soetaert, W. (2011). One-step production of unacetylated sophorolipids by anacetyltransferase negative Candida bombicola. Biotechnology and Bioengineering, 108(12), 2923-2931), strain NCZQX05 was transformed with crDNA containing the SbLEU2 gene and a HygR selection marker gene fragment with a 2×300bp homologous arm. The HygR gene replaced the SbLEU2 gene. Figure 8 A). This method produced approximately 10 3 10 colonies. Using an optimized electroporation protocol (Benatuil, L., Perez, JM, Belk, J. & Hsieh, C.-M. An improved yeast transformation method for the generation of very large human antibody libraries. Protein Eng Des Sel 23, 155-159), the number of transformants increased to over 10. 5 ( Figure 8B). The editing of SbADE2 in the NCZQX05 strain was tested by transformation of three linear DNA fragments. More than 5 x 105transformants were obtained after transformation of the HygR selection marker gene fragment with the crDNA targeting SbLEU2 (for selection marker gene integration) and the crDNA targeting SbADE2 and 600 bp homologous recombination repair donor DNA (for SbADE2 deletion). Diagnostic PCR showed 100% efficiency of SbADE2 deletion and selection marker gene integration. 3 Figure 8 C). In contrast, when the selection marker gene lacks the crDNA targeting SbLEU2, only 79 colonies were obtained, which indicates that induction of double-strand break stimulates homologous recombination for selection marker gene integration. By comparing the effect of selection marker gene fragment transformation DNA amount (1-5 μg) on the number of transformants and editing efficiency, it was determined that transformation of 2 μg of selection marker gene fragment is sufficient for high efficiency editing. Figure 8 D).

[0178] Example 6: Plasmid-free iterative genome editing in Yarrowia lipolytica

[0179] (1) Plasmid construction

[0180] The SbLEU2 gene with 300 bp flanking sequences (leu2::LEU2) was amplified from S. bombicola genome with primer pair P189 / P192 and two DNA fragments containing vector backbone were amplified from pUCIScURA3-crKanMX-2 (primer pair P195 / P203, P194 / P204) by T5 cloning to construct pUCISbLEU2-crHyg-2.

[0181] (2) DNA fragment preparation

[0182] Selection marker gene fragments were amplified from pUCIHygR-crSbLEU2-1 with primer pair P189 / P192 (for selection marker DNA fragment leu2::HygR without crDNA) and P189 / P127 (for selection marker DNA fragment leu2::HygR-crDNA.LEU2 with crDNA) and purified. Selection marker gene fragments were amplified from pUCISbLEU2-crHyg-2 with primer pair P189 / P192 (for selection marker DNA fragment leu2::LEU2 without crDNA) and P189 / P127 (for selection marker DNA fragment leu2::LEU2-crDNA.HygR with crDNA) and purified.

[0183] ​The preparation of long homologous recombination repair donor DNA (about 600 bp) and crDNA is described in reference example 5.

[0184] (3) Iterative editing

[0185] Using the modified electroporation method in example 5, the iterative replacement of the target gene editing and selection marker DNA was achieved by co-transformation of crDNA targeting SbADE2, SbUGTA1 or SbKU70 with the corresponding homologous recombination repair donor DNA, and a selection marker DNA fragment. The transformation components are shown in Table 8.

[0186] Table 8 Transformation components

[0187]

[0188] (4) Results analysis

[0189] An iterative gene editing was performed in S. bombicola using a similar strategy as in example 1, but using 600 bp (2 x 300 bp) homologous recombination repair donor DNA for homologous recombination repair. Two different selection marker genes were alternately integrated at a genomic fixed site (SbLEU2 locus), carrying hygromycin resistance gene HygR and leucine auxotrophy selection gene SbLEU2, respectively. Two versions of selection marker gene editing were also constructed and compared, one containing crDNA targeting the integration site of the selection marker gene, and the other without crDNA.

[0190] The editing of SbADE2 gene was first performed using the HygR selection marker gene fragment integrated into SbLEU2 locus, crDNA targeting SbADE2 gene (crDNA.SbADE2) and 600 bp SbADE2 knockout homologous recombination repair donor DNA (donor.SbADE2). Both versions of HygR selection marker gene were 100% integrated, while the SbADE2 gene knockout efficiency was close to 100%. Similar to example 1, the addition of crDNA to the selection marker gene fragment significantly increased the number of transformants, also indicating that the CRISPR / Cas12a induced double-strand break significantly enhanced the homologous recombination repair mediated integration of the selection marker gene. Without the need for marker gene recovery, the editing of SbUGTA1 gene was immediately performed using the SbLEU2 selection marker gene expression cassette integrated into the same locus, crDNA targeting SbUGTA1 gene (crDNA.SbUGTA1) and 600 bp SbUGTA1 knockout homologous recombination repair donor DNA (donor.UGTA1). The addition of crDNA to the selection marker gene fragment simultaneously improved the integration efficiency of SbLEU2 selection marker gene and the knockout efficiency of SbUGTA1 gene Figure 9A). Next, the SbKU70 gene was edited, using the same HygR selection marker gene fragment, crDNA targeting the KU70 gene (crDNA.KU70), and 600 bp SbKU70 knockout homologous recombination repair donor DNA (donor.KU70) as the first round of editing, and the addition of crDNA to the selection marker gene fragment also improved the integration efficiency of the HygR selection marker gene and the SbKU70 gene knockout efficiency Figure 9 B). The above experimental results prove the feasibility of iterative gene editing in S. bombicola independent of plasmid transformation.

[0191] Example 7: Plasmid-free multi-gene editing of Ogataea parasp..

[0192] At the same time, three crDNAs targeting SbADE2, SbUGTA1 and SbKU70 were transformed, and the corresponding homologous recombination repair donor DNA and selection marker gene fragment containing crDNA were prepared by the same method as in Example 5, and the transformation components are shown in Table 9. When targeting two sites of SbADE2 and SbKU70, 100% efficiency was achieved for simultaneous editing of the two sites; and when targeting two sites of SbADE2 and SbUGTA1, 90% efficiency was achieved for simultaneous editing of the two sites. However, when targeting three sites (SbADE2, SbUGTA1 and SbKU70), no colonies were obtained for simultaneous editing of the three genes, which may be due to the low activity of the crRNA targeting SbUGTA1. This example demonstrates that multiple editing can be achieved by co-transformation of multiple crDNA / homologous recombination repair donor DNA and a single selection marker gene Figure 10 ).

[0193] Table 9 Transformation components

[0194]

[0195]

[0196] Example 8: Improving the homologous recombination ability of Ogataea parasp..

[0197] (1) Construction of strains NCZQX07 and NCZQX08

[0198] Two flanking sequences (322 and 300 bp, respectively) of the SbKU70 gene were amplified from the genomic DNA of S. bombicola using primer pair P237-240, and fused by overlap extension PCR to generate the homologous recombination repair donor DNA cassette for SbKU70 gene deletion. As described in Example 5, 5 μg of the homologous recombination repair donor DNA, 5 μg (about 93 pmol) of the crDNA targeting SbKU70 (crDNA.SbKU70), and 2 μg of the HygR selection marker gene fragment (leu2::HygR-crDNA.LEU2) were co-transformed into strain NCZQX05 to generate strain NCZQX08. Only 2 μg of the HygR selection marker gene fragment (leu2::HygR-crDNA.LEU2) was transformed into the NCZQX05 strain to obtain the control strain NCZQX07. Transformants were selected on YPD+Hyg plates. Diagnostic PCR was performed using primer pairs P241 / P242 and P199 / P200, P201 / P202, respectively, to verify the SbKU70 deletion and the integration of HygR at the SbLEU2 locus.

[0199] (2) Construction of strains NCZQX09 and NCZQX10

[0200] The ScRAD52 and SbRAD52 genes were amplified from the genomic DNA of S. cerevisiae and S. bombicola, respectively. The ScRAD52 gene was amplified using primer pair P246 / P247, and the SbRAD52 gene was amplified using primer pair P251 / P252. Both genes were fused with the SbGPD1 promoter and terminator (obtained from the genomic DNA using primer pairs P154 / P245, P248 / P149), and two homologous flanking sequences were amplified from the S. bombicola genome using primer pairs P243 / P244, P249 / P250. 4 μg of either fusion fragment was co-transformed with 5 μg (about 93 pmol) of the crDNA targeting the intergenic region between STBO_02450 and STBO_02451 (crDNA.STBO_02450) and 2 μg of the HygR selection marker gene fragment (leu2::HygR-crDNA.LEU2) into strain NCZQX05 to generate strains NCZQX09 and NCZQX10. Diagnostic polymerase chain reaction was performed using primer pairs P255 / P256 and P205 / P201, respectively, to confirm the insertion of the RAD52 gene cassette near the STBO_02450 gene and the integration of HygR at the SbLEU2 locus.

[0201] (3) Preparation of DNA fragments

[0202] The preparation of the screening marker DNA fragment (leu2::LEU2-crDNA.HygR, leu2::HygR-crDNA.LEU2) was as described in Example 6.

[0203] The preparation of the long homologous recombination repair DNA fragment (600 bp) for SbADE2 gene knockout was as described in Example 5. The different length short homologous recombination repair DNA fragments for SbADE2 gene knockout were prepared by annealing extension. Specifically, the 80 bp homologous recombination repair donor DNA was obtained by annealing extension using primers P215 / P216; the 100 bp homologous recombination repair donor DNA was obtained by annealing extension using primers P213 / P214; and the 120 bp homologous recombination repair donor DNA was obtained by annealing extension using primers P211 / P212. The concentration of the two oligonucleotides in 25 μL of PCR mixture was 32 μM, and 18 cycles of amplification were performed. Then, 7 μL (224 pmol, ~16.8 μg) or 10 μL (320 pmol, ~24 μg) of the homologous recombination repair donor DNA was directly used for transformation.

[0204] The preparation of crDNA was as described in Example 4. crDNA.SbADE2 was prepared by annealing two 87 nt reverse complementary oligonucleotides (P207 / P208). However, 87 bp crDNA.SbADE2-1 was prepared by overlap extension of two oligonucleotides (P261 / P262). The concentration of the two oligonucleotides in 25 μL of PCR mixture was 32 μM, and 18 cycles of amplification were performed. Then, 5.812 μL (185.984 pmol, ~10 μg) of the homologous recombination repair donor DNA was directly used for transformation.

[0205] (4) DNA fragment transformation

[0206] The improved electroporation method described in Example 5 was used to transform crDNA targeting ADE2, 600 bp or 120 bp homologous recombination repair DNA, and screening marker gene fragments into NCZQX05, NCZQX07, NCZQX08, NCZQX09, and NCZQX10. The transformation components are shown in Table 10, and the number of transformants and the editing efficiency of the SbADE2 gene were compared.

[0207] Table 10 Transformation components

[0208]

[0209] (5) Gene editing method using short homologous recombination repair donor DNA for NCZQX10

[0210] Based on the above optimized configuration, the application establishes a method for gene editing using short homologous recombination repair donor DNA. Using the optimized electroporation method, by simultaneously transforming the crDNA and the corresponding homologous recombination repair donor DNA for SbADE2, and screening the marker DNA fragment to realize the replacement of the target gene and the screening marker DNA. The transformation components are shown in Table 11.

[0211] Table 11 Transformation components

[0212]

[0213] (6) Result analysis

[0214] Firstly, the effect of the length of the homologous recombination repair DNA fragment in the S. bombicola strain NCZQX05 on the SbADE2 editing efficiency was compared. 5 μg of crDNA, corresponding homologous recombination repair donor DNA and 2 μg of screening marker gene fragment were used for transformation. Using 5 μg of long homologous recombination repair donor DNA (2 x 300 bp), 10 3 transformants were obtained, and the SbADE2 gene editing efficiency reached 100% (10 / 10), while using 120 bp short homologous recombination repair donor DNA, even if the amount of homologous recombination repair donor DNA was increased, the number of transformants was still less than 10 Figure 11 ).

[0215] Knocking out the endogenous SbKU70 gene or overexpressing the ScRAD52 gene of S. cerevisiae obtained the NCZQX08 and NCZQX09 strains, and the homologous recombination activity of these two strains was not enhanced. Overexpression of the endogenous SbRAD52 gene obtained the NCZQX10 strain, and its homologous recombination activity was significantly enhanced. Using 120 bp short homologous recombination repair donor DNA, the number of transformants was more than 10 4 , and the editing efficiency of SbADE2 gene reached 100% Figure 12 ). The NCZQX10 strain has high homologous recombination ability. By quantitatively comparing the gene editing efficiency and colony forming units (CFU), it has been proved that overexpression of the endogenous gene SbRAD52 can significantly enhance the homologous directed repair (HDR) ability. In this genetic background, only short homologous recombination repair donor DNA fragments with a total length of 120 bp (2 x 60 bp) on both sides of the homologous arm can realize efficient plasmid-free genome editing. This breakthrough greatly simplifies the complexity of homologous recombination repair donor construction.

[0216] To explore the HDR activity limit of the strain, the length of the homologous recombination repair donor DNA was further shortened to 80 bp, and the editing efficiency of the ADE2 gene was basically not affected Figure 13 ), indicating that the NCZQX10 strain has excellent homologous recombination ability and can use a short homologous arm of 40 bp for efficient gene editing. In the NCZQX10 strain overexpressing SbRAD52, 80 bp homologous arms are sufficient to support precise HDR processes, breaking the lower limit requirement of homologous arm length in traditional cognition. Enhancement of the intrinsic recombination ability of the strain: The NCZQX10 strain exhibits an abnormally active homologous recombination mechanism, and the recognition and utilization efficiency of the endogenous repair system for micro-homologous sequences are greatly enhanced. Establishment of a high-efficiency editing platform: This result confirms that the NCZQX10 strain has a unique ability to use very short homologous recombination repair donor DNA (≤80 bp) to achieve high-precision editing, providing a technical basis for rapid, multi-site genome modification.

[0217] Overexpression of SbRAD52 not only solves the HDR efficiency bottleneck of short homologous recombination repair donor DNA (120 bp), but also endows the strain with the excellent property of using ultra-short homologous arms (80 bp) to maintain high-efficiency editing. The ultra-high homologous recombination activity exhibited by the NCZQX10 strain makes it an ideal tool strain for simplified, scarless gene editing in S. bombicola and potentially other non-model yeasts.

[0218] Example 9: Optimization of the addition amount of screening marker DNA fragment to improve gene editing efficiency

[0219] The crDNA.SbUGTA1 was prepared by annealing two 87 nt reverse complementary oligonucleotides using the method described in Example 4. The donor.SbUGTA1 of 120 bp was prepared using the method described in Example 8. The screening marker gene fragment leu2::LEU2-crDNA.HygR was prepared using the method described in Example 6.

[0220] The improved electroporation method described in Example 5 was used to transform NCZQX10 with crDNA targeting SbUGTA1, homologous recombination repair DNA (120 bp), and different amounts of screening marker gene fragments. The transformation components are shown in Table 12, and the number of transformants and the editing efficiency of the SbUGTA1 gene were compared.

[0221] Table 12 Transformation components

[0222]

[0223] The experimental results show that the knockout efficiency of SbUGTA1 in Examples 6 and 7 is not high. In this example, the amount of the screening marker gene fragment is reduced from 3 μg to 0.25 μg, and although the number of transformants is also reduced from 2.9 x 10 4 to 2.6 x 10 3 , the editing efficiency of SbUGTA1 is increased from 30% to 100%. This example proves that the editing efficiency of the target gene can be increased by increasing the molar ratio of the target gene editing fragment (crDNA and homologous recombination repair donor DNA) to the screening marker gene fragment.

[0224] Example 10: Fusion of crDNA and homologous recombination repair donor DNA

[0225] (1) Preparation of DNA fragments

[0226] (A) (B) (C) of crSbADE2-1+donorDNA are obtained by two rounds of PCR.

[0227] Specifically, (A) crSbADE2-1+donorDNA is prepared by first preparing 25 μL of PCR mixture containing oligonucleotides P207, P263, P215 and P216 at a concentration of 32 μM each, amplifying for 18 cycles, and then taking 0.5 μL as a template for the second round of PCR, using primer pair P264 / P265 for PCR amplification, to obtain a 187 bp product A.

[0228] (B) crSbADE2-1+donorDNA is prepared by first preparing 25 μL of PCR mixture containing oligonucleotides P266, P267, P215 and P216 at a concentration of 32 μM each, amplifying for 18 cycles, and then taking 0.5 μL as a template for the second round of PCR, using primer pair P264 / P265 for PCR, to obtain a 168 bp product B.

[0229] (C) crSbADE2-1+donorDNA is prepared by first preparing 25 μL of PCR mixture containing oligonucleotides P268, P269, P215 and P216 at a concentration of 32 μM each, amplifying for 18 cycles, and then taking 0.5 μL as a template for the second round of PCR, using primer pair or P264 / P270 for PCR, to obtain a 178 bp product C.

[0230] crSbADE2-1+donorDNA Thio-modified DNA fragment is a DNA fragment with thio-modification (Phosphorothioate, abbreviated as ph, or represented by *) obtained by using thio-modified primers at both ends in the second round of PCR. Specifically, (A) crSbADE2-1+donorDNA (Ph) is obtained by using primer pair P271 / P272 in the second round of PCR to amplify the DNA product A (Ph) with 187 bp of thio-modification at both ends.

[0231] (B) crSbADE2-1+donorDNA (Ph) is obtained by using primer pair P271 / P272 in the second round of PCR to amplify the DNA product B (Ph) with 168 bp of thio-modification at both ends.

[0232] (C) crSbADE2-1+donorDNA (Ph) is obtained by using primer pair P271 / P273 in the second round of PCR to amplify the DNA product C (Ph) with 178 bp of thio-modification at both ends.

[0233] The screening marker gene fragment leu2::LEU2-crDNA.HygR was prepared by the method described in Example 6.

[0234] (2) DNA fragment transformation

[0235] The combination fragment of the crDNA targeting ADE2 and the homologous recombination repair DNA, and the screening marker gene fragment were transformed into NCZQX10 by the improved electroporation method described in Example 5, and the transformation components are shown in Table 13, and the number of transformants and the editing efficiency of the SbADE2 gene were compared.

[0236] Table 13 Transformation components

[0237]

[0238] Based on the optimization configuration of Example 9, the replacement of the target gene and the screening marker DNA was realized by transforming the thio-modified DNA fragment of the fusion DNA fragment targeting ADE2 (crDNA+donorDNA (Ph)) and the screening marker DNA fragment. The transformation components are shown in Table 14.

[0239] Table 14 Transformation components

[0240]

[0241] (3) Result analysis

[0242] The embodiment adopts three ways to combine crDNA and homologous recombination repair donor DNA, and the lengths of the designed fragments are 187 bp, 168 bp and 178 bp, respectively Figure 15 A). Comparing the editing efficiencies of the three DNA fragments, it is determined that the editing efficiency of A fragment is the highest, reaching 90%; the editing efficiency of B fragment is only 10%; and the editing efficiency of C fragment is 40% Figure 15 B). Since the length of the edited DNA is compatible with high-throughput DNA synthesis, it lays a foundation for constructing an edited DNA library and performing genome-scale gene editing.

[0243] After introducing thio modification into the three DNA fragments, and using the screening marker addition amount in embodiment 9, it is found that the editing efficiencies of the three DNA fragments are all improved, especially the editing efficiency of B fragment is improved to 70%. Using A fragment, the editing efficiency is still the highest, reaching 100%; and the editing efficiency of C fragment is also improved to 80% Figure 15 C).

[0244] Obviously, the above embodiments are only examples for the purpose of clarity, and are not a limitation on the embodiments. For those skilled in the art, other different forms of changes or variations can be made on the basis of the above description. Here, it is not necessary and impossible to exhaust all the embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A plasmid-free gene editing system based on T7 RNA polymerase, characterized in that, The plasmid-free gene editing system includes the following components: (1) T7 RNA polymerase; (2) Cas12a nuclease; (3) Screening for marker genes; (4) A short double-stranded DNA expression cassette, wherein the short double-stranded DNA expression cassette comprises, in sequence, a T7 promoter, a first positive repeat sequence, a spacer sequence, and a second positive repeat sequence.

2. The plasmid-free gene editing system according to claim 1, characterized in that: The T7 RNA polymerase and the Cas12a nuclease are modified with nuclear localization sequences at their ends.

3. The plasmid-free gene editing system according to claim 1, characterized in that: The plasmid-free gene editing system also includes the RAD52 protein.

4. The application of the plasmid-free gene editing system according to any one of claims 1-3 in fungal gene editing.

5. A gene editing method, characterized in that: The plasmid-free gene editing system and the expression cassette containing donor DNA described in any one of claims 1-3 are transformed into a host bacterium, wherein the host bacterium is a fungus.

6. The gene editing method according to claim 5, characterized in that: The T7 RNA polymerase and the Cas12a nuclease are integrated and expressed in the host bacteria.

7. The gene editing method according to claim 5, characterized in that: It also includes the step of splicing the short double-stranded DNA expression cassette and the expression cassette containing donor DNA.

8. The gene editing method according to claim 7, characterized in that, The splicing includes any of the following: (1) Link the donor DNA expression cassette downstream of the second positive repeat sequence of the short double-stranded DNA expression cassette; (2) Link the donor DNA expression cassette downstream of the spacer sequence of the short double-stranded DNA expression cassette; (3) Link the donor DNA expression cassette upstream of the first positive repeat sequence of the short double-stranded DNA expression cassette.

9. The gene editing method according to claim 7, characterized in that: The molar ratio of the donor DNA expression cassette, the short double-stranded DNA expression cassette or splice fragment to the screening marker gene is (1-1000):

1.

10. The gene editing method according to claim 5, characterized in that: It also includes the step of thiomodifying the ends of the donor DNA or the short double-stranded DNA expression cassette.

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