CN117683803A - Gene editing system with high homologous recombination efficiency for pichia pastoris and application - Google Patents
Gene editing system with high homologous recombination efficiency for pichia pastoris and application Download PDFInfo
- Publication number
- CN117683803A CN117683803A CN202211103030.6A CN202211103030A CN117683803A CN 117683803 A CN117683803 A CN 117683803A CN 202211103030 A CN202211103030 A CN 202211103030A CN 117683803 A CN117683803 A CN 117683803A
- Authority
- CN
- China
- Prior art keywords
- gene
- pichia pastoris
- cas9
- exonuclease
- homologous recombination
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241000235058 Komagataella pastoris Species 0.000 title claims abstract description 64
- 230000006801 homologous recombination Effects 0.000 title claims abstract description 39
- 238000002744 homologous recombination Methods 0.000 title claims abstract description 39
- 238000010362 genome editing Methods 0.000 title claims abstract description 24
- 108091033409 CRISPR Proteins 0.000 claims abstract description 55
- 108060002716 Exonuclease Proteins 0.000 claims abstract description 48
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 39
- 230000004927 fusion Effects 0.000 claims abstract description 28
- 239000012634 fragment Substances 0.000 claims abstract description 19
- 230000010354 integration Effects 0.000 claims abstract description 17
- 239000013604 expression vector Substances 0.000 claims description 30
- 101150006234 RAD52 gene Proteins 0.000 claims description 21
- 239000013612 plasmid Substances 0.000 claims description 21
- 101100355599 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) mus-11 gene Proteins 0.000 claims description 20
- 102000053062 Rad52 DNA Repair and Recombination Human genes 0.000 claims description 20
- 108700031762 Rad52 DNA Repair and Recombination Proteins 0.000 claims description 20
- 101150071637 mre11 gene Proteins 0.000 claims description 20
- 230000002018 overexpression Effects 0.000 claims description 10
- 238000010367 cloning Methods 0.000 claims description 9
- 150000007523 nucleic acids Chemical group 0.000 claims description 9
- 101150112849 EXO1 gene Proteins 0.000 claims description 6
- 108010052305 exodeoxyribonuclease III Proteins 0.000 claims description 6
- 241000588724 Escherichia coli Species 0.000 claims description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 5
- 238000010354 CRISPR gene editing Methods 0.000 claims description 4
- 238000012408 PCR amplification Methods 0.000 claims description 4
- 210000000349 chromosome Anatomy 0.000 claims description 4
- 101150028135 MPH1 gene Proteins 0.000 claims description 3
- 241000235648 Pichia Species 0.000 claims description 3
- 210000004899 c-terminal region Anatomy 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 102000013165 exonuclease Human genes 0.000 abstract description 20
- 230000014509 gene expression Effects 0.000 abstract description 17
- 238000000034 method Methods 0.000 abstract description 17
- 230000034431 double-strand break repair via homologous recombination Effects 0.000 abstract description 16
- 238000010276 construction Methods 0.000 abstract description 4
- 230000008569 process Effects 0.000 abstract description 4
- 102000004169 proteins and genes Human genes 0.000 abstract description 4
- 230000006798 recombination Effects 0.000 abstract description 4
- 238000005215 recombination Methods 0.000 abstract description 4
- 108020004414 DNA Proteins 0.000 description 18
- 108020005004 Guide RNA Proteins 0.000 description 15
- 230000037361 pathway Effects 0.000 description 14
- 101150039661 FAA1 gene Proteins 0.000 description 9
- 230000006780 non-homologous end joining Effects 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 8
- 230000000694 effects Effects 0.000 description 6
- 108020004705 Codon Proteins 0.000 description 5
- 102000053602 DNA Human genes 0.000 description 5
- 102100033996 Double-strand break repair protein MRE11 Human genes 0.000 description 5
- 101000591400 Homo sapiens Double-strand break repair protein MRE11 Proteins 0.000 description 5
- 101100127688 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) FAA1 gene Proteins 0.000 description 5
- 230000001965 increasing effect Effects 0.000 description 5
- 108020004707 nucleic acids Proteins 0.000 description 5
- 102000039446 nucleic acids Human genes 0.000 description 5
- 238000003752 polymerase chain reaction Methods 0.000 description 5
- 101150105372 POX1 gene Proteins 0.000 description 4
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 150000002191 fatty alcohols Chemical class 0.000 description 4
- 238000003209 gene knockout Methods 0.000 description 4
- 230000008439 repair process Effects 0.000 description 4
- KIAPWMKFHIKQOZ-UHFFFAOYSA-N 2-[[(4-fluorophenyl)-oxomethyl]amino]benzoic acid methyl ester Chemical compound COC(=O)C1=CC=CC=C1NC(=O)C1=CC=C(F)C=C1 KIAPWMKFHIKQOZ-UHFFFAOYSA-N 0.000 description 3
- 102100036826 Aldehyde oxidase Human genes 0.000 description 3
- 108010006654 Bleomycin Proteins 0.000 description 3
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 3
- 101100009781 Danio rerio dmbx1a gene Proteins 0.000 description 3
- 101000928314 Homo sapiens Aldehyde oxidase Proteins 0.000 description 3
- 101000976645 Homo sapiens Zinc finger protein ZIC 3 Proteins 0.000 description 3
- 241001506991 Komagataella phaffii GS115 Species 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 101100127690 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) FAA2 gene Proteins 0.000 description 3
- 101100194320 Zea mays PER1 gene Proteins 0.000 description 3
- 102100023495 Zinc finger protein ZIC 3 Human genes 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 229960001561 bleomycin Drugs 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- 230000002708 enhancing effect Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 101150044508 key gene Proteins 0.000 description 3
- 238000012269 metabolic engineering Methods 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 238000005457 optimization Methods 0.000 description 3
- 230000007017 scission Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
- 102000053642 Catalytic RNA Human genes 0.000 description 2
- 108090000994 Catalytic RNA Proteins 0.000 description 2
- 241000701959 Escherichia virus Lambda Species 0.000 description 2
- 102100029075 Exonuclease 1 Human genes 0.000 description 2
- 101000959452 Homo sapiens Alcohol dehydrogenase class-3 Proteins 0.000 description 2
- 101000918264 Homo sapiens Exonuclease 1 Proteins 0.000 description 2
- 241001138401 Kluyveromyces lactis Species 0.000 description 2
- 102000009572 RNA Polymerase II Human genes 0.000 description 2
- 108010009460 RNA Polymerase II Proteins 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 2
- 101100394762 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) HFD1 gene Proteins 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000003197 gene knockdown Methods 0.000 description 2
- 238000007857 nested PCR Methods 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 108091092562 ribozyme Proteins 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 102100039702 Alcohol dehydrogenase class-3 Human genes 0.000 description 1
- 102100034044 All-trans-retinol dehydrogenase [NAD(+)] ADH1B Human genes 0.000 description 1
- 101710193111 All-trans-retinol dehydrogenase [NAD(+)] ADH4 Proteins 0.000 description 1
- 101100179978 Arabidopsis thaliana IRX10 gene Proteins 0.000 description 1
- 101100233722 Arabidopsis thaliana IRX10L gene Proteins 0.000 description 1
- 238000010453 CRISPR/Cas method Methods 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 101150067325 DAS1 gene Proteins 0.000 description 1
- 102100035102 E3 ubiquitin-protein ligase MYCBP2 Human genes 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 101150115938 GUT1 gene Proteins 0.000 description 1
- 101100264215 Gallus gallus XRCC6 gene Proteins 0.000 description 1
- 241001099156 Komagataella phaffii Species 0.000 description 1
- 102000011931 Nucleoproteins Human genes 0.000 description 1
- 108010061100 Nucleoproteins Proteins 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 244000146510 Pereskia bleo Species 0.000 description 1
- 102000002490 Rad51 Recombinase Human genes 0.000 description 1
- 108010068097 Rad51 Recombinase Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 108020005091 Replication Origin Proteins 0.000 description 1
- 101100010928 Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2) tuf gene Proteins 0.000 description 1
- 101100008874 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) DAS2 gene Proteins 0.000 description 1
- 101100516268 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) NDT80 gene Proteins 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 101150001810 TEAD1 gene Proteins 0.000 description 1
- 101150074253 TEF1 gene Proteins 0.000 description 1
- 102100029898 Transcriptional enhancer factor TEF-1 Human genes 0.000 description 1
- 102100036976 X-ray repair cross-complementing protein 6 Human genes 0.000 description 1
- 108010084455 Zeocin Proteins 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- -1 and during repair Proteins 0.000 description 1
- 101150038500 cas9 gene Proteins 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 231100000244 chromosomal damage Toxicity 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 101150088049 dna2 gene Proteins 0.000 description 1
- 230000005782 double-strand break Effects 0.000 description 1
- 230000001819 effect on gene Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 108010075712 fatty acid reductase Proteins 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 238000012224 gene deletion Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000003262 industrial enzyme Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 101150085005 ku70 gene Proteins 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- CWCMIVBLVUHDHK-ZSNHEYEWSA-N phleomycin D1 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC[C@@H](N=1)C=1SC=C(N=1)C(=O)NCCCCNC(N)=N)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C CWCMIVBLVUHDHK-ZSNHEYEWSA-N 0.000 description 1
- 108010001814 phosphopantetheinyl transferase Proteins 0.000 description 1
- 229920001470 polyketone Polymers 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
- 235000007586 terpenes Nutrition 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The application provides a gene editing system with high homologous recombination efficiency of pichia pastoris and application, wherein the construction method comprises the following steps: and the exonuclease and CAS9 genes are fused and expressed, so that the recombination efficiency of pichia pastoris is enhanced, and the seamless knockout and multi-fragment integration efficiency of single genes and multiple genes are improved. The invention proves that the fusion expression of the homologous recombination related protein and the Cas9 protein can accelerate the homologous recombination repair process, thereby improving the homologous recombination efficiency.
Description
Technical Field
The application relates to a gene editing system with high homologous recombination efficiency of pichia pastoris and application thereof, belonging to the technical field of microbial metabolic engineering application.
Background
Pichia pastoris (syn. Komagataella phaffii) is one of the exogenous gene expression systems widely used at present, and has the characteristics of high stability, high expression, high secretion and high density growth. At present, some important recombinant proteins, industrial enzymes and food additives such as polyketone, terpenes, fatty acid and the like can be synthesized in pichia pastoriset al, meta.eng., 2018.50:2-15). The compound biosynthetic pathway often involves multiple genes, so that the cell factory for constructing the compound of interest is often a singleA time-consuming process, an efficient and accurate gene editing method is urgently needed.
The CRISPR/Cas system is used as a high-efficiency genome editing technology and mainly comprises 2 parts: cas9 protein and sgRNA (single guide RNA). When Cas9 cleaves the gene target, a DNA Double strand break gap (DSBs) can be created in front of the PAM sequence. Cells are repaired primarily by means of homologous recombination repair (HR) and non-homologous end joining (NHEJ). Compared with NHEJ, HR can be controlled and accurately repaired by taking donor DNA (donor) as a template at a DNA target point, so that the steps of gene deletion, insertion, integration and the like can be specifically completed. However, pichia pastoris mainly relies on NHEJ mode for genome repair, and homologous recombination is inefficient. Previous studies have shown that enhancing either the HR pathway key gene (RAD 52) or inhibiting the NHEJ pathway key gene (e.g., KU 70/80) can increase the efficiency of homologous recombination (Cai et al nucleic Acids res.,2021,49 (13), 7791-7805.Weninger et al.J.Cell.Biochem.2018,119 (4), 3183-3198), but knocking out the NHEJ pathway key gene can have some adverse effects, e.g., the occurrence of chromosome-terminal genetic instability in pichia pastoris KU70 mutants. Furthermore, the addition of methanol to the medium makes the cells susceptible to chromosomal damage or disruption, while the endogenous NHEJ pathway can help to restore viability. Strategies to increase the efficiency of homologous recombination have therefore focused mainly on enhancing the endogenous homologous recombination capacity.
The choice of DSBs repair pathway between NHEJ and HR depends largely on whether the DSB ends are processed (Ceccaldi et al trends Cell biol., 2016.26:52-64.). Typically, the HR initial excision is triggered by the Sae2 protein and is accomplished by the Mre11-Rad50-Xrs2 (MRX) complex in yeast. Mre11 has 3' -5' exonuclease activity, and cleavage of DSBs produces short 3' -end single stranded DNA, exposure of ssDNA being necessary for initiation of HDR. The long-distance exonucleases Exo1 and Dna2 further cleave the single-stranded ends of the extended DSBs. The protein a (RPA) coated 3' end suspensions were then replicated. The Rad51 protein then replaces RPA, forming a nucleoprotein filament for searching and pairing with the homologous sequence. In NHEJ, the DSBs ends are co-stabilized by Ku (yKu 70/yKu) and MRX complexes, with little or no need for DSBs end cleavage processing, and the DSBs ends are directly religated. HR requires an initial short range excision followed by a long range excision. The key to HR repair is therefore whether DSBs can be rapidly cleaved by exonuclease to form ssDNA. Thus, the present project aims to accelerate cleavage of DSBs to form ssDNA by fusion expression of a key exonuclease at the initial stage of the homologous recombination pathway, facilitating progress toward homologous recombination repair.
Disclosure of Invention
According to one aspect of the present application, the present invention provides a gene editing system with high homologous recombination efficiency for Pichia pastoris, aiming at the key problems to be solved.
The method is realized by adopting the following technical scheme:
a gene editing system with high homologous recombination efficiency of pichia pastoris, the gene editing system comprises an exonuclease gene and a CAS9 gene fusion expression vector, wherein the CAS9 gene is connected with the exonuclease gene; the exonuclease gene is selected from one of escherichia coli exonuclease III (EcEXO III) gene, pichia pastoris EXO1 gene and pichia pastoris MRE11 gene.
Optionally, the nucleic acid sequence of the escherichia coli exonuclease III (EcEXO III) gene is shown as SEQ ID NO. 3; the EXO1 gene of the Pichia pastoris is shown as SEQ ID NO. 4; the nucleic acid sequence of the Pichia pastoris MRE11 gene is shown as SEQ ID NO. 5.
Alternatively, the exonuclease gene and CAS9 gene fusion expression vector is a plasmid or chromosome.
Optionally, in the expression vector containing the exonuclease gene and the CAS9 gene in fusion, the exonuclease gene is expressed at the C end or the N end of the CAS9 gene of the CRISPR/Cas9 gene editing system plasmid.
Alternatively, the exonuclease gene and CAS9 gene fusion expression vector is a C-terminal fusion expression vector of Pichia pastoris MRE11 gene and CAS9 gene.
Alternatively, the exonuclease gene and CAS9 gene fusion expression vector is an N-terminal fusion expression vector of Pichia pastoris EXO1 gene and CAS9 gene.
Optionally, the preparation method of the fusion expression vector containing the exonuclease gene and the CAS9 gene comprises the following steps: respectively carrying out PCR amplification on the CAS9 gene, the plasmid skeleton and the exonuclease gene; and then obtaining the fusion expression vector containing the exonuclease gene and the CAS9 gene through seamless cloning.
The application also provides a gene editing system with high homologous recombination efficiency of the pichia pastoris, and application of the gene editing system in pichia pastoris gene editing.
Alternatively, the application is seamless knockout or multi-fragment integration of the gene sequence of a pichia pastoris strain.
Optionally, the pichia pastoris is a pichia pastoris strain over-expressed by RAD 52;
alternatively, when the gene sequence of the pichia pastoris strain is subjected to multi-fragment integration, the pichia pastoris is a pichia pastoris strain with the mph1 gene knocked out on the basis of the RAD52 over-expression strain.
A specific embodiment is set forth below:
five exonucleases (Exonecut, short for Exo) are screened, fusion expression is carried out on the five exonucleases and the N end and the C end of CAS9 respectively to construct CRISPR/Cas9 system plasmids, FAA1 genes of pichia pastoris strains are knocked out, and the Exonuclease with the best effect is selected.
The construction method comprises the following steps: constructing a sgRNA expression vector pPICZ-Cas9-gFAA1-Exo of a target FAA1 gene, introducing the sgRNA expression vector into a Pichia pastoris strain, knocking out the FAA1 gene, counting the total number of transformants, selecting the transformants and calculating the positive rate of the transformants.
Wherein, the expression vector pPICZ-Cas9-gFAA1-Exo is constructed on the basis of pPICZ-Cas9-gFAA 1. pPICZ-Cas9-gFAA1 expression vector, gRNA expression cassette and selectable marker bleomycin (Zeocin) resistance gene Bleo R And a kluyveromyces lactis origin of replication panARS capable of plasmid amplification in pichia pastoris. The promoter in the gRNA expression cassette is RNA pol II type promoter P HTX1 For bi-directional expression of CAS9 and gRNA, which recognizes the ribozyme sequences HH and HDV, completely excises the flanking redundant sequences after gRNA transcription. Exonuclease derived from Exo of T7 phage (T7 Exo) (SEQ ID NO: 1), exonuclease of Red recombination system of lambda phage (lambda Redexo) (SEQ ID NO: 2) andcoli exonuclease III (EcExo III) (SEQ ID NO: 3), codon optimized for these three genes. In addition, there are 2 endogenous genes, EXO1 (SEQ ID NO: 4) and MRE11 (SEQ ID NO: 5), which are utilized with CAS9 sequences (GGGGS) without codon optimization 3 (SEQ ID NO: 6) flexible linker peptide linkage. These five genes are linked at the N-and C-terminus of CAS9, respectively.
The test strain was pichia pastoris GS115 strain. GS115 genomic sequences were obtained by accession to NCBI (https:// www.ncbi.nlm.nih.gov /), genomic sequence number: GCA_001746955.1 (SEQ ID NOS: CP014715.1, CP014716.1, CP014717.1, CP014718.1 of the four chromosomes included in the genome).
CAS9-MRE11 was applied for seamless knockout and multi-segment recombination.
The gene sequence of the pichia pastoris strain is subjected to seamless knockout and multi-fragment integration by adopting the CAS9-MRE11 combination with the best effect. Seamless knockouts mainly include single gene (FAA 1), two genes (FAA 2 and HFD 1), and three genes (FAA 2, POX1, and HFD 1) seamless knockouts. The strain is Pichia pastoris RAD52 over-expression strain, and the RAD52 over-expression strain is P GAP -KpRAD52-T AOX1 The expression cassette was inserted at the GS115HIS4 site (strain source reference Cai et al nucleic Acids Res.,2021,49 (13), 7791-7805).
The integration of multiple fragments was achieved by inserting a heterologous fatty alcohol synthesis pathway into the FAA1 gene locus, which pathway comprises three donor strains overlapping each other, the desired strain being Pichia pastoris RAD52 overexpressing strain and RAD52-mph1Δ strain, the RAD52-mph1Δ strain being a mph1 gene knockdown based on the RAD52 overexpressing strain, which contributes to the integration of multiple fragments (strain source reference Cai et al nucleic Acids Res.,2021,49 (13), 7791-7805). The method comprises the following specific steps:
(1) Constructing a new targeting gRNA expression vector;
(2) Constructing a donor DNA fragment for gene knockout and expression by an overlap extension PCR method, wherein the length of a homologous arm is set to 1000bp;
(3) Introducing 500ng of gRNA expression vector and 500ng or 1000ng of donor DNA molecule into Pichia pastoris cells by a shock transformation method;
(4) The correct transformants were verified.
The method of the application can obtain the following beneficial effects:
(1) The pichia pastoris metabolic engineering method has high homologous recombination capability, and the fact that the pichia pastoris homologous recombination efficiency can be improved by expressing the exonuclease related to homologous recombination is proved, wherein the CAS9 fusion expression MRE11 gene is the key for enhancing the pichia pastoris homologous recombination capability.
(2) The invention further expands the application potential of pichia pastoris as a microbial cell factory, so that the pichia pastoris is more convenient, rapid and accurate in metabolic engineering and synthetic biology research.
Drawings
FIG. 1 is a schematic representation of the CRISPR/Cas9 expression system of example 1; wherein A and B are schematic diagrams of the construction process of fusion expression vectors of exonuclease genes and N ends and C ends of CAS9 genes; c is a seamless knockout schematic diagram of the FAA1 gene.
FIG. 2 shows the homologous recombination results of the exonuclease in GS115 strain with FAA1 gene knockout in example 1; wherein A and B are the positive rate and the number of transformants in which the exonuclease gene is fused to the N-and C-termini of the CAS9 gene.
FIG. 3 shows the result of homologous recombination of the FAA1 gene knockout of Mre11 in the RAD52 gene-overexpressing strain of example 2.
FIG. 4 shows the results of homologous recombination by knockout of two and three genes in the strain over-expressing the RAD52 gene for Mre11 of example 2; wherein A is a schematic diagram of knocking out two genes and three genes; b and C are homologous recombination results of knockout two genes and three genes.
FIG. 5 shows the result of the integration of the Mre11 multiple fragments into homologous recombination efficiency in example 3; wherein A is a multi-segment integration schematic diagram of the fatty alcohol synthesis pathway, and B is a multi-segment integration result.
Detailed Description
The invention is further illustrated in the following, in conjunction with the accompanying drawings and detailed embodiments.
The experimental methods used in the following examples are all conventional methods unless otherwise specified; the reagents, materials, etc. used in the examples described below are commercially available unless otherwise specified. The parameters used in the examples below were all manufacturer recommended parameters unless otherwise specified.
The pPICZ-Cas9-gFAA1-Exo plasmid templates referred in the application all take pPICZ-Cas9-gFAA1 constructed in the laboratory previously as a framework, and the two-gene and three-gene plasmid templates all take pPICZ-Cas9-gFAA2-POX1-HFD1 constructed in the laboratory previously as a framework (the method is referred to by Cai et al nucleic Acids Res.,2021,49 (13), 7791-7805).
EXAMPLE 1 exonuclease and CAS9 fusion expression to improve homologous recombination efficiency
The pPICZ-Cas9-gFAA1 plasmid has a humanized codon optimized CAS9 gene derived from P derived from Pichia pastoris HTX1 The bi-directional promoter initiates both CAS9 and gRNA. P (P) HTX1 The RNA pol II type promoter can recognize ribozyme sequences HH and HDV, and completely cut off the flanking redundant sequences after gRNA transcription. Meanwhile, in order to enable the vector containing CAS9 and gRNA expression modules to exist stably and freely in Pichia pastoris, an replication origin panARS (SEQ ID NO: 1) derived from Kluyveromyces lactis is constructed in the expression vector. To fusion express FIGS. 1A and B with the exonuclease gene at the N-and C-terminus of CAS9, pPICZ-Cas9-gFAA1 was split into two parts for PCR amplification, one part being the CAS9 gene and the other part being the plasmid backbone, with the exonuclease gene being amplified separately. Exonuclease genes include Exo (T7 Exo) derived from T7 phage (SEQ ID NO: 1), exonuclease (lambda RedExo) of the Red recombination system of lambda phage (SEQ ID NO: 2) and exonuclease III (EcExo III) of E.coli (SEQ ID NO: 3), and these three genes were amplified by PCR using plasmids as templates after codon optimization. In addition, there are 2 endogenous genes, MRE11 (SEQ ID NO: 5) and EXO1 (SEQ ID NO: 4), which do not require codon optimization, with GS115 genomic DNA as a template. The DNA sequence (SEQ ID NO: 6) of the (GGGGS) 3-linked peptide is added to a primer of the target gene.
Using Takara CoHS DNA Polymerase cloning of the desired band, PCR method referenceHS DNA Polymerase instructions for use. Annealing temperature was set according to the primers required for cloning and extension time according to +.>The extension rate of HS DNA Polymerase was 1min/kb and the length of the cloned fragment was determined. The gene fragments obtained by PCR were ligated using the one-step cloning kit of Renzan (ClonExpress MultiS One Step Cloning Kit, C113). Procedure the pPICZA-gRNA-Exo plasmid was obtained by seamless cloning, with reference to the instructions of the Norpran kit.
Construction of a donor DNA fragment: the 1000bp sequences on the upstream and downstream of the FAA1 coding region of the gene were amplified respectively, and the complete donor DNA fragment was obtained by overlap extension PCR (FIG. 1C).
Conversion: 500ng of gRNA expression vectors pPICZ-Cas9-gFAA1-EXO, pPICZ-Cas9-gFAA1 and 1 mu g of donor DNA are transformed into wild Pichia pastoris GS115 by an electrotransformation mode, and the mixture is subjected to static culture for 3-4 days at 30 ℃ on YPD plates containing bleomycin, so that the total number of transformants on the plates is calculated. 20 colonies of transformants were selected and cultured overnight in YPD bleomycin broth. Extracting genome as a template for verifying PCR (polymerase chain reaction) for amplification, obtaining positive transformants with 2000bp as knocked-out FAA1 after electrophoresis, and calculating the proportion of the number of positive transformants to 20 strains as homologous recombination positive rate.
The test strain was pichia pastoris GS115 strain. GS115 genomic sequences were obtained by accession to NCBI (https:// www.ncbi.nlm.nih.gov /), genomic sequence number: GCA_001746955.1 (SEQ ID NOS: CP014715.1, CP014716.1, CP014717.1, CP014718.1 of the four chromosomes included in the genome).
As shown in FIG. 2A, the results of the experiment show that exonuclease Mre11 enhances the HR efficiency of FAA1 gene to the highest, from 13.3% of control CAS9 to 25% of CAS9-MRE11 (MRE 11-C), followed by EXO1-CAS9 (EXO 1-N) to 23.4%. The number of transformants in both groups decreased (fig. 2B), but it was considered that the number of transformants still reached several hundred, and thus it was still sufficient for subsequent selection of transformants. In addition, the CAS9-EcEXO III (EcEXO III-C) homologous recombination efficiency was almost the same as that of the control, with no decrease in the conversion rate.
Example 2 MRE11 and RAD52 promote seamless knockout
Rad52 is a eukaryotic homologous recombination-mediated protein, and during repair, rad52 may participate in the second chain end capturing and annealing processes, thus having the effect of promoting homologous recombination. Previously we found that overexpression of the Pichia pastoris own RAD52 gene alone can significantly improve the homologous recombination efficiency of FAA1, FAA2 and GUT1 genes. We therefore considered combining RAD52 and MRE11 to analyze the effect on gene editing. We analyzed the homologous recombination effects of MRE11 on monogenic, two-and three-genes in Pichia pastoris RAD52 overexpressing strains. The strain is Pichia pastoris RAD52 over-expression strain, and the RAD52 over-expression strain is P GAP -KpRAD52-T AOX1 The expression cassette was inserted at the GS115HIS4 site (Cai et al nucleic Acids Res.,. 2021,49 (13), 7791-7805).
Monogenic: using FAA1 as an example, 500ng of the gRNA expression plasmids pPICZ-Cas9-gFAA1-MRE11, pPICZ-Cas9-gFAA1 and 1. Mu.g of FAA1-donor DNA were transformed into Pichia pastoris RAD52 over-expression strain by means of electrotransformation, the remainder of the procedure being as in example 1.
Two gene knockouts: taking simultaneous knockout of FAA2 and HFD1 as an example, taking the pPICZ-Cas9-gFAA2-POX1-HFD1 plasmid as a template, dividing the plasmid into 2 parts for amplification except for the gRNA targeting region of the POX1 gene, and connecting the amplified plasmid with the MRE11 gene fragment through seamless cloning. 500ng of the gRNA expression vectors pPICZ-Cas9-gFAA2-HFD1-MRE11, pPICZ-Cas9-gFAA2-HFD1 and 500ng of each FAA2-donor, HFD1-donor DNA were transformed into Pichia pastoris RAD52 over-expressed strains by means of electrotransformation, and the rest of the procedure was the same as in example 1.
Three gene knockdown: taking simultaneous knockout of FAA2, POX1 and HFD1 as an example, taking the pPICZ-Cas9-gFAA2-POX1-HFD1 plasmid as a template, dividing the plasmid into 2 parts for PCR amplification, and connecting the 2 parts with the MRE11 gene fragment through seamless cloning. The remaining procedure was as in example 1, except that 500ng of the gRNA expression vectors pPICZ-Cas9-gFAA2-POX1-HFD1-MRE11, pPICZ-Cas9-gFAA2-POX1-HFD1 and 500ng of each of FAA2-donor, HFD1-donor and POX1-donor DNA were used to transform Pichia pastoris GS115-RAD 52.
As shown in FIG. 3, the results of the experiment show that exonuclease Mre11 increases the HR efficiency of the FAA1 gene from 88.3% for control CAS9 to 91.7% for CAS9-MRE11, with some decrease in transformant numbers. As can be seen from FIG. 4B, exonuclease Mre11 increased the HR efficiency of both genes by 10%, from 76.7% for control CAS9 to 86.7% for CAS9-MRE11, and the number of transformants increased by 48%. Exonuclease Mre11 increased the HR efficiency of the three genes (FAA 2, POX1 and HFD 1) to 6%, from 10.8% for control CAS9 to 16.7% for CAS9-MRE11 (FIG. 4C), with substantially the same number of transformants.
Example 3 CAS9-MRE11 promotes Multi-fragment integration
Multi-fragment integration is essential for long biosynthetic pathways. Thus, the fatty alcohol metabolic pathway is utilized as a multi-segment integrated test pathway. The pathway includes three exogenous genes: a fatty acid reductase encoding gene CAR, a phosphopantetheinyl transferase encoding gene NpgA to activate CAR, and a saccharomyces cerevisiae alcohol reductase encoding gene ADH5. By P ADH2 And T DAS1 P was used as promoter and terminator of NpgA TEF1 And T AOX1 As promoter and terminator of CAR, P was used TPI And T DAS2 As promoters and terminators for ADH5, three expression cassettes were overlapped with each other by about 500bp to form three donor DNAs using the FAA1 gene as an integration site (FIG. 5A).
The three donors were amplified using genomic DNA of Pichia pastoris fatty alcohol high-producing strain as a template, and 500ng of pPICZ-Cas9-gFAA1 or pPICZ-Cas9-Ppmre11-gFAA1 plasmid were respectively electrically transformed into Pichia pastoris RAD52 and RAD52-mph1Δ strains together with 500ng of each of the three donor DNA. The strain is a Pichia pastoris RAD52 over-expression strain and a RAD52-mph1Δ strain, the RAD52-mph1Δ strain is obtained by knocking out the mph1gene on the basis of the RAD52 over-expression strain, and the strain is beneficial to multi-fragment integration. The rest of the procedure is as in example 1.
As can be seen from FIG. 5B, in the RAD52 overexpressing strain, exonuclease Mre11 increases the HR efficiency of the integrated pathway from 66.7% for control CAS9 to 91.7% for CAS9-MRE 11. In RAD52-mph1Δ strain, exonuclease Mre11 increased the HR efficiency of the integration pathway from 71.7% for control CAS9 to 93.7% for CAS9-MRE11, increasing the number of transformants by 103.7% and 76%, respectively.
The results of the examples confirm that pichia pastoris can increase the homologous recombination capacity by fusion expression of homologous recombination pathway protein Mre11, and lay a foundation for development of pichia pastoris as a high-efficiency microbial cell factory.
The foregoing description is only a few examples of the present application and is not intended to limit the present application in any way, and although the present application is disclosed in the preferred examples, it is not intended to limit the present application, and any person skilled in the art may make some changes or modifications to the disclosed technology without departing from the scope of the technical solution of the present application, and the technical solution is equivalent to the equivalent embodiments.
Claims (10)
1. The gene editing system with high homologous recombination efficiency for pichia pastoris is characterized by comprising an exonuclease gene and a CAS9 gene fusion expression vector, wherein the CAS9 gene is connected with the exonuclease gene; the exonuclease gene is selected from one of escherichia coli exonuclease III (EcEXO III) gene, pichia pastoris EXO1 gene and pichia pastoris MRE11 gene.
2. The Pichia pastoris gene editing system with high homologous recombination efficiency according to claim 1, wherein the nucleic acid sequence of the E.coli exonuclease III (EcEXO III) gene is shown in SEQ ID NO. 3; the EXO1 gene of the Pichia pastoris is shown as SEQ ID NO. 4; the nucleic acid sequence of the Pichia pastoris MRE11 gene is shown as SEQ ID NO. 5.
3. The gene editing system with high homologous recombination efficiency according to claim 1, wherein the exonuclease gene and CAS9 gene fusion expression vector is a plasmid or a chromosome; preferably, the exonuclease gene and CAS9 gene fusion expression vector is a plasmid.
4. The pichia pastoris gene editing system of claim 1, wherein the exonuclease gene is fusion expressed at the C-terminus or N-terminus of the CRISPR/CAS9 gene editing system plasmid CAS9 gene in the exonuclease gene and CAS9 gene fusion expression vector.
5. The gene editing system with high homologous recombination efficiency according to claim 4, wherein the exonuclease gene and CAS9 gene fusion expression vector is a C-terminal fusion expression vector of pichia pastoris MRE11 gene and CAS9 gene.
6. The gene editing system with high homologous recombination efficiency according to claim 4, wherein the exonuclease gene and CAS9 gene fusion expression vector is an N-terminal fusion expression vector of pichia pastoris EXO1 gene and CAS9 gene.
7. The gene editing system with high homologous recombination efficiency according to claim 4, wherein the preparation method of the fusion expression vector comprising the exonuclease gene and the CAS9 gene comprises the following steps: respectively carrying out PCR amplification on the CAS9 gene, the plasmid skeleton and the exonuclease gene; and then obtaining the fusion expression vector containing the exonuclease gene and the CAS9 gene through seamless cloning.
8. Use of the gene editing system of high homologous recombination efficiency of Pichia pastoris according to any of claims 1 to 7 for gene editing of Pichia pastoris.
9. The use according to claim 8, wherein the use is seamless knockout or multi-fragment integration of the gene sequence of a pichia pastoris strain.
10. The use according to claim 8 or 9, wherein the pichia is a RAD52 overexpressed pichia strain;
preferably, when the gene sequence of the pichia pastoris strain is subjected to multi-fragment integration, the pichia pastoris is a pichia pastoris strain with the mph1 gene knocked out on the basis of the RAD52 over-expression strain.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211103030.6A CN117683803A (en) | 2022-09-09 | 2022-09-09 | Gene editing system with high homologous recombination efficiency for pichia pastoris and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211103030.6A CN117683803A (en) | 2022-09-09 | 2022-09-09 | Gene editing system with high homologous recombination efficiency for pichia pastoris and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117683803A true CN117683803A (en) | 2024-03-12 |
Family
ID=90132533
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211103030.6A Pending CN117683803A (en) | 2022-09-09 | 2022-09-09 | Gene editing system with high homologous recombination efficiency for pichia pastoris and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117683803A (en) |
-
2022
- 2022-09-09 CN CN202211103030.6A patent/CN117683803A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Lueking et al. | A system for dual protein expression in Pichia pastoris and Escherichia coli | |
| US9822357B2 (en) | Bidirectional promoter | |
| JP2011115182A (en) | Ura5 gene and method for stable genetic integration in yeast | |
| KR20180088733A (en) | Yeast cell | |
| US20200149055A1 (en) | Inducible promoter for gene expression and synthetic biology | |
| EP2285966A1 (en) | Multi-species polynucleotide control sequences | |
| AU2016329244B2 (en) | Novel episomal plasmid vectors | |
| CN117683803A (en) | Gene editing system with high homologous recombination efficiency for pichia pastoris and application | |
| CN109628448B (en) | Industrial saccharomyces cerevisiae stress resistance modification based on saccharomyces cerevisiae CRISPR library | |
| EP3810779A1 (en) | Genetic selection markers based on enzymatic activities of the pyrimidine salvage pathway | |
| CN113265383B (en) | Hansenula polymorpha gene editing system, application thereof and gene editing method | |
| US11466280B2 (en) | Gene targeting method | |
| CN114540356A (en) | Rhodosporidium toruloides promoter and application thereof | |
| WO2017163946A1 (en) | Induction-type promoter | |
| JP4671394B2 (en) | Promoter DNA from Candida utilis | |
| CN113056554A (en) | Recombinant yeast cells | |
| CA2589591A1 (en) | Malate synthase regulatory sequences for heterologous gene expression in pichia | |
| CN113151339B (en) | Gene mutation expression cassette and application thereof | |
| CN116555269B (en) | Bumblebee candida utilis inducible promoter and application thereof | |
| CN116286904B (en) | Bovine-derived CRISPR/boCas13a gene editing system, method and application | |
| EP3015549B1 (en) | Novel polypeptide, and use thereof | |
| KR101920036B1 (en) | The screening method for gene without frameshift mutation and nonsense mutation using E.coli and ampicillin resistance gene | |
| WO2022144374A2 (en) | Novel yeast strains | |
| WO2017002733A1 (en) | Cloning vector | |
| JP2884486B2 (en) | Mitochondrial ribosomal protein gene and use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |