WO2018083128A2 - Microbial genome editing - Google Patents

Microbial genome editing Download PDF

Info

Publication number
WO2018083128A2
WO2018083128A2 PCT/EP2017/077975 EP2017077975W WO2018083128A2 WO 2018083128 A2 WO2018083128 A2 WO 2018083128A2 EP 2017077975 W EP2017077975 W EP 2017077975W WO 2018083128 A2 WO2018083128 A2 WO 2018083128A2
Authority
WO
WIPO (PCT)
Prior art keywords
cas9
polynucleotide
temperature
homologous recombination
sequence
Prior art date
Application number
PCT/EP2017/077975
Other languages
French (fr)
Other versions
WO2018083128A3 (en
Inventor
John Van Der Oost
Richard Van Kranenburg
Elleke Fenna BOSMA
Ioannis MOUGIAKOS
Original Assignee
Wageningen Universiteit
Stichting Voor De Technische Wetenschappen
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Wageningen Universiteit, Stichting Voor De Technische Wetenschappen filed Critical Wageningen Universiteit
Publication of WO2018083128A2 publication Critical patent/WO2018083128A2/en
Publication of WO2018083128A3 publication Critical patent/WO2018083128A3/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]

Definitions

  • the present invention relates to the field of microbial genetic engineering. More particularly the invention concerns methods and systems for yielding enriched populations of cells containing a desired genome modification. The invention also concerns the field of gene editing employing specific sequence guided nucleases to achieve site-specific cutting of microbial genetic material.
  • microbes other than the widely-used model organisms such as Escherichia coli and Saccharomyces cerevisiae, are being evaluated for their respective abilities to act as production hosts.
  • Thermophilic organisms are of particular interest due to their multiple advantages over mesophilic organisms when being used as production hosts ( Bosma, E.F., et al., (2013) Current Biotechnology, 2, 360-379; Lin, L. and Xu, J.
  • thermophiles as production hosts is generally hampered by the lack of well-developed genome editing tools compared to the currently used mesophilic model organisms (Bosma, E.F., etal., (2013) Current Biotechnology, 2, 360-379; Taylor, M.P., et al., (201 1 ) Microbial biotechnology, 4, 438-448.)
  • ET 138 Bacillus smithii ET 138
  • This species grows between 37°C and 65°C and efficiently utilizes both C5 and C6 sugars (Bosma, E.F., et al., (2015) Applied and Environmental Microbiology, 81 , 1874-1883; Nakamura, L.K., et al., (1988) International Journal of Systematic Bacteriology, 38, 63-73).
  • the tool developed for this bacterium allows for the introduction of scar-free markerless gene deletions via a homologous recombination process and /acZ-based counter-selection relying on the toxicity of high concentrations of 5- bromo-4-chloro-3-indolyl-3-D-galactopyranoside (X-gal) (Bosma, E.F., et al., (2015) Microbial Cell Factories, 14, Art.nr.99; Van Spanning, R.J.M., et al., (1991 ) Journal of Bacteriology, 173, 6962-6970.).
  • the developed process is time-consuming, with the fastest possible route to gene deletion taking approximately 2 to 3 weeks from transformation to generation of a scar-free markerless knockout (Bosma, E.F., et al., (2015) Microbial Cell Factories, 14, Art.nr.99).
  • the counter-selection step is not stringent enough for removal of genes that are essential for the fitness and the metabolism of the strain.
  • Cas9 introduces a chromosomal double stranded DNA break (DSDB), which is lethal (Oh, J.-H. and van Pijkeren, J. -P. (2014) Nucleic acids research, 42, e131 ; Zeng, H., et al., (2015) Appl Microbiol Biotechnol, 99, 10575-10585; Mougiakos, I., et al., (2016). Trends in Biotechnology, 34, 575-587; Barrangou, R. and van Pijkeren, J. P. (2016) Curr Opin Biotechnol, 37, 61 -68).
  • DSDB chromosomal double stranded DNA break
  • NHEJ Non-Homologous End Joining
  • Indel insertion/deletion
  • the ET 138 genome contains the genes for the basic prokaryotic NHEJ-like system, consisting of DNA ligase LigD and DNA-end-binding protein Ku ( Bosma, E.F., et al., (2016) Standards in Genomic Sciences, 1 1 ; Shuman, S. and Glickman, M.S. (2007) Nat Rev Micro, 5, 852-861.24,25).
  • the level of activity of the NHEJ-like system in ET 138 is unknown.
  • Cas9 was employed to introduce DSDBs in prokaryotic genomes. These breaks modestly induced the recombination of a provided rescuing/editing template into the targeted chromosome, resulting in genetically modified cells (Jiang, W., et al., (2013) Nature Biotech, 31 , 233-239; Xu, T., et al., (2015) Appl Environ Microbiol, 81 , 4423-4431 ; Huang, H., et ai, (2015) Acta Biochimica et Biophysica Sinica, 47, 231 -243).
  • the edited cells avoided subsequent Cas9 targeting events, but in many studies the number of surviving/edited colonies was low with high percentage of mixed (both wild type and mutant) or escape mutant genotypes (Huang, H., et ai, (2016) ACS Synth Biol; Li, Q., et al, (2016) Biotechnol J, 1 1 , 961 -972; Wang, Y., et al, (2015) J Biotechnol, 200, 1 -5). The number of surviving colonies as well as the percentage of successfully edited cells was higher in studies that allowed homologous recombination of the editing templates to take place prior to the Cas9 targeting. This way Cas9 was employed for stringent counter-selection of the unedited genomes. For this approach either homologous recombination was faster than Cas9-targeting or cas9 expression was induced after homologous recombination (Li, Y., et al,
  • thermophilic archaeon has been employed for genome editing (Li, Y., et al, (2015) Nucleic Acids Research, 44) as well as chromosome-based genetic manipulations have been reported for a few naturally competent thermophiles (Zeldes, B.M., et al. (2015) Front Microbiol, 6, 1209), no reports are available on using Cas9-based editing in thermophilic organisms.
  • the inventors have discovered a novel method for microbial genome editing.
  • the invention provides a method of microbial genome editing, comprising the steps of:
  • nuclease or a polynucleotide comprising a sequence encoding the nuclease; wherein the nuclease forms a ribonuclease complex with the guide RNA, and wherein the ribonuclease complex makes site-specific double stranded DNA breaks (DSDB) in the microbial genome;
  • DSDB site-specific double stranded DNA breaks
  • HR homologous recombination
  • the methods described herein therefore advantageously employ a temperature switch, from a higher growth temperature phase favouring HR and not the site-directed nuclease activity, to a lower growth temperature phase whereby counter selection occurs by the site-directed nuclease activity removing microbes which do not have a desired modification introduced by HR.
  • the population of microbes surviving after the temperature switch counter selection is thereby enhanced for the desired modification.
  • Any microbial cells have the potential to be modified by the methods described herein. A requirement is that the microbes can grow across a temperature range in which a selected ribonuclease complex has nuclease activity at one of the microbial growth temperatures and substantially no nuclease activity at another microbial growth temperature.
  • the methods described herein allow the process of homologous recombination to be favoured at a first temperature such that the microbial genome can be modified with the desired mutation and a second temperature in which unmodified cells can be targeted by the ribonuclease complex to introduce a DSDB into the genomes of the unmodified cells.
  • a second temperature in which unmodified cells can be targeted by the ribonuclease complex to introduce a DSDB into the genomes of the unmodified cells.
  • NHEJ non-homologous end joining
  • these methods overall increase the population of microbial cells with the desired mutation whilst eliminating any unmodified microbial cells.
  • the methods described herein are used in microbes that have substantially no endogenous NHEJ repair mechanism.
  • the methods described herein may be applied to microbes that have an endogenous NHEJ repair mechanism.
  • the methods described herein may be applied to microbes that have an endogenous NHEJ repair mechanism but wherein the NHEJ repair mechanism is either conditionally reduced or the NHEJ activity is knocked out.
  • the methods described herein utilises a sequence of the homologous recombination polynucleotide that has at least one mis-match with the guide RNA, such that the guide RNA is no longer able to recognise the modified genome.
  • the ribonuclease complex will not recognise the modified genome. Therefore, no DSDB can be introduced by the ribonuclease complex and so the modified cells will survive. However, the cells with unmodified genomes will still have substantial complementarity to the guide RNA and consequently can be cleaved site-specifically by the ribonuclease complex.
  • the homologous recombination polynucleotide may have more than 1 mis-match with the guide RNA, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32 or more mis-matches with the guide RNA (from the 5' end or the 3' end).
  • Such multiplicity of mis-matches may be contiguous.
  • a multiplicity of mis-matches may be non-contiguous. There may be a mixture of contiguous and non-contiguous mis-matches where there are three or more mismatches.
  • the 32 nt at the 5'-end of the sgRNA molecule corresponds to the crRNA module.
  • the methods described herein utilise a sequence of the homologous recombination polynucleotide that has one or more contiguous or non-contiguous mismatches in the 13 base pairs between the guide RNA and the target DNA site proximal to the PAM, and/or seven or more contiguous mismatches in the 5'-terminal region of the protospacer.
  • Another way of characterising the degree of recognition between the homologous recombination polynucleotide and the guide of the ribonuclease is to express this in terms of sequence identity.
  • sequence of the homologous recombination peptide may have less than 99% identity when aligned fully with the sequence of the guide; in alternative embodiments this can be less than 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91 %. Also, less than 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%.
  • the way in which the ribonucleoase complex is prevented from acting to cleave the microbial genome is not so much to modify or eliminate the sequence targeted by the guide, but rather the PAM required by the ribonuclease complex.
  • the PAM is either modified or eliminated in order to blind the ribonuclease complex to the specific cutting site. Therefore, methods of the invention may include those using a sequence of the homologous recombination polynucleotide that does not include a PAM sequence recognised by the ribonuclease complex. Therefore, no DSDB can be introduced by the ribonuclease complex and so the HR modified cells will survive. However, the unmodified cells will still be recognised by the ribonuclease complex of editing nuclease and its guide and so consequently are cleaved site-specifically.
  • the methods described herein rely on homologous recombination to modify the genome of the microbe.
  • the upstream flank and downstream flanks are 0.5 kilobases (kb) to 1 .0 kb each in length.
  • kb kilobases
  • the homologous recombination polynucleotide can in some embodiments further comprise a polynucleotide sequence between the upstream and downstream flanking regions. This polynucleotide sequence could for example contain a modification that is to be introduced into the microbial genome.
  • homologous recombination relies upon the upstream and downstream flanks having substantial complementarity to the target regions, mismatches can be accommodated as well. Therefore, in some embodiments, homologous recombination is known to occur between DNA segments with extensive homology to the upstream and downstream flanks. In alternative embodiments, the upstream and downstream flanks have complete complementarity to the target regions. The upstream and downstream flanks need not be identical in size. However, in some instances the upstream and downstream flanks are identical in size. The efficiency of homologous recombination will vary depending on the likelihood of homologous recombination of the smallest fragment length of the flank.
  • homologous recombination even if the homologous recombination process is inefficient, advantageously the method described herein will select for any microbial cell that has the desired modification over the unmodified microbial cell.
  • Homologous recombination also allows large deletions (e.g. 50 kb or more) to be made encompassing complete gene clusters.
  • Homologous recombination is also used for recombineering, which is a well-known method to allow for recombination over smaller fragments (45-100 nt).
  • the methods described herein can optionally further comprise at least another homologous recombination polynucleotide or a polynucleotide comprising a sequence encoding a homologous recombination polynucleotide having a sequence substantially complementary to a second target region containing the target in the microbial genome.
  • the methods described herein utilise a homologous recombination polynucleotide that is DNA.
  • the DNA is single stranded.
  • the DNA is double stranded.
  • the DNA is double stranded and plasmid borne.
  • homologous recombination in the methods described herein may be used to remove a polynucleotide sequence from the microbial genome.
  • homologous recombination in the methods described herein may be used to insert one or more gene(s), or fragment(s) thereof, in to the microbial genome.
  • homologous recombination in the methods described herein can be used to modify or replace at least one nucleotide in the microbial genome. Consequently, the methods described herein can be used for any desired kind of genome modification.
  • Methods of the invention involve treating microbial cells in order to introduce essential elements of HR polynucleotide, guide, nuclease (or ribonuclease complex) whether directly or indirectly via an expression plasmid or vector.
  • components are introduced in to the microbial cells at the same temperature as the first incubation temperature or substantially the first incubation temperature.
  • the components can be introduced at sub-optimal growth temperatures for the microbe concerned. Potentially these temperatures may be characterised by minimal or no cell division or the microbial culture is in stasis.
  • polynucleotide and polypeptide components are already widely known in the art such as transformation, transduction or transfection.
  • the polynucleotide components are introduced by chemical transformation, heat-shock transformation or natural transformation.
  • polynucleotide components described herein are introduced into the cells as part of a polynucleotide vector, e.g. an expression plasmid.
  • the components can be introduced as part of several e.g. two or three polynucleotide vectors. The components can be introduced into the cells substantially separately, simultaneously or sequentially.
  • the methods described herein require introduction into the microbial cells at least one guide RNA or at least one polynucleotide comprising a sequence encoding a guide RNA.
  • the methods described herein can further comprise introducing at least a second guide RNA or at least one polynucleotide comprising a sequence encoding a guide RNA; wherein the second guide RNA is substantially complementary to a second target polynucleotide sequence(s) in a microbial genome.
  • the guide RNA comprises a crRNA and tracrRNA.
  • the guide RNA comprises a single chimeric guide RNA.
  • the guide RNA comprises a crRNA.
  • the guide RNAs can be of any length.
  • the guide RNA may comprise additional modification to the 5' terminus such as phosphorylation or hydroxylation.
  • the methods defined herein can use the B. coagulans phosphotransacetylase ⁇ pta) promoter Ppta without its ribosome binding sequence to mediate expression of a guide RNA from a plasmid.
  • the methods described herein can optionally further comprise incubating the cells at a third temperature; wherein the third temperature is greater than the second temperature; optionally wherein the third temperature is the same as the first temperature. If the third temperature is the same as the second temperature this effectively leads to prolonging the duration at which the microbes are incubated at the second temperature.
  • the third temperature could be the same as the first temperature such that the microbial cells are incubated at the first temperature twice, once before the second temperature and once after the second temperature. Optionally, this could be via intervening temperatures as described herein.
  • the culture medium can be changed at any time for fresh culture medium. This may happen at least once; possibly two or more times.
  • the culture medium changes may take place together with a change in temperature of the microbial culture.
  • nuclease activity by the ribonuclease complex is less than the homologous recombination activity in the microbial cells when incubated at the first temperature.
  • the respective rates of ribonuclease activity introducing DSDBs and HR are such that HR prevails and so as a result of microbial growth at the first temperature for a period of time results in enrichment of cells for the HR modified genome.
  • the intervening temperature is selected from: 40 ° C, 41 ° C, 42 ° C, 43 ° C, 44 ° C, 45 ° C, 46 ° C, 47 ° C, 48 ° C, 49 ° C, 50 ° C, 51 ° C, 52 ° C, 53 ° C, 54 ° C, 55 ° C. Included in all of the above whole numbers is any intervening one tenth fraction of a degree, e.g. 0.1 , 0.2, etc.
  • the methods described herein have the first incubation temperature of between 20 ° C and 70 ° C, preferably 45 ° C to 55 ° C.
  • the methods described herein have the first incubation temperature as higher than 39 ° C, preferably higher than 40 ° C, more preferably higher than 41 ° C, as and even more preferably higher than 42 ° C.
  • the first incubation temperature is a minimum temperature of 40 ° C, 41 ° C, 42 ° C, 43 ° C, 44 ° C, or 45 ° C.
  • the first incubation temperature is a maximum temperature of 55 ° C, 56 ° C, 57 ° C, 58 ° C, 59 ° C, 60 ° C, 61 ° C, 62 ° C, 63 ° C, 64 ° C, or 65 ° C.
  • the first incubation temperature can be selected from: 20 ° C, 21 ° C, 22 ° C, 23 ° C, 24 ° C, 25 ° C, 26 ° C, 27 ° C, 28 ° C, 29 ° C, 30 ° C, 31 ° C, 32 ° C, 33 ° C, 34 ° C, 35 ° C, 36 ° C, 37 ° C, 38 ° C, 39 ° C, 40 ° C, 41 ° C, 42 ° C, 43 ° C, 44 ° C, 45 ° C, 46 ° C, 47 ° C, 48 ° C, 49 ° C, 50 ° C, 51 ° C, 52 ° C, 53 ° C, 54 ° C, 55 ° C, 56 ° C, 57 ° C, 58 ° C, 59 ° C, 60 ° C, 61 ° C, 62 ° C, 63 ° C, 64 ° C, 65 ° C, 66 ° C, 60
  • the methods described herein have the second incubation temperature of between 20 ° C and 50 ° C, preferably 35 ° C to 45 ° C.
  • the second incubation temperature is lower than 45 ° C, preferably lower than 44 ° C, more preferably lower than 43 ° C, and even more preferably lower than 42 ° C.
  • the second incubation temperature is a minimum temperature of 30 ° C, 31 ° C, 32 ° C, 33 ° C, 34 ° C, 35 ° C, 36 ° C or 37 ° C.
  • the second incubation temperature is a maximum temperature of 40 ° C, 41 ° C, 42 ° C, 43 ° C, 44 ° C or 45 ° C.
  • the second incubation temperature can be selected from: 20 ° C, 21 ° C, 22 ° C, 23 ° C, 24 ° C, 25 ° C, 26 ° C, 27 ° C, 28 ° C, 29 ° C, 30 ° C, 31 ° C, 32 ° C, 33 ° C, 34 ° C, 35 ° C, 36 ° C, 37 ° C, 38 ° C, 39 ° C, 40 ° C, 41 ° C, 42 ° C, 43 ° C, 44 ° C, 45 ° C, 46 ° C, 47 ° C, 48 ° C, 49 ° C or 50 ° C. Included in all of the above whole numbers is any intervening one tenth fraction of a degree, e.g. 0.1 , 0.2, etc.
  • the methods described herein can optionally further comprise incubation at a fourth temperature.
  • the fourth incubation temperature step allows for the modified microbial cells to be cured of an introduced plasmid.
  • the plasmid introduced into the microbial cells is unable to replicate above a specific temperature.
  • any introduced plasmid may be cured by culture in non-selective conditions.
  • the duration of incubation may be tailored to suit particular microbial strains and to ensure the desired degree of completion of HR and/or DSDB formation.
  • incubation times at the variously selected temperatures of operating the invention may be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 hours, for example. More incubation times may be employed as needed.
  • the duration of the incubation temperatures may vary between 6 to 20 hours for each incubation step; preferably 8 to 16 hours.
  • microbes modified by the methods described herein may be grown in any medium.
  • Particularly suitable media that may be used for particular microbes are well known in the art. Specific media used will depend on the microbes used in the methods described herein.
  • microbes may be grown in media comprising TVMY medium and optionally further comprising any carbon source selected from: xylose, uracil and glucose, or any combination thereof.
  • microbes may be grown in media comprising LB2 medium, optionally further comprising any carbon source selected from: xylose, uracil and glucose, or any combination thereof.
  • the methods described herein are for any desired microbial genome modification.
  • a microbe includes bacteria, archaea, fungi, yeast and protozoa.
  • the microbes are mesophilic or thermophilic.
  • a mesophilic microbe is a microbe that has optimal growth at a temperature below 42 ° C, preferably between 20 ° C and 42 ° C and a thermophilic microbe is defined as a microbe that has optimal growth at or above 42 ° C, preferably between 42 ° C and 70 ° C.
  • the thermophilic microbes are hyperthermophiles, in other words, they are able to grow in temperatures greater than 70 ° C, preferably between 70 ° C and 100 ° C.
  • the bacteria are capable of growth or survive an incubation step at a temperature below 42°C, preferably the bacteria are selected from: Acidithiobacillus species including Acidithiobacillus caldus; Aeribacillus species including Aeribacillus pallidus; Alicyclobacillus species including Alicyclobacillus acidocaldarius, Alicyclobacillus acidoterrestris, Alicyclobacillus cycloheptanicus; Anoxybacillus species including Anoxybacillus caldiproteolyticus, Anoxybacillus flavithermus, Anoxybacillus rupiensis, Anoxybacillus tepidamans; Bacillus species including Bacillus caldolyticus, Bacillus caldotenax, Bacillus caldovelox, Bacillus coagulans, Bacillus clausii, Bacillus licheniformis, Bacillus
  • Thermovibrio species including Thermovibrio ammonificans, Thermovibrio ruber, Thermovirga species including Thermovirga // ' en/7 and Thermus species including Thermus aquaticus, Thermus caldophilus, Thermus flavus, Thermus scotoductus, Thermus thermophilus;
  • a method described herein can be used to modify bacteria capable of growth at a temperature above 42°C.
  • the bacteria are selected from: Acidithiobacillus species including Acidithiobacillus caldus; Actinobacillus species including Actinobacillus succinogenes; Anaerobic-spirillum species including Anaerobiospirillum succiniciproducens; Bacillus species including Bacillus alcaliphilus, Bacillus amyloliquefaciens, Bacillus circulans, Bacillus cereus, Bacillus clausii, Bacillus firmus, Bacillus halodurans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus subtilis, Bacillus thuringiensis; Basfia species including Basfia succiniciproducens; Brevibacillus species including Brevibacillus brevis; Brevi
  • a method defined herein could be used to modify the genome of yeast or fungi.
  • the fungal species can grow at a temperature above 42°C, preferably the fungi is selected from: an Aspergillus species including, but not limited to, Aspergillus nidulans, Aspergillus niger, Aspergillus terreus, Aspergillus oryzae and Aspergillus terreus, more preferably the Aspergillus species is Aspergillus nidulans or Aspergillus niger.
  • the fungal species capable of growing at a temperature above 42°C could be a Candida species.
  • microbes identified in bold typeface have been found to be particularly suitable/applicable in use for the present invention.
  • thermophilic microbes selected from: Thermophilic bacilli, Aeribacillus, Alicyclobacillus, Anoxybacillus, Bacillus, Geobacillus; Paenibacillus species; Thermophilic Clostridia, including Anaerobacter, Anaerobacterium, Caldicellulosiruptor, Clostridium, Moorella, Thermoanaerobacter, Thermoanaerobacterium, Thermobrachium, Thermohalobacter species or one or more Thermophilic Lactobacillus species and mesophilic bacteria selected from Bacillus species, Escherichia coli, and Lactobacillus species.
  • Suitable nucleases to be used in the methods described herein are selectable at the option of the average skilled person. A choice may depend upon the optimal growth temperature of the particular microbe being used. Methods described herein preferable use Cas9, preferably Streptococcus pyogenes Cas9, or C2C1. Alternatively, methods described herein can use Cpf1 . Further alternative nucleases suitable for the methods described herein are C2C3 or Argonaute. It is also contemplated that the methods described herein may use other nucleases such as zinc finger nucleases (ZFNS), meganucleases or transcription activator effector like nucleases (TALENS).
  • ZFNS zinc finger nucleases
  • TALENS transcription activator effector like nucleases
  • Preferred nucleases can be selected from the following tables:
  • Cas9 Alistipes sp.
  • Cas9 Prevotella oulorum Cas9 Prevotella ruminicola
  • the methods described herein can use modified nuclease variants that are nickases.
  • a nickase can be created via a mutation in either one of the HNH or the RuvC catalytic domains of the nucleases.
  • amino acid residues of the nucleases may be substituted conservatively or non-conservatively.
  • Conservative amino acid substitutions refer to those where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not alter the functional properties of the resulting polypeptide.
  • nucleic acid sequences may be substituted conservatively or non- conservatively without affecting the function of the polypeptide.
  • Conservatively modified nucleic acids are those substituted for nucleic acids which encode identical or functionally identical variants of the amino acid sequences.
  • each codon in a nucleic acid can be modified to yield a functionally identical molecule. Accordingly, each silent variation (i.e. synonymous codon) of a polynucleotide or polypeptide, which encodes a polypeptide of the present invention, is implicit in each described polypeptide sequence.
  • polynucleotide sequences may be substituted conservatively or non-conservatively without affecting the function of the polypeptide.
  • Conservatively modified polynucleotides are those substituted for nucleic acids which encode identical or functionally identical variants of the amino acid sequences.
  • each codon in a nucleic acid except AUG and UGG; typically the only codons for methionine or tryptophan, respectively
  • each silent variation i.e. synonymous codon of a polynucleotide or polypeptide, which encodes a polypeptide of the present invention, is implicit in each described polypeptide sequence.
  • the invention defines a selectable replicable plasmid comprising a guide RNA or a polynucleotide comprising a sequence encoding a guide RNA under the control of a first heterologous promoter and with a terminator; a non-codon or codon optimised polynucleotide comprising a sequence encoding a nuclease under the control of a second heterologous promoter with a terminator; and homologous recombination polynucleotide or a polynucleotide comprising a sequence encoding a homologous recombination polynucleotide.
  • Suitable promoters and terminators that may be used in a plasmid may depend upon the particular microbes used in the methods described herein. Suitable promoters and terminators are well known in the art.
  • the selectable replicable plasmid defined herein can optionally comprise inducible promoters. Suitable inducible promoters that may be used in a plasmid may depend upon the particular microbes used in the methods described herein. Suitable promoters and terminators are well known in the art.
  • a selectable replicable plasmid defined herein comprises a first heterologous promoter that is B.
  • the invention provides a kit for performing the methods as defined herein, wherein the kit comprises a plasmid as herein defined and instructions for use.
  • the invention provides a clonal library obtainable by the method as described herein, wherein the clonal library comprises a plurality of clones harbouring the modified microbial genome that is resistant to site-specific DSDB by the ribonuclease complex.
  • the invention provides a microbial host cell modified by the methods as defined herein.
  • FIG. 1 A schematic overview of the basic pWUR_Cas9nt construct.
  • the non-codon optimized spCas9 gene was employed for the construction of the pWUR_Cas9nt vector, since the S. pyogenes and B. smithii GC-content and codon usage are highly similar.
  • the spCas9 was placed under the control of P xyn A.
  • a Rho- independent terminator from B. subtilis was introduced after the stop codon of the gene.
  • the spCas9 module is followed by an sgRNA- expressing module that encompasses a spacer which does not target the genome of B. smithii ET 138.
  • the sgRNA module was placed under the transcriptional control of P p t a from Bacillus coagulans -without its RBS- (Bosma et al., MCF, 2015; Kovacs, A.T., et al., (2010) Applied and Environmental Microbiology, 76, 4085-4088) and it is followed by a second Rho- independent terminator from B. subtilis.
  • the spCas9 and sgRNA modules were synthesized as one fragment, which was subsequently cloned into the pNW33n through the BspHI and Hindi 11 restriction sites.
  • Lane 1 corresponds to the marker (1 kb+ DNA ladder, ThermoFisher)
  • Lanes 2 to 4 correspond to ET 138 wild type cultures transformed with the pWUR_Cas9nt and grown at 55, 45, 37°C respectively
  • Lanes 5 to 7 correspond to ET 138 wild type cultures transformed with the pNW33n and grown at 55, 45 and 37°C respectively
  • Lanes 7, 8, 9, 1 1 , 12 correspond to different negative controls
  • Lane 10 corresponds to the positive control, for which pWUR_Cas9nt was used as the PCR template.
  • FIG. 1 A. Sequential transfer scheme of wild type ET 138 cultures to evaluate spCas9 expression and targeting efficiency at different temperatures.
  • ET 138 cells were transformed with the pWUR_Cas9sp1 , pWUR_Cas9sp2, pWUR_Cas9sp3, pWUR_Cas9nt and pNW33n vectors and plated on LB2 agar plates with chloramphenicol (Day 1 ). After overnight (ON) incubation at 55°C, single colonies were restreaked on LB2 agar plates with chloramphenicol and incubated ON at 55°C and 45°C (Day 2).
  • Colonies from the 45°C plates were transferred to LB2 agar plates with chloramphenicol for ON incubation at 37°C (Day 3). The plates from days 2 and 3 were then used for inoculation of liquid medium (Day 4); 1 colony per 55°C, 45°C and 37°C plate was transferred to LB2 medium with xylose, glucose and chloramphenicol for ON incubation at 55°C, 45°C and 37°C respectively, while 1 extra colony per 45°C plate was transferred to the same medium for overnight incubation at 42°C. B.
  • Results of the targeting experiment showing ⁇ measurements from cultures of wild type ET 138 transformed with the 3 different pyrF targeting cas9 sp constructs, the non- targeting cas9 sp construct and pNW33n.
  • the growth of the cells with the pyrF targeting constructs is greatly affected at 37°C, which is not observed for cells containing the non-targeting constructs.
  • FIG. 3 Schematic representation of the different homologous recombination and spCas9- mediated mutations described.
  • the first single cross over event (SCO) can occur by insertion of the editing plasmid into the chromosome either through the upstream homologous region (UHR) -as depicted here- or through the downstream homologous region (DHR).
  • UHR upstream homologous region
  • DHR downstream homologous region
  • a scar-less, markerless pyrF gene deletion was established; after insertion of the editing vector into the chromosome, via homologous recombination with the plasmid-borne editing template (2x 1 -kbp flanks, immediately flanking the pyrF gene and thus removing it from start to stop codon), a second SCO event results in either WT revertants or edited cells.
  • the spCas9 targeting of the WT cells acts as counter selection for the pyrF mutants.
  • B. Gene knock-out via insertion of stop codons and a restriction site. The followed process was similar to the gene deletion described above.
  • the hsdR restriction gene was inactivated by inserting stop codons and a restriction site between codons 212 and 221 that were contained in a 2 kbp long HR-fragment that expands 289 bp upstream and 1 .65 kb downstream from the start codon of the hsdR gene on the genome of the ET 138. In-between the 2 stop codons, an EcoRV restriction site was added, generating a frame shift and facilitating the screening process. The spacer was designed to target the original sequence without stop codons and restriction site.
  • FIG. 5 A. Agarose gel electrophoresis showing the resulting products from colony PCR on ET 138 colonies transformed with pWUR_Cas9spR_hr for the detection of hsdR knock-out mutants from the 3-day long editing process in LB2xg medium.
  • the 2.75 kbp PCR fragments that resulted using genome specific primers (Lanes 1 ,3,5,7,9) were digested with the EcoRV restriction enzyme. Each digestion mixture was loaded next to its corresponding PCR- fragment (Lanes 2,4,6,8,10).
  • the expected size of the PCR fragment for the knock-out (non-edited) mutants was 2.3 kbp.
  • Figure 9 ⁇ ⁇ measurements from the 7-day long pyrF deletion culturing process.
  • B. smithii ET 138 AldhL AsigF cells transformed with pWUR_Cas9sp1 , pWUR_Cas9sp1_hr, pWUR_Cas9nt or pWUR_Cas9nt_hr were cultured in TVMY X g U medium. After growth at 55°C for 24 hours, the cultures were sequentially transferred every 24 hours to fresh media while gradually lowering the culturing temperature from 55°C to 37°C, with an intermediate transfer at 45°C. After 3 more transfers at 37°C cells were transferred back to 55°C with an intermediate transfer at 45°C.
  • the pWURCas9_sp1 cultures at 37°C showed almost no growth, indicating efficient spCas9 targeting, while the pWUR_Cas9nt and pWUR_Cas9nt_hr control cultures grew at all the temperatures as expected for B. smithii ET 138 cultures.
  • the pWUR_Cas9sp1_hr cultures showed poor growth in the first 2 culturing steps at 37°C, but the growth was reconstituted from the 3rd culturing step at 37°C onwards to control levels, indicating the development of either sgRNA and spCas9 escape mutants, or ApyrF deletion mutants that can avoid the spCas9 targeting.
  • FIG. 10 Phenotypic evaluation of 5-FOA sensitivity and uracil auxotrophy of B. smithii ET 138 pyrF wild-types and mutant. Cells were grown overnight on TVMY medium with the following additions: Plates annotated "Ta” contained 2 g/L 5-FOA and 50 mg/L uracil; Plates annotated “Tb” contained 2 g/L 5-FOA and no uracil; Plates annotated “Td” contained no 5- FOA and no uracil.
  • Streptococcus pyogenes Cas9 genome editing was applied for the first time to a moderate thermophile i.e. Bacillus smithii, including a gene deletion, a gene knockout via insertion of premature stop codons and a gene insertion. On average it took 1 week from transformation to clean deletion, knock out or knock in - including the plasmid curing step - with an editing efficiency of 90% for the gene deletion and the gene knock out and 20% for the gene insertion.
  • the major advantage of this system is the limited requirement in genetic parts: only one plasmid and no inducible or well-characterized promoters to drive the spCas9 and sgRNA expression. Since B. smithii grows between 37°C and 65°C, the mesophilic, well-characterized Streptococcus pyogenes Cas9 (spCas9) was used and its expression controlled in ET 138. spCas9 was found to be inactive in ET 138 at temperatures above 42°C and so its activity was tightly controlled by altering the cultivation temperature. The inventors created a clean gene deletion, a gene disruption and a gene insertion by using a system based on one single plasmid.
  • the system employs a plasmid-borne homologous recombination template for introducing the desired modifications to the genome at elevated temperatures, while the non- edited cells are subsequently eliminated by the spCas9 counter-selection tool at 37°C. Moreover, the system could be readily applied for genome editing of other non-model organisms with limited genetic toolbox parts.
  • Example 1 In vivo expression validation of spCas9 at different temperatures
  • Inventors designed and constructed the modular pWUR_Cas9nt construct, that encompasses the cas9 gene of S. pyogenes (referred to as cas9 sp herein) and an sgRNA-expressing module for which the spacer is predicted not to target any site of the B. smithii genome, (i.e. nt, for non-targeting).
  • the backbone of the pWUR_Cas9nt is the pNW33n vector, which was the only available vector for B.
  • the sgRNA-module was placed under the control of the B. coagulans phosphotransacetylase (pfa) promoter P pfa (Bosma, E.F., et al., (2015) Microbial Cell Factories, 14, Art.nr.99; Kovacs, A.T., et al., (2010) Applied and Environmental Microbiology, 76, 4085-4088) without its RBS.
  • pCas9-expression and toxicity levels in ET 138 were tested by transforming a single batch of competent cells, with the non-targeting pWUR_Cas9nt vector and with the empty pNW33n control vector.
  • the transformed cells were plated on LB2 plates supplemented with chloramphenicol. Plates were incubated overnight only at 55°C, as previous incubation attempts at lower temperatures were not successful (data not shown).
  • One single colony per transformation was used for sequential transfers in LB2 xg broth, transferring the cultures from 55°C to 37°C, with two intermediate steps at 45°C and 42°C.
  • Total RNA was isolated from each pWUR_Cas9nt culture after 18 hours of incubation at every temperature and performed semi-quantitative reverse transcription (rt)-PCR using specific primers for the cas9 sp gene. Transcription of cas9 sp was observed for all temperatures ( Figure 1 C). The growth of the cas9 S p-expressing cultures was similar to the pNW33n control cultures at all temperatures (see below), indicating that the expression of cas9 sp is not toxic for the ET 138 cells at any of the tested temperatures.
  • TVMY medium and LB2 medium were used as described previously ((Bosma, E.F., et al., (2015) Microbial Cell Factories, 14, Art.nr.99).
  • TVMY xgu is TVMY supplemented with 0,5 g/l xylose, 0,5 g/l glucose and 50 mg/L uracil.
  • TVMY xg is TVMY supplemented with 0,5 g/l xylose and 0,5 g/l glucose.
  • LB2 xg is LB2 supplemented with 0,5 g/l xylose and 0,5 g/l glucose.
  • Substrates were added separately as 50% autoclaved solutions after autoclavation of the medium.
  • Uracil was added as 50 mg/ml filter sterilized solution in 1 M NaOH after autoclavation of the medium and addition of the substrates.
  • £ coli strains were grown in LB medium at 37°C. For plates, 30 g of agar (Difco) per liter of medium was used for B. smithii in all experiments; 15 g of agar (Difco) per liter of LB was used for £ coli. If required, chloramphenicol was added in concentrations of 25 ⁇ g/mL for £ coli DH5a, 15 ⁇ g/mL for £. coli TG90, and 7 ⁇ g/mL for B. smithii.
  • IdhL gene re-inserted to restore its
  • Genomic DNA isolation, transformations, colony PCR, sequence and phenotypic verification Genomic DNA from B. smithii strains was isolated using the MasterPureTM Gram Positive DNA Purification Kit (Epicentre). Heat shock transformation of E. coli strains was performed according to the NEB supplier's protocol. Transformation of B. smithii strains was performed as described previously in Bosma, E.F., et al., (2015) Microbial Cell Factories, 14, Art.nr.99. Plasmids for transforming B. smithii were extracted from E. coli via maxiprep isolation (Genomed Jetstar 2.0).
  • smithii ET138 AldhL AsigF ApyrF cells were plated on TVMY medium with 30 g/L agar and the following additions: a) 2 g/L 5-FOA and 50 mg/L uracil, b) 2 g/L 5-FOA and no uracil, d) no 5-FOA and no uracil.
  • Plasmids and primers used are shown in Table 2 and 3.
  • Q5 polymerase NEB was used for all PCR reactions for cloning purposes.
  • NEB T4 ligase was used for assembling the pWURJacZ, pWUR_Cas9nt, pWUR_Cas9nt_hr, pWUR_Cas 9sp1 , pWUR_Cas9sp1_hr, pWUR_Cas9sp2, pWUR_Cas9sp2_hr, pWUR_Cas9sp3 and pWUR_Cas9sp3_hr vectors.
  • the NEBuilder® HiFi DNA Assembly Master Mix was used for assembling the pWUR_Cas9spR_hr, pWUR_Cas9spKI_hr1 and pWUR_Cas9spKI_hr2 constructs. All the used restriction enzymes were obtained from NEB. Purification of PCR products was performed after running them on a 0.8% agarose gel using the ZymocleanTM Gel DNA Recovery Kit.
  • a DNA fragment comprised of the P xyn A and the lacZ gene was synthesized by GeneArt and inserted into pNW33n using digestion with BspHI and Kpnl and subsequent ligation and cloning into E. coli DH5a, creating plasmid pWURJacZ.
  • the P xyn A sequence was used exactly as originally described (Currie et al., (2013) Biotechnology for Biofuels, 6, 32), using the sequence until the start codon of the corresponding gene in the original host.
  • a synthetic gene string was synthesized by GeneArt containing the elements depicted in Figure 1 B except the P xyn A promoter.
  • P xyn A was amplified from pWURJacZ using primers BG6538 and BG6541.
  • Primer BG6541 replaces the final 6 bp of P xyn A by an Xbal site, changing the final -1 to -6 sequence from GTAAGA to TCTAGA and keeping the total length the same as in the original promoter.
  • Primer BG6538 adds a BspHI site on the start of the P xyn A.
  • the entire synthesized spCas9 module without promoter for spCas9 was amplified using primers BG6542 and BG6543, keeping the Xbal and Hindi 11 sites already present in the module. Subsequently, vector pNW33n was digested with BspHI and Hind III, the P xyn A PCR-product with Xbal and BspHI and the spCas9 module PCR-product with Xbal and Hind III. The three elements were ligated in a 3-point ligation and cloned into E.
  • coli TG90 (TG90 is a derivative of TG1 , carrying the pcnB80 zadvJn W allele; it can be re-constructed using the methods in Gonzy-Treboul, G., et al., (1992) J. Mol. Biol. 224:967-979). Plasmid was extracted and the correct sequence was verified by sequencing, creating plasmid pWUR_Cas9nt ( Figure 1A).
  • oligo sets were annealed to create the 3 spacers, after which the annealed spacers were inserted into the construct as follows. Oligo sets were BG6017+6021 for spacer 1 , BG6018+6022 for spacer 2, BG6019+6023 for spacer 3. Each set was annealed by adding 5 ⁇ _ 10 mM oligo sets together with 10 ⁇ _ NEB buffer 2.1 and 74 ⁇ _ MQ water. Mixtures were heated to 94°C for 5 min and gradually cooled down to 37°C at 0.03°C/sec using a PCR machine.
  • Annealed oligos and plasmid pWUR_Cas9nt were digested with BspEI and BsmBI (NEB).
  • BspEI digestion was performed at 37°C for 15 min, after which BsmBI was added and the mixture was further incubated at 55°C for 15 min.
  • ligation was performed using NEB T4 ligase and mixtures were transformed to £. coli TG90. All constructs were verified by sequencing and all were correct except spacer 2, which is missing 7 nt from the P p t a that is driving spacer expression (Figure 7). As we were not sure whether this would have any influence, we decided to nevertheless use this spacer for future work. Constructs were named pWUR_Cas9sp1 until pWUR_Cas9sp3 according to their corresponding spacer.
  • flanks were cloned from genomic DNA of ET 138 using primers BG 5798+5799 (upstream, 958 bp) and BG5800+5801 (downstream, 979 bp), introducing Sail and Xbal restriction sites.
  • the flanks were fused in an overlap extension PCR using primers BG5798+5801 making use of the complementary overhangs in primers BG5799 and BG5800.
  • flanks and pNW33n were digested with Sail and Xbal, ligated and transformed into E. coli DH5a.
  • primers BG6850+6849 were used, which both introduce a BspHI site.
  • the five spCas9- plasmids and the amplified pyrF-flanks were digested with BspHI, followed by alkaline phosphatase treatment of the vectors (Thermo Scientific), ligated and transformed in E. coli TG90. Since only one restriction site was used, the flanks could have been inserted in both orientations.
  • a 4-fragment NEBuilder® HiFi DNA Assembly was designed and executed for the construction of the hsdR-modifying plasmid pWUR_Cas9spR_hr.
  • the backbone of the vector was PCR amplified from pWUR_Cas9sp1 using the Q5 DNA polymerase (NEB) and primers BG7836+7837.
  • the HR fragment upstream of the targeted site in the hsdR gene was PCR amplified from the B. smithii ET 138 genome using the Q5 DNA polymerase (NEB) and primers BG7838+7839.
  • the HR fragment downstream of the targeted site in the hsdR gene was PCR amplified from the B.
  • the cas9sp and sgRNA containing fragment was PCR amplified from the pWUR_Cas9sp1 vector using the Q5 DNA polymerase (NEB) and primers BG7842+7843.
  • Two 4-fragment NEBuilder® HiFi DNA Assemblies were designed and executed for the construction of the IdhL-restoration plasmids pWUR_Cas9spKI-hr1 and pWUR_Cas9spKI- hr2.
  • the backbone of both vectors was PCR amplified from the pWUR_Cas9sp1 vector using the Q5 DNA polymerase (NEB) and primers BG8134+7837.
  • the HR fragment upstream of and including the IdhL gene was PCR amplified from the B.
  • the HR fragment downstream of the IdhL gene was PCR amplified from the B. smithii ET 138 genome using the Q5 DNA polymerase (NEB), primers BG8138+8139 for the pWUR_Cas9spKI-hr1 vector and primers BG8138+8140 for the pWUR_Cas9spKI-hr2 vector.
  • the cas9sp and sgRNA containing fragment of both vectors was PCR amplified from the pWUR_Cas9sp1 vector using the Q5 DNA polymerase (NEB) and primers BG8141 +7842.
  • the spCas9 module contains S. pyogenes Cas9 under the T. saccharolyticum P promoter followed by a B. subtilis-demed Rho-independent terminator, followed by a spacer under the B. coagulans Ppta promoter followed by another B. suM/Zs-derived Rho-independent terminator ( Figure 1 A).
  • Cm R chloramphenicol resistance gene (chloramphenicol acetyltransferase); BGSC: Bacillus Genetic Stock Centre, USA; us: upstream; ds: downstream; bp: base pairs.
  • RNA isolation was performed by the phenol extraction based on Van Hijum et al., 2005. Overnight 10 mL cultures were centrifuged at 4°C and 4816 x g for 15 min and immediately used for RNA isolation. After removal of the medium, cells were resuspended in 0.5 mL ice cold TE buffer (pH 8.0) and kept on ice. All samples were divided into two 2 mL screw-capped tubes containing 0.5 g zirconium beads, 30 ⁇ _ 10% SDS, 30 ⁇ _ 3 M sodium acetate (pH 5.2), and 500 ⁇ _ Roti-Phenol (pH 4.5-5.0, Roth).
  • RNA phase was transferred to a new tube, to which 400 ⁇ _ chloroform-isoamyl alcohol (Roth) was added, after which samples were centrifuged at 4°C and 18,400 x g for 3 min.
  • 300 ⁇ _ of the aqueous phase was transferred to a new tube and mixed with 300 ⁇ _ of the Lysis buffer from the High Pure RNA Isolation Kit (Roche). Subsequently, the rest of the procedure from this kit was performed according to the manufacturer's protocol, except for the DNAse incubation step, which was performed for 45 min. Integrity and concentration of the isolated RNA was checked on Nanodrop-1000.
  • Reverse Transcriptase PCR was performed using Superscript III Reverse Transcriptase kit (Invitrogen) according to the manufacturer ' s protocol.
  • Superscript III Reverse Transcriptase kit (Invitrogen) according to the manufacturer ' s protocol.
  • RNA RNA
  • 200 ng of random primers were used for synthesis of the first-strand cDNA.
  • the products were used as a template for PCR using spCas9-specific forward and reverse primers BG6237 and BG6232, resulting in a 255 bp product. Products were visualized on a 2% agarose gel ran for 20 min.
  • Example 2 In vivo validation of spCas9 activity at different temperatures
  • B. smithii genome encompasses the genes for the basic prokaryotic NHEJ-like system (Bosma, E.F., et al., (2016) Standards in Genomic Sciences, 1 1 ; Shuman, S. and Glickman, M.S. (2007) Nat Rev Micro, 5, 852-861 ).
  • the first approach was to construct a spCas9-based genome editing tool focused on determining the in vivo temperature limits of spCas9 and the capacity of the ET 138 NHEJ-like mechanism to repair the spCas9-induced DSDBs.
  • the pyrF gene encodes the orotidine 5'-phosphate decarboxylase and is part of the operon for pyrimidine biosynthesis. Removal of the gene causes uracil-auxotrophy and resistance to the toxic uracil-analogue 5-fluorootic acid (5-FOA). It is a frequently used selection and counter-selection system in many organisms including thermophiles (Tripathi, S. A et al., (2010) Applied and Environmental Microbiology, 76, 6591 -6599; Kita, A., et al., (2013) Bioscience, Biotechnology and Biochemistry, 77, 301 -306; Chung, D., et al., (2012) PLoS ONE, 7).
  • a clean pyrF deletion mutant was made being ET 138 ldhL sigF pyrF by adding the fused 1 -kb up- and downstream pyrF flanks to pNW33n and applying 5- FOA pressure to select for double cross-over mutants.
  • a total of 9 rounds of subculturing on selective media containing uracil and 5-FOA was required before pure knockouts were obtained with a knockout efficiency of around 50% (data not shown), making the process rather time-consuming.
  • 3 vectors were constructed based on the pWUR_Cas9nt sequence, designated pWUR_Cas9sp1 , pWUR_Cas9sp2, and pWUR_Cas9sp3, each containing a different pyrF- targeting spacer (sp1 -3).
  • a single batch of wild type ET 138 competent cells was transformed with the 3 targeting vectors, the non-targeting control pWUR_Cas9nt and the empty vector control pNW33n.
  • the aforementioned results indicate that the designed spCas9 system is active and introducing lethal DSDBs to the ET 138 genome but only at temperatures below 42°C. It also indicates that the NHEJ system in ET 138 is inactive under the tested conditions or not active enough to counteract the spCas9 activity.
  • the sequencing results of the pWUR_Cas9sp2 construct revealed the deletion of 7 nucleotides near the 3' end of the P p t a ( Figure 7). The results of the targeting experiment show that the uncharacterized nature of the Ppta, do not hinder the targeting efficiency of spCas9.
  • Example 3 Efficient gene deletion using a HR-CRISPR-Cas9 counter-selection system
  • a Cas9-based editing system for ET 138 was created, exploiting its efficient homologous recombination mechanism (Bosma, E.F., et al., (2015) Microbial Cell Factories, 14, Art.nr.99) and the temperature-induced spCas9 activity at 37°C.
  • the experimental setup consisted of a single plasmid that combines the recombination template and the spCas9- and sgRNA- expressing modules. Providing the cells with the appropriate plasmid-borne editing template at 55°C and then inducing the expression of active spCas9 at 37°C through a sequential culturing process, is expected to form a powerful homologous recombination and counter- selection system.
  • pWUR_Cas9sp1 was selected as the pyrF-targeting vector for further experiments, which was always compared to the non-targeting control pWUR_Cas9nt.
  • a fusion of the two pyrF-flanks was added, creating the pWUR_Cas9nt_hr and pWUR_Cas9sp1_hr vectors (i.e. hr, for homologous recombination) ( Figure 3A).
  • PCR on genomic DNA from the pWUR_Cas9nt and pWURCas9_sp1 cultures showed no pyrF knockout bands at any culturing temperature due to the lack of homologous recombination template in the constructs.
  • pWURCas9_sp1 cultures at 37°C showed almost no growth while the pWUR_Cas9nt cultures at all the temperatures showed the expected growth for ET 138 culture.
  • PCR on genomic DNA from the liquid cultures containing the vectors with pyrF flanks showed the absence of knockout bands for the pWUR_Cas9nt_hr cultures from the first culturing step at 37°C onwards, but very strong pyrF knockout bands for the pWUR_Cas9sp1_hr cultures for the same culturing steps mentioned above ( Figure 4A).
  • the striking difference in the density of the knockout bands between the targeting pWUR_Cas9sp1_hr and the non-targeting pWUR_Cas9nt_hr cultures indicates successful spCas9 activity and pyrF-targeting by the pWUR_Cas9sp1_hr construct.
  • the process for the pWUR_Cas9sp1_hr- containing strain was repeated and reduced the number of culturing steps at 37°C from 4 to 1 while keeping the culturing time of each step in a window between 8 and 16 hours.
  • 3 different media were used in order to observe possible medium-dependent variations in the efficiency of the system: TVMY selection medium supplemented with xylose, glucose and uracil (TVMYxgu), TVMY selection medium supplemented with xylose, glucose but not with uracil (TVMYxg) and LB2 medium supplemented with xylose and glucose (LB2 xg ).
  • ET 138 has a type I Restriction-Modification (R-M) system.
  • R-M Restriction-Modification
  • Methylation analysis of the PacBio genome sequencing data showed the existence of the single motif "Cm6AGNNNNNNTGT [SEQ ID NO: 51 ]/ACm6AN N N N N N CTG [SEQ ID NO: 52]" with N6-methyladenine (m6A) modifications (unpublished data).
  • the hsdR gene was knocked out. Between the origin of replication (on) and the cas9 sp gene of the pWUR_Cas9nt vector, we introduced a 2 kbp long HR-fragment that expands 289 bp upstream and 1 .65 kb downstream from the start codon of the hsdR gene on the genome of the ET 138. In this HR-fragment we replaced the 25 nt long part between odons 212 and 221 , including the last nucleotide of codon 212, of the hsdR gene with a 8 nt long sequence comprised of 2 stop codons and the EcoRV restriction site, generating a frame shift and facilitating the screening process (Figure 3B).
  • hsdR gene Since the hsdR gene is 2952 nucleotides (984 codons) long, only a fifth of it will be translated due the introduction of the stop codons.
  • sgRNA module for spCas9 targeting of the unmodified genomes, completing the construction of the pWUR_Cas9spR_hr editing vector.
  • B. smithii ET138 AldhL AsigF cells were transformed with the new vector and sequentially cultured as before, gradually lowering the temperature from 55°C to 37°C, with an intermediate transfer at 45°C, and then back up to 55°C. The duration of each culturing step was within a window of 8 to 16 hours.
  • LB2 and TVMY Five transformants per medium were subjected to colony PCR, after which the PCR fragments were digested with EcoRV. All colonies from the LB2-culturing process were successfully modified (Figure 5A) giving 100% editing efficiency, whereas 2 of the colonies from the TVMY process were modified giving 40% editing efficiency ( Figure 5B). This is in contrast with the result from the pyrF deletion process, where there were no modified colonies resulting from the LB2-culturing process. Plasmid curing was performed as before and the correct mutations were verified by sequencing.
  • Example 5 Metabolic engineering using spCas9: knock-in of the IdhL gene
  • the inventors evaluated the applicability of our Cas9-based system in markerless gene chromosomal integrations by knocking into the genome of ET 138 AldhL AsigF AhsdR the 942 bp long genomic fragment between the start and the stop codons of the lactate dehydrogenase (IdhL) gene.
  • the reconstitution of the lactate production in the resulting ET 138 AsigF AhsdR strain would allow for efficient growth under anaerobic conditions, while retaining the advantages of a sporulation- and R-M-deficient strain.
  • Two versions were constructed of a pWUR_Cas9-based vector that target the ET 138 AldhL AsigF AhsdR genome at the same position between the IdhL stop codon and the beginning of the adjacent rho-independent transcriptional terminator.
  • HR was facilitated with 1 kb flanks (pWUR_Cas9spKI-hr1 ) or 0.75kb flanks (pWUR_Cas9spKI_hr2).
  • the region between the IdhL stop codon and its rho-independent transcriptional terminator was inverted, avoiding spCas9 targeting (Figure 3C).
  • the region between the start and stop codon was provided with the wild-type IdhL sequence to allow its knocking in.
  • ET138 AldhL AsigF AhsdR was transformed with the 2 pWUR_Cas9spKI_hr versions and sequentially cultured the transformants as described before, gradually lowering the temperature from 55 to 37°C, with or without an intermediate transfer at 45°C, and then back up to 55°C. Each culturing step was within a window of 8 to 16 hours. 2 types of selection media, LB2 and TVMY were used. The colony PCR results of the TVMY culturing processes showed that none out of the tested colonies had the knock-in genotype.
  • a culturing period with an additional intermediate step at 45°C allows for efficient homologous recombination and double cross over events to occur, leading to the appearance of the mutants that Cas9 will select for. This is in line with the observations in L. reuterii (Oh & Van Pijkeren, 2014, op. cit). In addition, it may be that the stress of the temperature drop increases the efficiency of the homologous recombination mechanism.
  • Example 6 Application of spCas9 in Geobacillus thermoglucosidans spCas9 is used as counter selection tool in Geobacillus.
  • Upstream and downstream fragments of 0.75 kb were generated by PCR using the following primer combinations at an annealing temperature of 58°C:
  • 2178 (5'-TCACGTTAAGGGATTTTGGTCATGACATGTAAGATAGATAGGGCATC-3' [SEQ ID NO: 53]); and 2179 (5'-CATCTGTTTTCCCAAAGGAGATATGTATTCATTATTATTAG-3' [SEQ ID NO: 54]) and the following primer combinations at an annealing temperature of 66°C:
  • 2180 (5'-ACATATCTCCTTTG G GAAAAC AG ATG CAGG AG G-3' [SEQ ID NO: 55]); and 2181 (5'-ATTATCCTCAGCTCACTAGCGCCATTTTTGCGGATCCCCCTTATTTTTTATC-3' [SEQ ID NO: 56]) and by using chromosomal DNA of G. thermoglucosidans sigF (see WO2016/012296) as a template.
  • the vector backbone was amplified in two parts using pWUR_Cas9nt as a template, introducing the targeting spacer sequence 5'-AGGAGAATATAGCTAACGTC-3 [SEQ ID NO: 57].
  • One fragment was generated using the following primer combination using an annealing temperature of 60°C:
  • 2171 (5'-GTTATACAGGAGAATATAGCTAACGTCGTTTTAGAGCTAGAAATAGCAAGT TAAAATAAG-3' [SEQ ID NO: 60]); and 2172 (5'-TCATGACCAAAATCCCTTAAC-3' [SEQ ID NO: 61 ])
  • the four PCR fragments were created with Phusion Flash High-Fidelity PCR master mix (ThermoFisher) according to the manufacturer's instructions. The fragments were separated on a 1 % agarose gel and extracted from gel into 10 ⁇ _ h O using a Zymo DNA Clean and concentrator spin column (Zymo Research). The four fragments were assembled into a single plasmid by fusing the 25-bp overlapping regions using the NEBuilder HiFi DNA Assembly Cloning Kit (New England BioLabs). Plasmid DNA was concentrated using a Zymo DNA Clean and Concentrator spin column (Zymo Research) and eluted into and transformed to electrocompetent E.
  • coli TG90 (Gonzy-Treboul, G., Karmzyn-Campelli, C, Stragier, P. 1992. J. Mol. Biol. 224:967-979). Transformants were plated on LB agar plates supplemented with 10 mg/L chloramphenicol and incubated at 37°C. A single colony was selected for plasmid extraction using the ZymoPURETM Plasmid Midiprep Kit (Zymo Research). Plasmid integrity was confirmed by sequence analysis.
  • the integration vector was transformed to Geobacillus thermoglucosidans AsigF (see WO2016/012296) by electroporation as described elsewhere (see e.g. WO2016/012296) and plated on TGP plates supplemented with 8 mg/L chloramphenicol. Plates were incubated overnight at 55°C. A single colony was selected and grown overnight at 55°C in TGP broth supplemented with 8 mg/L chloramphenicol. Subsequently, 1 ml transfers were performed to 10 ml fresh prewarmed TGP medium supplemented with 8 mg/L chloramphenicol.
  • EXAMPLE 7 Application of spCas9 in Bacillus coagulans spCas9 is evaluated as a counter selection tool in Bacillus coagulans.
  • the Bacillus coagulans DSM 1 759-bp sigF gene (NCBI GenelD: 29812540) was chosen as deletion target. Upstream and downstream fragments of 0.85 kb were generated by PCR using the following primer pair combinations both at an annealing temperature of 58°C and by using chromosomal DNA of B.
  • coagulans DSM 1 as a template:
  • 2561 (5'-TCACGTTAAG G GATTTTGGTCATGAGTGAGTCTG G CTATTGACCTG G-3' [SEQ ID NO: 64]); and 2562 (5'-ATGAAAAAAGCGCACGTCGGCACGACTCCTTAATTG-3' [SEQ ID NO: 65])
  • the vector backbone was amplified in two parts using pWUR_Cas9nt as a template, introducing the targeting spacer sequence 5'-CGATGAGTTAACGAAAAAGC-3' [SEQ ID NO: 68] or the non-targeting spacer sequence 5'-GAAAGACCCGTATCCAAGAA-3' [SEQ ID NO: 69].
  • the targeting spacer one fragment was generated using primer combination: 2173 (5'-ATGGCGCTAGTGAGCTG-3' [SEQ ID NO: 58]); and
  • the other fragment was generated using primer combination:
  • the PCR fragments were created with Phusion Flash High-Fidelity PCR master mix (ThermoFisher) according to the manufacturer's instructions, using an annealing temperature of 58°C for all reactions.
  • the two PCR products containing the sigF flanks were fused to the two PCR products of the vector containing the targeting spacer.
  • the two PCR products containing the sigF flanks were fused to the two PCR products of the vector containing the non-targeting spacer.
  • the fragments were assembled into a single plasmid by fusing the 25-bp overlapping regions using the NEBuilder HiFi DNA Assembly Cloning Kit (New England BioLabs).
  • Plasmid DNA was concentrated using a Zymo DNA Clean and Concentrator spin column (Zymo Research) and eluted into 10 ⁇ _ hbO and transformed to electrocompetent E. coli TG90 (Gonzy-Treboul, G., Karmzyn-Campelli, C, Stragier, P. 1992. J. Mol. Biol. 224:967-979). Transformants were plated on LB agar plates supplemented with 10 mg/L chloramphenicol and incubated at 37°C. A single colony was selected for plasmid extraction using the ZymoPURETM Plasmid Midiprep Kit (Zymo Research). Plasmid integrity was confirmed by sequence analysis.
  • Both integration vectors pMH243 and pMH249 were transformed to Bacillus coagulans by electroporation as described elsewhere (Kovacs, A. T., van Hartskamp, M., Kuipers, O. P., & van Kranenburg, R. 2010. Applied and Environmental Microbiology, 76(12), 4085-4088) and plated on BC plates supplemented with 7 mg/L chloramphenicol (Kovacs, A. T., van Hartskamp, M., Kuipers, O. P., & van Kranenburg, R. 2010. Applied and Environmental Microbiology, 76(12), 4085-4088). Plates were incubated overnight at 45°C.
  • a single colony was selected and grown overnight at 45°C in BC broth supplemented with 7 mg/L chloramphenicol. Subsequently, 0.2 ml transfers were performed to 10 ml fresh, prewarmed BC medium supplemented with 7 mg/L chloramphenicol. Incubations were for 8 h at 65°C, for recombination to occur, and for 24 hours at 45°C. After this, 20 ml was transferred to 10 ml fresh, prewarmed BC medium supplemented with 7 mg/L chloramphenicol and cultures were incubated overnight at 37°C.
  • EXAMPLE 8 Application of spCas9 in Bacillus thermoamylovorans spCas9 is evaluated as counter selection tool in Bacillus thermoamylovorans.
  • Upstream and downstream fragments of 0.85 kb were generated by PCR using primer combinations 2553 (5'-TCACGTTAAGGGATTTTGGTCATGAGTGAGTCTGCAAGT
  • pWUR_Cas9nt as a template, introducing the targeting spacer sequence 5'-ACTAATTA AGAAAAGTCAGG-3' [SEQ ID NO: 80].
  • TTTTAAGAATAATC-3' [SEQ ID NO: 81 ]).
  • the other fragment was generated using primer combination 2557 (5'-GTTATACACTAATTAAGAAAAGTCAGGGTTTTAGAGCTAGAAATAG CAAGTTAAAATAAG-3' [SEQ ID NO: 82]) and 2172 (5'-TCATGACCAAAATCCCTTAAC-3' [SEQ ID NO: 61 ]).
  • the PCR fragments were created with Phusion Flash High-Fidelity PCR master mix (ThermoFisher) according to the manufacturer's instructions, using an annealing temperature of 58°C for all reactions.
  • the four fragments were assembled into a single plasmid by fusing the 25-bp overlapping regions using the NEBuilder HiFi DNA Assembly Cloning Kit (New England BioLabs). Plasmid DNA was concentrated using a Zymo DNA Clean and Concentrator spin column (Zymo Research) and eluted into 10 ⁇ _ h O and transformed to electrocompetent E. coli TG90 (Gonzy-Treboul, G., Karmzyn-Campelli, C, Stragier, P. 1992. J. Mol. Biol. 224:967-979). Transformants were plated on LB agar plates supplemented with 10 mg/L chloramphenicol and incubated at 37°C.
  • a single colony was selected for plasmid extraction using the ZymoPURETM Plasmid Midiprep Kit (Zymo Research). Plasmid integrity was confirmed by sequence analysis.
  • the integration vector was transformed to Bacillus thermoamylovorans by electroporation as described elsewhere (see WO2016/012296), except for the resistance during electroporation being 400 instead of 600 ohm, and plated on TGP plates supplemented with 7 mg/L chloramphenicol. Plates were incubated overnight at 55°C. A single colony was selected and grown overnight at 55°C in TGP broth supplemented with 7 mg/L chloramphenicol.
  • 10 ml of the frozen glycerol stock was revived in 10 ml fresh prewarmed TGP medium supplemented with 7 mg/L chloramphenicol and grown overnight at 50°C.
  • the glycerol stock storage step is not required and should be considered as optional at any step of the temperature downshift or upshift at 45°C or higher.
  • 1 ml transfers were performed to 10 ml fresh prewarmed TGP medium supplemented with 7 mg/L chloramphenicol. Incubations were for 8 h at 45°C, for overnight at 41 °C and for 8 h 37°C. After this, 0.5 ml was transferred to 10 ml fresh prewarmed TGP medium and the culture was grown overnight at 55°C.
  • the culture was plated at 55°C and colony PCR was performed on 82 colonies to check for knockouts using primers 572 (5 - AGCGGTATTGGAGAAATTTG-3' [SEQ ID NO: 83]) and 573 (5'- CGTCACAGCCCATTCATAG-3' [SEQ ID NO: 84]).
  • E. coli DH5a transformation efficiency was tested at different temperatures, particularly at 42°C and compared with transformation efficiency at 37°C and 40°C.
  • chemically competent cells NEB were transformed from the same batch with 50 pmol of the pUC19 vector, recovered in 1 ml SOB and plated on LB agar plates with 100 ⁇ g ml ampicillin for overnight incubation at the corresponding temperature. All the transformations gave approximately the same number of colonies (10000 colonies/nmol of pUC19) regardless of the incubation temperature. The only observed difference was the size of the colonies; the higher the incubation temperature, the smaller the colonies. This result clearly indicates that E.
  • coli are transformable at near thermophilic temperatures (for which spCas9 is inactive) and therefore the herein described tool for genome editing is applicable to mesophiles.
  • Mesophilic cells can be transformed with the editing constructs, recovered, plated on selective medium and cultured at any temperature that spCas9 is not active (42°C and above) allowing for homologous recombination events to occur. Subsequently, dropping the temperature to mesophilic levels (around 37°C) would induce the spCas9-based counter-selection process. Then by plating the cells again on selective medium at the mesophilic temperature, the colonies can be screened for the surviving mutants that are not targeted by the spCas9.
  • the following are nucleotide sequences of plasmids as described herein:

Landscapes

  • Genetics & Genomics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Mycology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

In an absence of efficient non-homologous end joining (NHEJ) repair mechanisms in the majority of microbes, double stranded DNA break (DSDB) typically leads to cell death. In methods of microbial gene editing using plasmid transformation, both homologous recombination and Cas9 site-specific gene editing events can be used together. Single or multiple plasmid approaches are used. In a method of counter-selection of microbes for a desired genetic change, a two-phase approach is used whereby a switch is made from a higher growth temperature phase favouring homologous recombination (HR) - as opposed to a Cas9 site-directed nuclease activity- to a lower growth temperature phase at which the Cas9 site directed nuclease activity takes place. This has the effect whereby the Cas9 site-directed nuclease activity has counter selecting activity, removing microbes which do not have a desired modification introduced beforehand by HR. The population of microbes surviving after the temperature switch counter selection is thereby enhanced for the desired modification.

Description

MICROBIAL GENOME EDITING
FIELD OF THE INVENTION The present invention relates to the field of microbial genetic engineering. More particularly the invention concerns methods and systems for yielding enriched populations of cells containing a desired genome modification. The invention also concerns the field of gene editing employing specific sequence guided nucleases to achieve site-specific cutting of microbial genetic material.
BACKGROUND OF THE INVENTION
Microbial fermentation of renewable resources into green fuels and chemicals is playing a major part in the development of the bio-based economy. The production costs of these environmentally friendly processes have to be reduced before they become competitive with the traditional fossil fuel-based industries. To this end, microbes other than the widely-used model organisms, such as Escherichia coli and Saccharomyces cerevisiae, are being evaluated for their respective abilities to act as production hosts. Thermophilic organisms are of particular interest due to their multiple advantages over mesophilic organisms when being used as production hosts ( Bosma, E.F., et al., (2013) Current Biotechnology, 2, 360-379; Lin, L. and Xu, J. (2013) Biotechnology Advances, 31 , 827-837; Olson, D.G., et al., (2015) Current Opinion in Biotechnology, 33, 130-141 .). For example, their ability to grow and ferment at thermophilic temperatures reduces the contamination risk and the cooling costs, increases substrate and product solubility while the fermentation process runs at the optimum temperature for enzymatic lignocellulose degradation, allowing for efficient simultaneous saccharification and fermentation (Ou, M., et al., (2009) Applied Biochemistry and Biotechnology, 1 , 76-82; Bhalla, A., et al., (2013) Bioresource Technology, 128, 751 -759). However, the use of non-model thermophiles as production hosts is generally hampered by the lack of well-developed genome editing tools compared to the currently used mesophilic model organisms (Bosma, E.F., etal., (2013) Current Biotechnology, 2, 360-379; Taylor, M.P., et al., (201 1 ) Microbial biotechnology, 4, 438-448.)
Previous work has established basic genome editing tools for the facultative thermophilic strain Bacillus smithii ET 138 (referred to as ET 138 herein) (Bosma, E.F., et al., (2015) Microbial Cell Factories, 14, Art.nr.99). This species grows between 37°C and 65°C and efficiently utilizes both C5 and C6 sugars (Bosma, E.F., et al., (2015) Applied and Environmental Microbiology, 81 , 1874-1883; Nakamura, L.K., et al., (1988) International Journal of Systematic Bacteriology, 38, 63-73). The tool developed for this bacterium allows for the introduction of scar-free markerless gene deletions via a homologous recombination process and /acZ-based counter-selection relying on the toxicity of high concentrations of 5- bromo-4-chloro-3-indolyl-3-D-galactopyranoside (X-gal) (Bosma, E.F., et al., (2015) Microbial Cell Factories, 14, Art.nr.99; Van Spanning, R.J.M., et al., (1991 ) Journal of Bacteriology, 173, 6962-6970.). However, the developed process is time-consuming, with the fastest possible route to gene deletion taking approximately 2 to 3 weeks from transformation to generation of a scar-free markerless knockout (Bosma, E.F., et al., (2015) Microbial Cell Factories, 14, Art.nr.99). Moreover, the counter-selection step is not stringent enough for removal of genes that are essential for the fitness and the metabolism of the strain. For the two successfully engineered genes, only 12.5-33% of the colonies had the mutant genotype, whereas for other genes only wild-type revertants and false positives were obtained, generating mostly (66-88%) or in some cases only wild-type revertants or false positives (Bosma, E.F., et al., (2015) Microbial Cell Factories, 14, Art.nr.99). To further develop ET 138 into a host organism of industrial relevance, faster and more efficient tools are required.
One of the fastest and most efficient methods currently available for genome editing is a system based on the Cas9 gene from the type II CRISPR-Cas bacterial adaptive immune system (Barrangou, R., et al., (2007) Science, 315, 1709-1712; Brouns, S.J., et al., (2008) Science, 321 , 960-964; van der Oost, J., et al., (2009) Trends Biochem Sci, 34, 401 -407). A short appropriately designed single guide RNA (sgRNA) molecule can direct Cas9 endonuclease to the desired target on the genome (Jinek, M., et al., (2013). eLife, 2, e00471.). There, Cas9 introduces a chromosomal double stranded DNA break (DSDB), which is lethal (Oh, J.-H. and van Pijkeren, J. -P. (2014) Nucleic acids research, 42, e131 ; Zeng, H., et al., (2015) Appl Microbiol Biotechnol, 99, 10575-10585; Mougiakos, I., et al., (2016). Trends in Biotechnology, 34, 575-587; Barrangou, R. and van Pijkeren, J. P. (2016) Curr Opin Biotechnol, 37, 61 -68). In eukaryotes, the active Non-Homologous End Joining (NHEJ) mechanism repairs Cas9-induced DSDBs in an error-prone manner, creating insertion/deletion (indel) mutations. These mutations usually render the gene inactive through frameshifting and simultaneously prevent further Cas9-recognition and subsequent cleavage due to the alteration of the target site (Jinek, M., et al., (2013). eLife, 2, e00471 .; Kim, S., et al., (2014) Genome Research, 24, 1012-1019; Mali, P., et al., (2013) Nat Meth, 10, 957-963; Cong, L, et al., (2013) Science, 339, 819-823). Whilst for most prokaryotes the NHEJ system is generally not present or not active (Bowater, R. and Doherty, A.J. (2006) PLoS genetics, 2, 93-99), the ET 138 genome contains the genes for the basic prokaryotic NHEJ-like system, consisting of DNA ligase LigD and DNA-end-binding protein Ku ( Bosma, E.F., et al., (2016) Standards in Genomic Sciences, 1 1 ; Shuman, S. and Glickman, M.S. (2007) Nat Rev Micro, 5, 852-861.24,25). However, the level of activity of the NHEJ-like system in ET 138 is unknown. The combination of Cas9 activity with editing templates, such as recombineering oligonucleotides or plasmid-borne sequences for homologous recombination, has been recently exploited for prokaryotic genome editing (Mougiakos, I., et al, (2016). Trends in Biotechnology, 34, 575-587; Barrangou, R. and van Pijkeren, J. P. (2016). Curr Opin Biotechnol, 37, 61 -68; Peters, J.M., et al., (2015) Curr Opin Microbiol, 27, 121 -126; Jiang, W., et al, (2013) Nature Biotech, 31 , 233-239). Cas9 was employed to introduce DSDBs in prokaryotic genomes. These breaks modestly induced the recombination of a provided rescuing/editing template into the targeted chromosome, resulting in genetically modified cells (Jiang, W., et al., (2013) Nature Biotech, 31 , 233-239; Xu, T., et al., (2015) Appl Environ Microbiol, 81 , 4423-4431 ; Huang, H., et ai, (2015) Acta Biochimica et Biophysica Sinica, 47, 231 -243). The edited cells avoided subsequent Cas9 targeting events, but in many studies the number of surviving/edited colonies was low with high percentage of mixed (both wild type and mutant) or escape mutant genotypes (Huang, H., et ai, (2016) ACS Synth Biol; Li, Q., et al, (2016) Biotechnol J, 1 1 , 961 -972; Wang, Y., et al, (2015) J Biotechnol, 200, 1 -5). The number of surviving colonies as well as the percentage of successfully edited cells was higher in studies that allowed homologous recombination of the editing templates to take place prior to the Cas9 targeting. This way Cas9 was employed for stringent counter-selection of the unedited genomes. For this approach either homologous recombination was faster than Cas9-targeting or cas9 expression was induced after homologous recombination (Li, Y., et al,
(2015) Metabolic Engineering, 31 , 13-21 ; Wang, Y., et ai, (2016) ACS Synth Biol, 5, 721 -732; Tong, Y., et al, (2015) ACS Synthetic Biology, 4, 1020-1029). Moreover, the vast majority of the studies required either a multiple-plasmid system or very tightly controlled promoters (Wang, Y., et al, (2016) ACS Synth Biol, 5, 721 -732; Barrangou, R. and van Pijkeren, J.-P.
(2016) Current Opinion in Biotechnology, 37, 61-68; Jiang, Y., et al. (2015) Appl Environ Microbiol 81 , 2506-2514.). Currently, only one plasmid, one selection marker and no inducible promoters are available for ET 138, limiting the options for such systems. Many of the well-known and widely applied genome editing tools, including CRISPR-Cas9 editing, are not amenable to thermophiles. The enzymatic machineries of these tools have not proven stable at temperatures higher than 37°C. Whereas the native CRISPR-Cas type I system of a thermophilic archaeon has been employed for genome editing (Li, Y., et al, (2015) Nucleic Acids Research, 44) as well as chromosome-based genetic manipulations have been reported for a few naturally competent thermophiles (Zeldes, B.M., et al. (2015) Front Microbiol, 6, 1209), no reports are available on using Cas9-based editing in thermophilic organisms. SUMMARY OF THE INVENTION
The inventors have discovered a novel method for microbial genome editing.
In one aspect the invention provides a method of microbial genome editing, comprising the steps of:
(a) introducing into cells:
(i) at least one guide RNA or at least one polynucleotide comprising a sequence encoding a guide RNA; wherein the or each guide RNA is substantially complementary to a target polynucleotide sequence(s) in a microbial genome;
(ii) a nuclease, or a polynucleotide comprising a sequence encoding the nuclease; wherein the nuclease forms a ribonuclease complex with the guide RNA, and wherein the ribonuclease complex makes site-specific double stranded DNA breaks (DSDB) in the microbial genome;
(iii) a homologous recombination polynucleotide or a polynucleotide comprising a sequence encoding an homologous recombination (HR) polynucleotide having a sequence substantially complementary to a target region containing the target in the microbial genome, and having regions upstream and downstream that flank the target;
(b) incubating the cells at a first temperature whereby homologous recombination occurs between the HR polynucleotide and the target region, wherein the modified microbial genome is resistant to site-specific DSDB by the ribonuclease complex; and
(c) incubating the cells at a second temperature whereby ribonuclease complex mediated site-specific DSDB occurs in unmodified cells;
wherein the first incubation temperature is greater than the second incubation temperature.
The methods described herein therefore advantageously employ a temperature switch, from a higher growth temperature phase favouring HR and not the site-directed nuclease activity, to a lower growth temperature phase whereby counter selection occurs by the site-directed nuclease activity removing microbes which do not have a desired modification introduced by HR. The population of microbes surviving after the temperature switch counter selection is thereby enhanced for the desired modification. Any microbial cells have the potential to be modified by the methods described herein. A requirement is that the microbes can grow across a temperature range in which a selected ribonuclease complex has nuclease activity at one of the microbial growth temperatures and substantially no nuclease activity at another microbial growth temperature. In this way the methods described herein allow the process of homologous recombination to be favoured at a first temperature such that the microbial genome can be modified with the desired mutation and a second temperature in which unmodified cells can be targeted by the ribonuclease complex to introduce a DSDB into the genomes of the unmodified cells. Due to an absence of an efficient non-homologous end joining (NHEJ) repair mechanism in the majority of microbes, DSDB typically leads to cell death. Thus, these methods overall increase the population of microbial cells with the desired mutation whilst eliminating any unmodified microbial cells. Preferably, the methods described herein are used in microbes that have substantially no endogenous NHEJ repair mechanism. Alternatively, the methods described herein may be applied to microbes that have an endogenous NHEJ repair mechanism. In further embodiments, the methods described herein may be applied to microbes that have an endogenous NHEJ repair mechanism but wherein the NHEJ repair mechanism is either conditionally reduced or the NHEJ activity is knocked out.
In one aspect, the methods described herein utilises a sequence of the homologous recombination polynucleotide that has at least one mis-match with the guide RNA, such that the guide RNA is no longer able to recognise the modified genome. This means that the ribonuclease complex will not recognise the modified genome. Therefore, no DSDB can be introduced by the ribonuclease complex and so the modified cells will survive. However, the cells with unmodified genomes will still have substantial complementarity to the guide RNA and consequently can be cleaved site-specifically by the ribonuclease complex.
The homologous recombination polynucleotide may have more than 1 mis-match with the guide RNA, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32 or more mis-matches with the guide RNA (from the 5' end or the 3' end). Such multiplicity of mis-matches may be contiguous. Alternatively, a multiplicity of mis-matches may be non-contiguous. There may be a mixture of contiguous and non-contiguous mis-matches where there are three or more mismatches. The 32 nt at the 5'-end of the sgRNA molecule corresponds to the crRNA module.
In some embodiments, the methods described herein utilise a sequence of the homologous recombination polynucleotide that has one or more contiguous or non-contiguous mismatches in the 13 base pairs between the guide RNA and the target DNA site proximal to the PAM, and/or seven or more contiguous mismatches in the 5'-terminal region of the protospacer. Another way of characterising the degree of recognition between the homologous recombination polynucleotide and the guide of the ribonuclease is to express this in terms of sequence identity. Therefore, the sequence of the homologous recombination peptide may have less than 99% identity when aligned fully with the sequence of the guide; in alternative embodiments this can be less than 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91 %. Also, less than 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%.
In another aspect of the method of the invention, the way in which the ribonucleoase complex is prevented from acting to cleave the microbial genome is not so much to modify or eliminate the sequence targeted by the guide, but rather the PAM required by the ribonuclease complex. The PAM is either modified or eliminated in order to blind the ribonuclease complex to the specific cutting site. Therefore, methods of the invention may include those using a sequence of the homologous recombination polynucleotide that does not include a PAM sequence recognised by the ribonuclease complex. Therefore, no DSDB can be introduced by the ribonuclease complex and so the HR modified cells will survive. However, the unmodified cells will still be recognised by the ribonuclease complex of editing nuclease and its guide and so consequently are cleaved site-specifically.
The methods described herein rely on homologous recombination to modify the genome of the microbe. Preferably, the upstream flank and downstream flanks are 0.5 kilobases (kb) to 1 .0 kb each in length. However, recombination using larger or shorter fragments is possible as well. The homologous recombination polynucleotide can in some embodiments further comprise a polynucleotide sequence between the upstream and downstream flanking regions. This polynucleotide sequence could for example contain a modification that is to be introduced into the microbial genome.
Whilst homologous recombination relies upon the upstream and downstream flanks having substantial complementarity to the target regions, mismatches can be accommodated as well. Therefore, in some embodiments, homologous recombination is known to occur between DNA segments with extensive homology to the upstream and downstream flanks. In alternative embodiments, the upstream and downstream flanks have complete complementarity to the target regions. The upstream and downstream flanks need not be identical in size. However, in some instances the upstream and downstream flanks are identical in size. The efficiency of homologous recombination will vary depending on the likelihood of homologous recombination of the smallest fragment length of the flank. However, even if the homologous recombination process is inefficient, advantageously the method described herein will select for any microbial cell that has the desired modification over the unmodified microbial cell. Homologous recombination also allows large deletions (e.g. 50 kb or more) to be made encompassing complete gene clusters. Homologous recombination is also used for recombineering, which is a well-known method to allow for recombination over smaller fragments (45-100 nt). The methods described herein can optionally further comprise at least another homologous recombination polynucleotide or a polynucleotide comprising a sequence encoding a homologous recombination polynucleotide having a sequence substantially complementary to a second target region containing the target in the microbial genome.
In preferred embodiments, the methods described herein, utilise a homologous recombination polynucleotide that is DNA. In some embodiments the DNA is single stranded. In other embodiments, the DNA is double stranded. In further embodiments, the DNA is double stranded and plasmid borne.
Homologous recombination in the methods described herein may be used to remove a polynucleotide sequence from the microbial genome. Alternatively, homologous recombination in the methods described herein may be used to insert one or more gene(s), or fragment(s) thereof, in to the microbial genome. As a further alternative, homologous recombination in the methods described herein can be used to modify or replace at least one nucleotide in the microbial genome. Consequently, the methods described herein can be used for any desired kind of genome modification.
Methods of the invention involve treating microbial cells in order to introduce essential elements of HR polynucleotide, guide, nuclease (or ribonuclease complex) whether directly or indirectly via an expression plasmid or vector. Various options exist for the introduction of components, whether separately, simultaneously or combined. In preferred embodiments, components are introduced in to the microbial cells at the same temperature as the first incubation temperature or substantially the first incubation temperature. Alternatively, the components can be introduced at sub-optimal growth temperatures for the microbe concerned. Potentially these temperatures may be characterised by minimal or no cell division or the microbial culture is in stasis.
A range of methods of introducing polynucleotide and polypeptide components into microbial cells are already widely known in the art such as transformation, transduction or transfection. In preferred embodiments, the polynucleotide components are introduced by chemical transformation, heat-shock transformation or natural transformation. In other embodiments polynucleotide components described herein are introduced into the cells as part of a polynucleotide vector, e.g. an expression plasmid. Alternatively, the components can be introduced as part of several e.g. two or three polynucleotide vectors. The components can be introduced into the cells substantially separately, simultaneously or sequentially.
The methods described herein require introduction into the microbial cells at least one guide RNA or at least one polynucleotide comprising a sequence encoding a guide RNA. Alternatively, the methods described herein can further comprise introducing at least a second guide RNA or at least one polynucleotide comprising a sequence encoding a guide RNA; wherein the second guide RNA is substantially complementary to a second target polynucleotide sequence(s) in a microbial genome. The option exists for making a multiplicity of DSDBs in the unmodified genome, in which case a multiplicity of corresponding guide RNAs will be required.
In some embodiments, the guide RNA comprises a crRNA and tracrRNA. Alternatively, the guide RNA comprises a single chimeric guide RNA. In further alternatives, the guide RNA comprises a crRNA. The guide RNAs can be of any length.
In other embodiments, the guide RNA may comprise additional modification to the 5' terminus such as phosphorylation or hydroxylation. The methods defined herein can use the B. coagulans phosphotransacetylase {pta) promoter Ppta without its ribosome binding sequence to mediate expression of a guide RNA from a plasmid.
The methods described herein can optionally further comprise incubating the cells at a third temperature; wherein the third temperature is greater than the second temperature; optionally wherein the third temperature is the same as the first temperature. If the third temperature is the same as the second temperature this effectively leads to prolonging the duration at which the microbes are incubated at the second temperature. Alternatively, the third temperature could be the same as the first temperature such that the microbial cells are incubated at the first temperature twice, once before the second temperature and once after the second temperature. Optionally, this could be via intervening temperatures as described herein.
In any of the methods described herein, the culture medium can be changed at any time for fresh culture medium. This may happen at least once; possibly two or more times. The culture medium changes may take place together with a change in temperature of the microbial culture. In some embodiments, in the methods described herein there is substantially no nuclease activity by the ribonuclease complex when the microbial cells are incubated at first incubation temperature. Alternatively, there may be some but not significant nuclease activity by the ribonuclease complex when the microbial cells are incubated at the first incubation temperature. In such situations nuclease activity by the ribonuclease complex is less than the homologous recombination activity in the microbial cells when incubated at the first temperature. The respective rates of ribonuclease activity introducing DSDBs and HR are such that HR prevails and so as a result of microbial growth at the first temperature for a period of time results in enrichment of cells for the HR modified genome.
In any of the methods, there may be incubation of the microbial cultures at one or more intervening temperatures between the first incubation temperature and the second incubation temperature, wherein the or each intervening temperature is lower than the first incubation temperature and higher than the second incubation temperature. Preferably, the intervening temperature is selected from: 40 °C, 41 °C, 42 °C, 43 °C, 44 °C, 45 °C, 46 °C, 47 °C, 48 °C, 49 °C, 50 °C, 51 °C, 52 °C, 53 °C, 54 °C, 55 °C. Included in all of the above whole numbers is any intervening one tenth fraction of a degree, e.g. 0.1 , 0.2, etc.
In some of the methods described herein there may be a gradual lowering of the incubation temperatures from the first incubation temperature to the second incubation temperature via a linear gradient, for example. Equally it could be a stepped gradient.
In another aspect, the methods described herein, have the first incubation temperature of between 20 °C and 70 °C, preferably 45 °C to 55 °C. Alternatively, the methods described herein have the first incubation temperature as higher than 39 °C, preferably higher than 40 °C, more preferably higher than 41 °C, as and even more preferably higher than 42 °C. In other embodiments, the first incubation temperature is a minimum temperature of 40 °C, 41 °C, 42 °C, 43 °C, 44 °C, or 45 °C. In other embodiments, the first incubation temperature is a maximum temperature of 55 °C, 56 °C, 57 °C, 58 °C, 59 °C, 60 °C, 61 °C, 62 °C, 63 °C, 64 °C, or 65 °C. Alternatively, the first incubation temperature can be selected from: 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, 40 °C, 41 °C, 42 °C, 43 °C, 44 °C, 45 °C, 46 °C, 47 °C, 48 °C, 49 °C, 50 °C, 51 °C, 52 °C, 53 °C, 54 °C, 55 °C, 56 °C, 57 °C, 58 °C, 59 °C, 60 °C, 61 °C, 62 °C, 63 °C, 64 °C, 65 °C, 66 °C, 67 °C, 68 °C, 69 °C or 70 °C. Included in all of the above whole numbers is any intervening one tenth fraction of a degree, e.g. 0.1 , 0.2, etc. In yet another aspect, the methods described herein have the second incubation temperature of between 20 °C and 50 °C, preferably 35 °C to 45 °C. A method as claimed in any preceding claim, wherein the second incubation temperature is lower than 45 °C, preferably lower than 44 °C, more preferably lower than 43 °C, and even more preferably lower than 42 °C. In other embodiments, the second incubation temperature is a minimum temperature of 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C or 37 °C. In other embodiments, the second incubation temperature is a maximum temperature of 40 °C, 41 °C, 42 °C, 43 °C, 44 °C or 45 °C. Alternatively, the second incubation temperature can be selected from: 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, 40 °C, 41 °C, 42 °C, 43 °C, 44 °C, 45 °C, 46 °C, 47 °C, 48 °C, 49 °C or 50 °C. Included in all of the above whole numbers is any intervening one tenth fraction of a degree, e.g. 0.1 , 0.2, etc.
The methods described herein can optionally further comprise incubation at a fourth temperature. In some embodiments, the fourth incubation temperature step allows for the modified microbial cells to be cured of an introduced plasmid. In these embodiments the plasmid introduced into the microbial cells is unable to replicate above a specific temperature. Alternatively, any introduced plasmid may be cured by culture in non-selective conditions. The duration of incubation may be tailored to suit particular microbial strains and to ensure the desired degree of completion of HR and/or DSDB formation. Therefore incubation times at the variously selected temperatures of operating the invention may be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 hours, for example. More incubation times may be employed as needed. The duration of the incubation temperatures may vary between 6 to 20 hours for each incubation step; preferably 8 to 16 hours.
The microbes modified by the methods described herein may be grown in any medium. Particularly suitable media that may be used for particular microbes are well known in the art. Specific media used will depend on the microbes used in the methods described herein.
In some embodiments, microbes may be grown in media comprising TVMY medium and optionally further comprising any carbon source selected from: xylose, uracil and glucose, or any combination thereof.
Alternatively, microbes may be grown in media comprising LB2 medium, optionally further comprising any carbon source selected from: xylose, uracil and glucose, or any combination thereof. The methods described herein are for any desired microbial genome modification. A microbe includes bacteria, archaea, fungi, yeast and protozoa. In preferred applications of the method of the invention, the microbes are mesophilic or thermophilic. A mesophilic microbe is a microbe that has optimal growth at a temperature below 42 °C, preferably between 20 °C and 42 °C and a thermophilic microbe is defined as a microbe that has optimal growth at or above 42 °C, preferably between 42 °C and 70 °C. In some embodiments, the thermophilic microbes are hyperthermophiles, in other words, they are able to grow in temperatures greater than 70 °C, preferably between 70 °C and 100°C.
Methods of the invention described herein may be used to modify genomes of bacterial cells. In particular embodiments, the bacteria are capable of growth or survive an incubation step at a temperature below 42°C, preferably the bacteria are selected from: Acidithiobacillus species including Acidithiobacillus caldus; Aeribacillus species including Aeribacillus pallidus; Alicyclobacillus species including Alicyclobacillus acidocaldarius, Alicyclobacillus acidoterrestris, Alicyclobacillus cycloheptanicus; Anoxybacillus species including Anoxybacillus caldiproteolyticus, Anoxybacillus flavithermus, Anoxybacillus rupiensis, Anoxybacillus tepidamans; Bacillus species including Bacillus caldolyticus, Bacillus caldotenax, Bacillus caldovelox, Bacillus coagulans, Bacillus clausii, Bacillus licheniformis, Bacillus methanolicus, Bacillus smithii including Bacillus smithii ET138, Bacillus subtilis, Bacillus thermocopriae, Bacillus thermolactis, Bacillus thermoamylovorans, Bacillus thermoleovorans; Caldibacillus species including Caldibacillus debilis; Caldicellulosiruptor species including Caldicellulosiruptor bescii, Caldicellulosiruptor hydrothermalis, Caldicellulosiruptor kristjanssonii, Caldicellulosiruptor kronotskyensis, Caldicellulosiruptor lactoaceticus, Caldicellulosiruptor obsidiansis, Caldicellulosiruptor owensensis, Caldicellulosiruptor saccharolyticus; Clostridium species including Clostridium clariflavum, Clostridium straminisolvens, Clostridium tepidiprofundi, Clostridium thermobutyricum, Clostridium thermocellum, Clostridium thermosuccinogenes, Clostridium thermopalmarium; Deinococcus species including Deinococcus cellulosilyticus, Deinococcus deserti, Deinococcus geothermalis, Deinococcus murrayi, Deinococcus radiodurans; Defluviitalea species including Defluviitalea phaphyphila, Desulfotomaculum species including Desulfotomaculum carboxydivorans, Desulfotomaculum nigrificans, Desulfotomaculum salinum, Desulfotomaculum solfataricum; Desulfurella species including Desulfurella acetivorans; Desulfurobacterium species including Desulfurobacterium thermolithotrophum; Geobacillus species including Geobacillus icigianus, Geobacillus caldoxylosilyticus, Geobacillus jurassicus, Geobacillus galactosidasius, Geobacillus kaustophilus, Geobacillus lituanicus, Geobacillus stearothermophilus, Geobacillus subterraneus, Geobacillus thermantarcticus, Geobacillus thermocatenulatus, Geobacillus thermodenitrificans, Geobacillus thermoglucosidans, Geobacillus thermoleovorans, Geobacillus toebii, Geobacillus uzenensis, Geobacillus vulcanii, Geobacillus zalihae; Hydrogenobacter species including Hydrogenobacter thermophiles; Hydrogenobaculum species including Hydrogenobaculum acidophilum; Ignavibacterium species including Ignavibacterium album; Lactobacillus species including Lactobacillus bulgaricus, Lactobacillus delbrueckii, Lactobacillus ingluviei, Lactobacillus thermotolerans; Marinithermus species including Marinithermus hydrothermalis; Moorella species including Moorella thermoacetica; Oceanithermus species including Oceanithermus desulfurans, Oceanithermus profundus; Paenibacillus species including Paenibacillus sp. J2, Paenibacillus marinum, Paenibacillus thermoaerophilus; Persephonella species including Persephonella guaymasensis, Persephonella hydrogeniphila, Persephonella marina; Rhodothermus species including Rhodothermus marinus, Rhodothermus obamensis, Rhodothermus profundi; Sulfobacillus species including Sulfobacillus acidophilus; Sulfurihydrogenibium species including Sulfurihydrogenibium azorense, Sulfurihydrogenibium kristjanssonii, Sulfurihydrogenibium rodmanii, Sulfurihydrogenibium yellowstonense; Symbiobacterium species including Symbiobacterium thermophilum, Symbiobacterium toebii; Thermoanaerobacter species including Thermoanaerobacter brockii, Thermoanaerobacter ethanolicus, Thermoanaerobacter italicus, Thermoanaerobacter kivui, Thermoanaerobacter marianensis, Thermoanaerobacter mathranii, Thermoanaerobacter pseudoethanolicus, Thermoanaerobacter wiegelii; Thermoanaerobacterium species including Thermoanaerobacterium aciditolerans, Thermoanaerobacterium aotearoense, Thermoanaerobacterium ethanolicus, Thermoanaerobacterium pseudoethanolicus, Thermoanaerobacterium saccharolyticum, Thermoanaerobacterium thermosaccharolyticum,
Thermoanaerobacterium xylanolyticum; Thermobacillus species including Thermobacillus composti, Thermobacillus xylanilyticus; Thermocrinis species including Thermocrinis albus, Thermocrinis ruber; Thermodulfatator species including Thermodesulfatator atlanticus, Thermodesulfatator autotrophicus, Thermodesulfatator indicus; Thermodesulfobacterium species including Thermodesulfobacterium commune, Thermodesulfobacterium hydrogeniphilum; Thermodesulfobium species including Thermodesulfobium narugense; Thermodesulfovibrio species including Thermodesulfovibrio aggregans, Thermodesulfovibrio thiophilus, Thermodesulfovibrio yellowstonii; Thermosipho species including Thermosipho africanus, Thermosipho atlanticus, Thermosipho melanesiensis; Thermotoga species including Thermotoga maritima, Thermotoga neopolitana, Thermotoga sp. RQ7; Thermovibrio species including Thermovibrio ammonificans, Thermovibrio ruber, Thermovirga species including Thermovirga //'en/7 and Thermus species including Thermus aquaticus, Thermus caldophilus, Thermus flavus, Thermus scotoductus, Thermus thermophilus;
Thiobacillus neapolitanus.
In another aspect, a method described herein can be used to modify bacteria capable of growth at a temperature above 42°C. In preferred embodiments, the bacteria are selected from: Acidithiobacillus species including Acidithiobacillus caldus; Actinobacillus species including Actinobacillus succinogenes; Anaerobic-spirillum species including Anaerobiospirillum succiniciproducens; Bacillus species including Bacillus alcaliphilus, Bacillus amyloliquefaciens, Bacillus circulans, Bacillus cereus, Bacillus clausii, Bacillus firmus, Bacillus halodurans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus subtilis, Bacillus thuringiensis; Basfia species including Basfia succiniciproducens; Brevibacillus species including Brevibacillus brevis; Brevibacillus laterosporus; Clostridium species including Clostridium acetobutylicum, Clostridium autoethanogenum, Clostridium beijerinkii, Clostridium carboxidivorans, Clostridium cellulolyticum, Clostridium ljungdahlii, Clostridium pasteurianum, Clostridum perfringens, Clostridium ragsdalei, Clostridium saccharobutylicum, Clostridium saccharoperbutylacetonium; Corynebacterium species including Corynebacterium glutamicum; Desulfitobacterium species including Desulfitobacterium dehalogenans, Desulfitobacterium hafniense; Desulfotomaculum species including Desulfotomaculum acetoxidans, Desulfotomaculum gibsoniae, Desulfotomaculum reducens, Desulfotomaculum ruminis; Enterobacter species including Enterobacter asburiae; Enterococcus species including Enterococcus faecalis; Escherichia species including Escherichia coli; Lactobacillus species including Lactobacillus acidophilus, Lactobacillus amylophilus, Lactobacillus amylovorus, Lactobacillus animalis, Lactobacillus arizonensis, Lactobacillus bavaricus, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus corynoformis, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sakei, Lactobacillus salivarius, Lactobacillus sanfriscensis; Mannheimia species including Mannheimia succiniciproducens; Paenibacillus species including Paenibacillus alvei, Paenibacillus beijingensis, Paenibacillus borealis, Paenibacillus dauci, Paenibacillus durus, Paenibacillus graminis, Paenibacillus larvae, Paenibacillus lentimorbus, Paenibacillus macerans, Paenibacillus mucilaginosus, Paenibacillus odorifer, Paenibacillus polymyxa, Paenibacillus stellifer, Paenibacillus terrae, Paenibacillus wulumuqiensis; Pediococcus species including Pediococcus acidilactici, Pediococcus claussenii, Pediococcus ethanolidurans, Pediococcus pentosaceus; Salmonella typhimurium; Sporolactobacillus species including Sporolactobacillus inulinus, Sporolactobacillus laevolacticus; Staphylococcus aureus; Streptococcus species including Streptococcus agalactiae, Streptococcus bovis, Streptococcus equisimilis, Streptococcus feacalis, Streptococcus mutans, Streptococcus oralis, Streptococcus pneumonia, Streptococcus pyogenes, Streptococcus salivarius, Streptococcus thermophilus, Streptococcus sobrinus, Streptococcus uberis; Streptomyces species including Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, Streptomyces lividans, Streptomyces parvulus, Streptomyces venezuelae, Streptomyces vinaceus; Tetragenococcus species including Tetragenococcus halophilus and Zymomonas species including Zymomonas mobilis.
In a further aspect, a method defined herein could be used to modify the genome of yeast or fungi. In particular embodiments, the fungal species can grow at a temperature above 42°C, preferably the fungi is selected from: an Aspergillus species including, but not limited to, Aspergillus nidulans, Aspergillus niger, Aspergillus terreus, Aspergillus oryzae and Aspergillus terreus, more preferably the Aspergillus species is Aspergillus nidulans or Aspergillus niger. Alternatively, the fungal species capable of growing at a temperature above 42°C, could be a Candida species.
The invention further relates to use of a method as defined herein to modify a yeast or fungal species that is able to grow below 42°C, preferably the fungi or yeast is selected from: Aspergillus species including Aspergillus fumigatus, Aspergillus nidulans, Aspergillus terreus, Aspergillus versicolor, Canariomyces species including Canariomyces thermophile; Chaetomium species including Chaetomium mesopotamicum, Chaetomium thermophilum; Candida species including Candida bovina, Candida sloofii, Candida thermophila, Candida tropicalis, Candida krusei (=lssatchenkia orientalis); Cercophora species including Cercophora coronate, Cercophora septentrionalis; Coonemeria species including Coonemeria aegyptiaca; Corynascus species including Corynascus thermophiles; Geotrichum species including Geotrichum candidum; Kluyveromyces species including Kluyveromyces fragilis, Kluyveromyces marxianus; Malbranchea species including Malbranchea cinnamomea, Malbranchea sulfurea; Melanocarpus species including Melanocarpus albomyces;Myceliophtora species including Myceliophthora fergusii, Myceliophthora thermophila; Mycothermus species including Mycothermus thermophiles (=Scytalidium thermophilum/Torula thermophila); Myriococcum species including Myriococcum thermophilum; Paecilomyces species including Paecilomyces thermophila; Remersonia species including Remersonia thermophila; Rhizomucor species including Rhizomucor pusillus, Rhizomucor tauricus; Saccharomyces species including Saccharomyces cerevisiae, Schizosaccharomyces species including Schizosaccharomyces pombe, Scytalidium species including Scytalidium thermophilum; Sordaris species including Sordaria thermophila; Thermoascus species including Thermoascus aurantiacus, Thermoascus thermophiles; Thermomucor species including Thermomucor indicae-seudaticae and Thermomyces species including Thermomyces ibadanensis, Thermomyces lanuginosus.
In the aforementioned lists, microbes identified in bold typeface have been found to be particularly suitable/applicable in use for the present invention.
Some preferred embodiments of the present invention include one or more thermophilic microbes selected from: Thermophilic bacilli, Aeribacillus, Alicyclobacillus, Anoxybacillus, Bacillus, Geobacillus; Paenibacillus species; Thermophilic Clostridia, including Anaerobacter, Anaerobacterium, Caldicellulosiruptor, Clostridium, Moorella, Thermoanaerobacter, Thermoanaerobacterium, Thermobrachium, Thermohalobacter species or one or more Thermophilic Lactobacillus species and mesophilic bacteria selected from Bacillus species, Escherichia coli, and Lactobacillus species.
Suitable nucleases to be used in the methods described herein are selectable at the option of the average skilled person. A choice may depend upon the optimal growth temperature of the particular microbe being used. Methods described herein preferable use Cas9, preferably Streptococcus pyogenes Cas9, or C2C1. Alternatively, methods described herein can use Cpf1 . Further alternative nucleases suitable for the methods described herein are C2C3 or Argonaute. It is also contemplated that the methods described herein may use other nucleases such as zinc finger nucleases (ZFNS), meganucleases or transcription activator effector like nucleases (TALENS).
Preferred nucleases can be selected from the following tables:
Figure imgf000018_0001
Protein Genus Species
Cas9 Acetobacter aceti
Cas9 Acetobacterium dehalogenans
Cas9 Acetobacter sp.
Cas9 Acholeplasma palmae
Cas9 Acidaminococcus
Cas9 Acidaminococcus intestini
Cas9 Acidaminococcus sp.
Cas9 Acidothermus cellulolyticus
Cas9 Acidovorax avenae
Cas9 Acidovorax ebreus
Cas9 Acidovorax sp.
Cas9 Actinobacillus capsulatus
Cas9 Actinobacillus minor
Cas9 Actinobacillus pleuropneumoniae
Cas9 Actinobacillus succinogenes
Cas9 Actinobacillus suis
Cas9 Aequorivita capsosiphonis
Cas9 Afipia sp.
Cas9 Alcaligenaceae
Cas9 Alicycliphilus denitrificans
Cas9 Alicycliphilus sp.
Cas9 Alicyclobacillus hesperidum
Cas9 Alicyclobacillus tengchongensis
Cas9 Alistipes inops
Cas9 Alistipes shahii
Cas9 Alistipes sp.
Cas9 Alkaliflexus imshenetskii
Cas9 Alloprevotella tannerae
Cas9 alpha proteobacterium
Cas9 Aminomonas paucivorans
Cas9 Anaerococcus tetradius
Cas9 Anaeromusa acidaminophila
Cas9 Anaerophaga thermohalophila
Cas9 Anaerotruncus sp.
Cas9 Anaerovibrio lipolyticus
Cas9 Andreprevotia chitinilytica
Cas9 Anoxybacillus sp.
Cas9 Apibacter mensalis Cas9 Aquabacterium parvum
Cas9 Aquimarina muelleri
Cas9 Arcobacter sp.
Cas9 Armatimonadetes bacterium
Cas9 Atopobium sp.
Cas9 Atopococcus tabaci
Cas9 Azospirillum halopraeferens
Cas9 Azospirillum lipoferum
Cas9 Azospirillum sp.
Cas9 Bacillaceae bacterium
Cas9 Bacillus cereus
Cas9 Bacillus cytotoxicus
Cas9 Bacillus niameyensis
Cas9 Bacillus okhensis
Cas9 Bacillus pseudalcaliphilus
Cas9 Bacillus smithii
Cas9 bacterium L21-Spi-D4
Cas9 bacterium LF-3
Cas9 Bacteroidales bacterium
Cas9 Bacteroides
Cas9 Bacteroides cellulosilyticus
Cas9 Bacteroides coprophilus
Cas9 Bacteroides coprosuis
Cas9 Bacteroides dorei
Cas9 Bacteroides eggerthii
Cas9 Bacteroides faecis
Cas9 Bacteroides fluxus
Cas9 Bacteroides fragilis
Cas9 Bacteroides gallinarum
Cas9 Bacteroides graminisolvens
Cas9 Bacteroides nordii
Cas9 Bacteroides pyogenes
Cas9 Bacteroides sp.
Cas9 Bacteroides uniformis
Cas9 Bacteroides vulgatus
Cas9 Bacteroidetes bacterium
Cas9 Bacteroidetes oral
Cas9 Barnesiella intestinihominis
Cas9 Barnesiella viscericola Cas9 Bdellovibrio exovorus
Cas9 Belliella baltica
Cas9 Bergeyella zoohelcum
Cas9 Betaproteobacteria bacterium
Cas9 Bibersteinia trehalosi
Cas9 Bifidobacterium bifidum
Cas9 Bifidobacterium bombi
Cas9 Bifidobacterium dentium
Cas9 Bifidobacterium merycicum
Cas9 Bifidobacterium sp.
Cas9 Bifidobacterium tsurumiense
Cas9 Bordetella pseudohinzii
Cas9 Bordetella sp.
Cas9 Brackiella oedipodis
Cas9 Bradyrhizobium sp.
Cas9 Brevibacillus laterosporus
Cas9 Bryobacter aggregatus
Cas9 Burkholderiales bacterium
Cas9 Butyricimonas virosa
Cas9 Butyrivibrio fibrisolvens
Cas9 Butyrivibrio hungatei
Cas9 Butyrivibrio sp.
Cas9 Caldimonas taiwanensis
Cas9 Campylobacter
Cas9 Campylobacter coli
Cas9 Campylobacter cuniculorum
Cas9 Campylobacter fetus
Cas9 Campylobacter jejuni
Cas9 Campylobacter lari
Cas9 Campylobacter peloridis
Cas9 Campylobacter sp.
Cas9 Campylobacter subantarcticus
Cas9 Candidatus Alistipes
Cas9 Candidatus Bacteroides
Cas9 Candidatus Hepatoplasma
Cas9 Candidatus Puniceispirillum
Cas9 Capnocytophaga canimorsus
Cas9 Capnocytophaga cynodegmi
Cas9 Capnocytophaga gingivalis Cas9 Capnocytophaga ochracea
Cas9 Capnocytophaga sp.
Cas9 Capnocytophaga sputigena
Cas9 Carnobacterium funditum
Cas9 Carnobacterium gallinarum
Cas9 Carnobacterium maltaromaticum
Cas9 Carnobacterium sp.
Cas9 Catellicoccus marimammalium
Cas9 Catenibacterium mitsuokai
Cas9 Catenibacterium sp.
Cas9 Chryseobacterium
Cas9 Chryseobacterium angstadtii
Cas9 Chryseobacterium aquaticum
Cas9 Chryseobacterium formosense
Cas9 Chryseobacterium gallinarum
Cas9 Chryseobacterium greenlandense
Cas9 Chryseobacterium gregarium
Cas9 Chryseobacterium indologenes
Cas9 Chryseobacterium palustre
Cas9 Chryseobacterium soli
Cas9 Chryseobacterium sp.
Cas9 Chryseobacterium vrystaatense
Cas9 Cloacibacillus evryensis
Cas9 Clostridiales bacterium
Cas9 Clostridium beijerinckii
Cas9 Clostridium botulinum
Cas9 Clostridium cadaveris
Cas9 Clostridium cellulolyticum
Cas9 Clostridium colicanis
Cas9 Clostridium perfringens
Cas9 Clostridium saccharogumia
Cas9 Clostridium sp.
Cas9 Clostridium spiroforme
Cas9 Collinsella sp.
Cas9 Comamonas granuli
Cas9 Coprobacter fastidiosus
Cas9 Coprobacter secundus
Cas9 Coprococcus catus
Cas9 Coraliomargarita sp. Cas9 Coriobacteriales bacterium
Cas9 Coriobacterium glomerans
Cas9 Corynebacterium diphtheriae
Cas9 Corynebacterium matruchotii
Cas9 Croceitalea dokdonensis
Cas9 Cytophagales bacterium
Cas9 Dechloromonas agitata
Cas9 Dechloromonas denitrificans
Cas9 Demequina sediminicola
Cas9 Desulfovibrio termitidis
Cas9 Devosia sp.
Cas9 Dielma fastidiosa
Cas9 Dinoroseobacter shibae
Cas9 Dolosigranulum pigrum
Cas9 Dorea longicatena
Cas9 Dysgonomonas macrotermitis
Cas9 Echinicola pacifica
Cas9 Effusibacillus pohliae
Cas9 Eggerthellaceae bacterium
Cas9 Eggerthella sp.
Cas9 Eggerthia catenaformis
Cas9 Elioraea tepidiphila
Cas9 Elizabethkingia anophelis
Cas9 Elizabethkingia meningoseptica
Cas9 Elizabethkingia sp.
Cas9 Elusimicrobium minutum
Cas9 Empedobacter brevis
Cas9 Empedobacter falsenii
Cas9 Endomicrobium proavitum
Cas9 Enterococcus cecorum
Cas9 Enterococcus columbae
Cas9 Enterococcus dispar
Cas9 Enterococcus faecalis
Cas9 Enterococcus faecium
Cas9 Enterococcus hirae
Cas9 Enterococcus italicus
Cas9 Enterococcus mundtii
Cas9 Enterococcus phoeniculicola
Cas9 Enterococcus sp. Cas9 Enterococcus villorum
Cas9 Epilithonimonas tenax
Cas9 Erysipelotnchaceae bacterium
Cas9 Eubacterium dolichum
Cas9 Eubacterium ramulus
Cas9 Eubacterium rectale
Cas9 Eubacterium sp.
Cas9 Eubacterium ventriosum
Cas9 Facklamia hominis
Cas9 Fibrobacter succinogenes
Cas9 Filifactor alocis
Cas9 Finegoldia magna
Cas9 Firmicutes bacterium
Cas9 Flavobacteriaceae bacterium
Cas9 Flavobacterium akiainvivens
Cas9 Flavobacterium branchiophilum
Cas9 Flavobacterium chungangense
Cas9 Flavobacterium columnare
Cas9 Flavobacterium daejeonense
Cas9 Flavobacterium frigidarium
Cas9 Flavobacterium psychrophilum
Cas9 Flavobacterium soli
Cas9 Flavobacterium sp.
Cas9 Flexibacter roseolus
Cas9 Fluviicola taffensis
Cas9 Fructobacillus ficulneus
Cas9 Fructobacillus fructosus
Cas9 Fructobacillus sp.
Cas9 Fusobacterium necrophorum
Cas9 Fusobacterium nucleatum
Cas9 Fusobacterium periodonticum
Cas9 Galbibacter marinus
Cas9 Gallibacterium anatis
Cas9 gamma proteobacterium
Cas9 Gardnerella vaginalis
Cas9 Gemella bergeriae
Cas9 Gemella cuniculi
Cas9 Gemella haemolysans
Cas9 Gemella morbillorum Cas9 Geobacillus icigianus
Cas9 Geobacillus kaustophilus
Cas9 Geobacillus sp.
Cas9 Geobacillus stearothermophilus
Cas9 Geobacillus subterraneus
Cas9 Gluconacetobacter diazotrophicus
Cas9 Gordonibacter pamelaeae
Cas9 Haematospirillum jordaniae
Cas9 Haemophilus influenzae
Cas9 Haemophilus parainfluenzae
Cas9 Haemophilus sputorum
Cas9 Halalkalibacillus halophilus
Cas9 Helcococcus kunzii
Cas9 Helcococcus sueciensis
Cas9 Helicobacter apodemus
Cas9 Helicobacter canadensis
Cas9 Helicobacter cinaedi
Cas9 Helicobacter fennelliae
Cas9 Helicobacter macacae
Cas9 Helicobacter muridarum
Cas9 Helicobacter mustelae
Cas9 Helicobacter pametensis
Cas9 Helicobacter pullorum
Cas9 Helicobacter rodentium
Cas9 Helicobacter sp.
Cas9 Helicobacter typhlonius
Cas9 llyobacter polytropus
Cas9 Indibacter alkaliphilus
Cas9 Jeotgalibaca dankookensis
Cas9 Joostella marina
Cas9 Kandleria vitulina
Cas9 Kiloniella laminariae
Cas9 Kingella kingae
Cas9 Kordia algicida
Cas9 Kordia jejudonensis
Cas9 Kurthia huakuii
Cas9 Lachnoanaerobaculu saburreum
m
Cas9 Lachnobacterium bovis Cas9 Lachnospiraceae bacterium
Cas9 Lachnospira multipara
Cas9 Lacinutrix jangbogonensis
Cas9 Lacticigenium naphtae
Cas9 Lactobacillus
Cas9 Lactobacillus acidifarinae
Cas9 Lactobacillus acidophilus
Cas9 Lactobacillus agilis
Cas9 Lactobacillus animalis
Cas9 Lactobacillus apinorum
Cas9 Lactobacillus apodemi
Cas9 Lactobacillus brevis
Cas9 Lactobacillus buchneri
Cas9 Lactobacillus cacaonum
Cas9 Lactobacillus casei
Cas9 Lactobacillus ceti
Cas9 Lactobacillus composti
Cas9 Lactobacillus concavus
Cas9 Lactobacillus coryniformis
Cas9 Lactobacillus curvatus
Cas9 Lactobacillus delbrueckii
Cas9 Lactobacillus diolivorans
Cas9 Lactobacillus equicursoris
Cas9 Lactobacillus farciminis
Cas9 Lactobacillus fermentum
Cas9 Lactobacillus floricola
Cas9 Lactobacillus florum
Cas9 Lactobacillus fuchuensis
Cas9 Lactobacillus futsaii
Cas9 Lactobacillus gasseri
Cas9 Lactobacillus gastricus
Cas9 Lactobacillus graminis
Cas9 Lactobacillus hammesii
Cas9 Lactobacillus harbinensis
Cas9 Lactobacillus heilongjiangensis
Cas9 Lactobacillus helsingborgensis
Cas9 Lactobacillus helveticus
Cas9 Lactobacillus hominis
Cas9 Lactobacillus horde! Cas9 Lactobacillus iners
Cas9 Lactobacillus jensenii
Cas9 Lactobacillus johnsonii
Cas9 Lactobacillus kalixensis
Cas9 Lactobacillus kimbladii
Cas9 Lactobacillus kullabergensis
Cas9 Lactobacillus kunkeei
Cas9 Lactobacillus lindneri
Cas9 Lactobacillus mali
Cas9 Lactobacillus melliventris
Cas9 Lactobacillus mindensis
Cas9 Lactobacillus mucosae
Cas9 Lactobacillus namurensis
Cas9 Lactobacillus nodensis
Cas9 Lactobacillus oligofermentans
Cas9 Lactobacillus otakiensis
Cas9 Lactobacillus ozensis
Cas9 Lactobacillus parabuchneri
Cas9 Lactobacillus paracasei
Cas9 Lactobacillus paracollinoides
Cas9 Lactobacillus parakefiri
Cas9 Lactobacillus pentosus
Cas9 Lactobacillus plantarum
Cas9 Lactobacillus psittaci
Cas9 Lactobacillus rennini
Cas9 Lactobacillus reuteri
Cas9 Lactobacillus rhamnosus
Cas9 Lactobacillus rossiae
Cas9 Lactobacillus ruminis
Cas9 Lactobacillus saerimneri
Cas9 Lactobacillus sakei
Cas9 Lactobacillus salivarius
Cas9 Lactobacillus sanfranciscensis
Cas9 Lactobacillus saniviri
Cas9 Lactobacillus secaliphilus
Cas9 Lactobacillus senmaizukei
Cas9 Lactobacillus sp.
Cas9 Lactobacillus tucceti
Cas9 Lactobacillus versmoldensis Cas9 Lactobacillus wasatchensis
Cas9 Lactobacillus zymae
Cas9 Leptotrichia sp.
Cas9 Leuconostoc gelidum
Cas9 Limnohabitans planktonicus
Cas9 Listeria fleischmannii
Cas9 Listeria innocua
Cas9 Listeria ivanovii
Cas9 Listeria monocytogenes
Cas9 Listeria seeligeri
Cas9 Loktanella vestfoldensis
Cas9 Lunatimonas lonarensis
Cas9 Mannheimia haemolytica
Cas9 Mannheimia sp.
Cas9 Mannheimia varigena
Cas9 Marinovum algicola
Cas9 Maritimibacter alkaliphilus
Cas9 Megasphaera sp.
Cas9 Mesonia mobilis
Cas9 Mesorhizobium
Cas9 Mesorhizobium sp.
Cas9 Methylocystis sp.
Cas9 Methylophilus sp.
Cas9 Methylovulum miyakonense
Cas9 Mobiluncus curtisii
Cas9 Mobiluncus mulieris
Cas9 Mucilaginibacter paludis
Cas9 Mucinivorans hirudinis
Cas9 Mucispirillum schaedleri
Cas9 Mycoplasma alvi
Cas9 Mycoplasma arginini
Cas9 Mycoplasma canis
Cas9 Mycoplasma cynos
Cas9 Mycoplasma gallisepticum
Cas9 Mycoplasma hyosynoviae
Cas9 Mycoplasma lipofaciens
Cas9 Mycoplasma mobile
Cas9 Mycoplasma ovipneumoniae
Cas9 Mycoplasma sp. Cas9 Mycoplasma spumans
Cas9 Mycoplasma synoviae
Cas9 Myroides injenensis
Cas9 Myroides odoratus
Cas9 Necropsobacter massiliensis
Cas9 Negativicoccus succinicivorans
Cas9 Neisseria
Cas9 Neisseria bacilliformis
Cas9 Neisseria cinerea
Cas9 Neisseria flavescens
Cas9 Neisseria lactamica
Cas9 Neisseria meningitidis
Cas9 Neisseria mucosa
Cas9 Neisseria sp.
Cas9 Neisseria wadsworthii
Cas9 Niabella aurantiaca
Cas9 Niabella soli
Cas9 Nitratifractor salsuginis
Cas9 Nitrobacter hamburgensis
Cas9 Nitrosomonas sp.
Cas9 Novosphingobium subterraneum
Cas9 Oceanobacillus manasiensis
Cas9 Oenococcus kitaharae
Cas9 Oligella urethralis
Cas9 Olivibacter sitiensis
Cas9 Olsenella profusa
Cas9 Olsenella sp.
Cas9 Olsenella uli
Cas9 Ottowia sp.
Cas9 Pannonibacter phragmitetus
Cas9 Parabacteroides johnsonii
Cas9 Parabacteroides sp.
Cas9 Parvibaculum lavamentivorans
Cas9 Parvimonas micra
Cas9 Parvimonas sp.
Cas9 Paste urella multocida
Cas9 Paste urella pneumotropica
Cas9 Pediococcus acidilactici
Cas9 Pediococcus damnosus Cas9 Pediococcus inopinatus
Cas9 Pediococcus parvulus
Cas9 Pediococcus pentosaceus
Cas9 Pediococcus stilesii
Cas9 Pedobacter cryoconitis
Cas9 Pedobacter glucosidilyticus
Cas9 Pelomonas sp.
Cas9 Peptoniphilaceae bacterium
Cas9 Peptoniphilus duerdenii
Cas9 Peptoniphilus obesi
Cas9 Peptoniphilus sp.
Cas9 Peptostreptococcus anaerobius
Cas9 Phascolarctobacteriu succinatutens m
Cas9 Planctomyces sp.
Cas9 Planococcus antarcticus
Cas9 Porphyromonadaceae bacterium
Cas9 Porphyromonas gingivalis
Cas9 Porphyromonas gulae
Cas9 Porphyromonas somerae
Cas9 Porphyromonas sp.
Cas9 Prevotella amnii
Cas9 Prevotella aurantiaca
Cas9 Prevotella baroniae
Cas9 Prevotella bivia
Cas9 Prevotella buccae
Cas9 Prevotella buccalis
Cas9 Prevotella corporis
Cas9 Prevotella denticola
Cas9 Prevotella disiens
Cas9 Prevotella falsenii
Cas9 Prevotella fusca
Cas9 Prevotella histicola
Cas9 Prevotella intermedia
Cas9 Prevotella loescheii
Cas9 Prevotella melaninogenica
Cas9 Prevotella nigrescens
Cas9 Prevotella oralis
Cas9 Prevotella oulorum Cas9 Prevotella ruminicola
Cas9 Prevotella saccharolytica
Cas9 Prevotella sp.
Cas9 Prevotella stercorea
Cas9 Prevotella timonensis
Cas9 Prevotella veroralis
Cas9 Pseudodonghicola xiamenensis
Cas9 Psychroflexus torquis
Cas9 Psychroflexus tropicus
Cas9 Psychroserpens sp.
Cas9 Rhodobacteraceae bacterium
Cas9 Rhodobacter capsulatus
Cas9 Rhodocyclaceae bacterium
Cas9 Rhodopse udomonas palustris
Cas9 Rhodospirillum rubrum
Cas9 Rhodovulum sp.
Cas9 Riemerella anatipestifer
Cas9 Rikenella microfusus
Cas9 Roseburia intestinalis
Cas9 Roseburia inulinivorans
Cas9 Roseovarius tolerans
Cas9 Rubritepida flocculans
Cas9 Ruminococcaceae bacterium
Cas9 Ruminococcus albus
Cas9 Ruminococcus flavefaciens
Cas9 Ruminococcus lactaris
Cas9 Ruminococcus sp.
Cas9 Runella limosa
Cas9 Saccharibacter sp.
Cas9 Salegentibacter sp.
Cas9 Salinispira pacifica
Cas9 Salsuginibacillus kocurii
Cas9 Scardovia wiggsiae
Cas9 Schleiferia thermophila
Cas9 Sedimenticola thiotaurini
Cas9 Sediminibacterium sp.
Cas9 Sediminimonas qiaohouensis
Cas9 Selenomonas sp.
Cas9 Sharpea azabuensis Cas9 Shimia marina
Cas9 Simonsiella muelleri
Cas9 Skermanella aerolata
Cas9 Solobacterium moorei
Cas9 Sphaerochaeta globosa
Cas9 Sphingobacterium spiritivorum
Cas9 Sphingobium baderi
Cas9 Sphingobium sp.
Cas9 Sphingomonas
Cas9 Sphingomonas changbaiensis
Cas9 Sphingomonas sanxanigenens
Cas9 Sphingomonas sp.
Cas9 Spirochaeta cellobiosiphila
Cas9 Spiroplasma apis
Cas9 Spiroplasma eriocheiris
Cas9 Spiroplasma litorale
Cas9 Spiroplasma turonicum
Cas9 Sporocytophaga myxococcoides
Cas9 Sporolactobacillus vineae
Cas9 Staphylococcus agnetis
Cas9 Staphylococcus haemolyticus
Cas9 Staphylococcus hyicus
Cas9 Staphylococcus intermedius
Cas9 Staphylococcus lugdunensis
Cas9 Staphylococcus microti
Cas9 Staphylococcus pasteuri
Cas9 Staphylococcus pseudintermedius
Cas9 Staphylococcus saprophyticus
Cas9 Staphylococcus schleiferi
Cas9 Staphylococcus simulans
Cas9 Staphylococcus sp.
Cas9 Staphylococcus warneri
Cas9 Stenoxybacter acetivorans
Cas9 Streptococcus
Cas9 Streptococcus agalactiae
Cas9 Streptococcus anginosus
Cas9 Streptococcus caballi
Cas9 Streptococcus canis
Cas9 Streptococcus castoreus Cas9 Streptococcus constellatus
Cas9 Streptococcus dentasini
Cas9 Streptococcus dentisani
Cas9 Streptococcus devriesei
Cas9 Streptococcus dysgalactiae
Cas9 Streptococcus equi
Cas9 Streptococcus equinus
Cas9 Streptococcus gallolyticus
Cas9 Streptococcus gordonii
Cas9 Streptococcus enry
Cas9 Streptococcus hongkongensis
Cas9 Streptococcus infantarius
Cas9 Streptococcus infantis
Cas9 Streptococcus iniae
Cas9 Streptococcus intermedius
Cas9 Streptococcus lutetiensis
Cas9 Streptococcus macacae
Cas9 Streptococcus macedonicus
Cas9 Streptococcus marimammalium
Cas9 Streptococcus massiliensis
Cas9 Streptococcus mitis
Cas9 Streptococcus mutans
Cas9 Streptococcus oligofermentans
Cas9 Streptococcus oralis
Cas9 Streptococcus orisratti
Cas9 Streptococcus ovis
Cas9 Streptococcus parasanguinis
Cas9 Streptococcus pasteurianus
Cas9 Streptococcus phocae
Cas9 Streptococcus plurextorum
Cas9 Streptococcus pneumoniae
Cas9 Streptococcus pseudopneumoniae
Cas9 Streptococcus pseudoporcinus
Cas9 Streptococcus pyogenes
Cas9 Streptococcus ratti
Cas9 Streptococcus salivarius
Cas9 Streptococcus sanguinis
Cas9 Streptococcus sinensis
Cas9 Streptococcus sobrinus Cas9 Streptococcus sp.
Cas9 Streptococcus suis
Cas9 Streptococcus thermophilus
Cas9 Streptococcus tigurinus
Cas9 Streptococcus uberis
Cas9 Streptococcus vestibularis
Cas9 Subdoligran ulum sp.
Cas9 Sulfitobacter donghicola
Cas9 Sulfuritalea hydrogenivorans
Cas9 Sulfurovum lithotrophicum
Cas9 Tannerella forsythia
Cas9 Tenacibaculum maritimum
Cas9 Thermithiobacillus tepidarius
Cas9 Thermopetrobacter sp.
Cas9 Thermophagus xiamenensis
Cas9 Thioalkalivibrio sp.
Cas9 Tissierellia bacterium
Cas9 Tistrella mobilis
Cas9 Treponema denticola
Cas9 Treponema endosymbiont
Cas9 Treponema lecithinolyticum
Cas9 Treponema maltophilum
Cas9 Treponema pedis
Cas9 Treponema putidum
Cas9 Treponema socranskii
Cas9 Treponema sp.
Cas9 uncultured bacterium
Cas9 uncultured Termite
Cas9 Ureibacillus thermosphaericus
Cas9 Veillonella atypica
Cas9 Veillonellaceae bacterium
Cas9 Veillonella dispar
Cas9 Veillonella magna
Cas9 Veillonella montpellierensis
Cas9 Veillonella parvula
Cas9 Veillonella sp.
Cas9 Verminephrobacter aporrectodeae
Cas9 Verminephrobacter eiseniae
Cas9 Virgibacillus sp. Cas9 Weeksella sp.
Cas9 Weeksella virosa
Cas9 Weissella halotolerans
Cas9 Weissella kandleri
Cas9 Wolinella succinogenes
Cas9 Woodsholea maritima
Cas9 Zunongwangia profunda
Cpf1 Acidaminococcus sp.
Cpf1 Anaerovibrio sp.
Cpf1 Arcobacter butzleri
Cpf1 Bacteroidetes oral
Cpf1 Butyrivibrio fibrisolvens
Cpf1 Butyrivibrio proteoclasticus
Cpf1 Butyrivibrio sp.
Cpf1 [Eubacterium] eligens
Cpf1 Flavobacterium branchiophilum
Cpf1 Flavobacterium sp.
Cpf1 Francisella philomiragia
Cpf1 Francisella tularensis
Cpf1 Helcococcus kunzii
Cpf1 Lachnospiraceae bacterium
Cpf1 Leptospira inadai
Cpf1 Moraxella bovoculi
Cpf1 Moraxella caprae
Cpf1 Moraxella lacunata
Cpf1 Oribacterium sp.
Cpf1 Porphyromonas crevioricanis
Cpf1 Porphyromonas macacae
Cpf1 Prevotella albensis
Cpf1 Prevotella brevis
Cpf1 Prevotella bryantii
Cpf1 Prevotella disiens
Cpf1 Proteocatella sphenisci
Cpf1 Pseudobutyrivibrio ruminis
Cpf1 Smithella sp.
Cpf1 Sneathia amnii
Cpf1 Succinivibrio dextrinosolvens
Cpf1 Succinivibrionaceae bacterium
Cpf1 Synergistes jonesii Cpf1 Treponema endosymbiont
C2c1 Alicyclobacillus acidoterrestris
C2c1 Alicyclobacillus contaminans
C2c1 Desulfovibrio inopinatus
C2c1 Desulfonatronum thiodismutans
C2c1 Opitutaceae bacterium
C2c1 Tuberibacillus calidus
C2c1 Bacillus thermoamylovorans
C2c1 Brevibacillus sp
C2c1 Bacillus sp
C2c1 Desulfatirhabdium butyrativorans
C2c1 Methylobacterium nodularis
In alternative embodiments, the methods described herein can use modified nuclease variants that are nickases. A nickase can be created via a mutation in either one of the HNH or the RuvC catalytic domains of the nucleases.
In all aforementioned aspects of the present invention, amino acid residues of the nucleases may be substituted conservatively or non-conservatively. Conservative amino acid substitutions refer to those where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not alter the functional properties of the resulting polypeptide. Similarly it will be appreciated by the skilled reader that nucleic acid sequences may be substituted conservatively or non- conservatively without affecting the function of the polypeptide. Conservatively modified nucleic acids are those substituted for nucleic acids which encode identical or functionally identical variants of the amino acid sequences. It will be appreciated by the skilled reader that each codon in a nucleic acid (except AUG and UGG; typically the only codons for methionine or tryptophan, respectively) can be modified to yield a functionally identical molecule. Accordingly, each silent variation (i.e. synonymous codon) of a polynucleotide or polypeptide, which encodes a polypeptide of the present invention, is implicit in each described polypeptide sequence.
Similarly, it will be appreciated by a person of average skill in the art that polynucleotide sequences may be substituted conservatively or non-conservatively without affecting the function of the polypeptide. Conservatively modified polynucleotides are those substituted for nucleic acids which encode identical or functionally identical variants of the amino acid sequences. It will be appreciated by the skilled reader that each codon in a nucleic acid (except AUG and UGG; typically the only codons for methionine or tryptophan, respectively) can be modified to yield a functionally identical molecule. Accordingly, each silent variation (i.e. synonymous codon) of a polynucleotide or polypeptide, which encodes a polypeptide of the present invention, is implicit in each described polypeptide sequence.
Figure imgf000037_0001
In another aspect, the invention defines a selectable replicable plasmid comprising a guide RNA or a polynucleotide comprising a sequence encoding a guide RNA under the control of a first heterologous promoter and with a terminator; a non-codon or codon optimised polynucleotide comprising a sequence encoding a nuclease under the control of a second heterologous promoter with a terminator; and homologous recombination polynucleotide or a polynucleotide comprising a sequence encoding a homologous recombination polynucleotide. Suitable promoters and terminators that may be used in a plasmid may depend upon the particular microbes used in the methods described herein. Suitable promoters and terminators are well known in the art.
The selectable replicable plasmid defined herein can optionally comprise inducible promoters. Suitable inducible promoters that may be used in a plasmid may depend upon the particular microbes used in the methods described herein. Suitable promoters and terminators are well known in the art. In preferred embodiments, a selectable replicable plasmid defined herein comprises a first heterologous promoter that is B. coagulans phosphotransacetylase (pta) promoter Ppfa without its ribosome binding sequence and the terminator is a Rho-independent terminator; and a second promoter is the xynA promoter from Thermoanaerobacterium saccharolyticum (PxynA) and the terminator is a Rho-independent terminator. In another aspect, the invention provides a kit for performing the methods as defined herein, wherein the kit comprises a plasmid as herein defined and instructions for use.
In yet another aspect, the invention provides a clonal library obtainable by the method as described herein, wherein the clonal library comprises a plurality of clones harbouring the modified microbial genome that is resistant to site-specific DSDB by the ribonuclease complex. In a further aspect the invention provides a microbial host cell modified by the methods as defined herein.
BRIEF DESCRIPTION OF THE FIGURES
The invention will now be described in detail with reference to examples and with reference to the accompanying drawings, in which:
Figure 1. A schematic overview of the basic pWUR_Cas9nt construct. A. The non-codon optimized spCas9 gene was employed for the construction of the pWUR_Cas9nt vector, since the S. pyogenes and B. smithii GC-content and codon usage are highly similar. In the pNW33n-based basic construct the spCas9 was placed under the control of PxynA. A Rho- independent terminator from B. subtilis (Kingsford et al., Genome biology, 2007) was introduced after the stop codon of the gene. The spCas9 module is followed by an sgRNA- expressing module that encompasses a spacer which does not target the genome of B. smithii ET 138. The sgRNA module was placed under the transcriptional control of Ppta from Bacillus coagulans -without its RBS- (Bosma et al., MCF, 2015; Kovacs, A.T., et al., (2010) Applied and Environmental Microbiology, 76, 4085-4088) and it is followed by a second Rho- independent terminator from B. subtilis. The spCas9 and sgRNA modules were synthesized as one fragment, which was subsequently cloned into the pNW33n through the BspHI and Hindi 11 restriction sites. B. To prevent double restriction sites and create a modular system, 5 point silent mutations (C192A, T387C, T101 1A, C3126A, G354A) were introduced to the gene (depicted as *). The depicted restriction sites are unique in the construct and introduced to facilitate the exchange of genetic parts. The spacer was easily exchanged to targeting spacers via BsmBi restriction digestion or Gibson assembly. The basic construct did not contain any HR-templates, but in the cases that these were added they were always inserted right upstream the spCas9-module and downstream the origin of replication. C. Total RNA was isolated from ET 138 wild type cells transformed with the pWUR_Cas9nt vector or pNW33n and grown at 55, 45 and 37°C. 6 cDNA libraries were produced with rt-PCR and used as templates for PCR with cas9sp-specific primers that amplify a 255bp long region. The PCR results are depicted in this agarose gel electrophoresis: Lane 1 corresponds to the marker (1 kb+ DNA ladder, ThermoFisher), Lanes 2 to 4 correspond to ET 138 wild type cultures transformed with the pWUR_Cas9nt and grown at 55, 45, 37°C respectively, Lanes 5 to 7 correspond to ET 138 wild type cultures transformed with the pNW33n and grown at 55, 45 and 37°C respectively, Lanes 7, 8, 9, 1 1 , 12 correspond to different negative controls and Lane 10 corresponds to the positive control, for which pWUR_Cas9nt was used as the PCR template.
Figure 2. A. Sequential transfer scheme of wild type ET 138 cultures to evaluate spCas9 expression and targeting efficiency at different temperatures. ET 138 cells were transformed with the pWUR_Cas9sp1 , pWUR_Cas9sp2, pWUR_Cas9sp3, pWUR_Cas9nt and pNW33n vectors and plated on LB2 agar plates with chloramphenicol (Day 1 ). After overnight (ON) incubation at 55°C, single colonies were restreaked on LB2 agar plates with chloramphenicol and incubated ON at 55°C and 45°C (Day 2). Colonies from the 45°C plates were transferred to LB2 agar plates with chloramphenicol for ON incubation at 37°C (Day 3). The plates from days 2 and 3 were then used for inoculation of liquid medium (Day 4); 1 colony per 55°C, 45°C and 37°C plate was transferred to LB2 medium with xylose, glucose and chloramphenicol for ON incubation at 55°C, 45°C and 37°C respectively, while 1 extra colony per 45°C plate was transferred to the same medium for overnight incubation at 42°C. B. Results of the targeting experiment showing Οϋβοο measurements from cultures of wild type ET 138 transformed with the 3 different pyrF targeting cas9sp constructs, the non- targeting cas9sp construct and pNW33n. The growth of the cells with the pyrF targeting constructs is greatly affected at 37°C, which is not observed for cells containing the non-targeting constructs.
Figure 3. Schematic representation of the different homologous recombination and spCas9- mediated mutations described. The first single cross over event (SCO) can occur by insertion of the editing plasmid into the chromosome either through the upstream homologous region (UHR) -as depicted here- or through the downstream homologous region (DHR). A. Gene deletion. A scar-less, markerless pyrF gene deletion was established; after insertion of the editing vector into the chromosome, via homologous recombination with the plasmid-borne editing template (2x 1 -kbp flanks, immediately flanking the pyrF gene and thus removing it from start to stop codon), a second SCO event results in either WT revertants or edited cells. The spCas9 targeting of the WT cells acts as counter selection for the pyrF mutants. B. Gene knock-out via insertion of stop codons and a restriction site. The followed process was similar to the gene deletion described above. The hsdR restriction gene was inactivated by inserting stop codons and a restriction site between codons 212 and 221 that were contained in a 2 kbp long HR-fragment that expands 289 bp upstream and 1 .65 kb downstream from the start codon of the hsdR gene on the genome of the ET 138. In-between the 2 stop codons, an EcoRV restriction site was added, generating a frame shift and facilitating the screening process. The spacer was designed to target the original sequence without stop codons and restriction site. C. Gene knock-in. The followed process was similar to the gene deletion and gene knock-out processes described above. The IdhL gene was re-inserted into mutant strain ET 138 ldhL sigF. This was achieved by adding the original IdhL gene sequence in- between 2x 1 kbp HR flanks. The spacer was designed to target the area between the IdhL stop codon and the beginning of the adjacent rho-independent transcriptional terminator. On the HR-flanks, the region between the IdhL stop codon and its rho-independent transcriptional terminator was inverted, avoiding the spCas9 targeting of edited cells. Figure 4. A. Agarose gel electrophoresis showing the results from PCR on the genomic DNA of a ET 138 ldhL sigF culture transformed with pWUR_Cas9sp1_hr and sequentially transferred to different temperatures (following the depicted temperature sequence) for detection of pyrF deletion mutants in the culture mixture. The pyrF deletion mutant band appears at the 1st 37°C culturing step (Lane 3) onwards. The last 2 lanes are the negative (WT) and positive ( pyrF) controls, that correspond to 2,9kb and 2,1 kb long DNA fragments respectively. B. Agarose gel electrophoresis showing the resulting products from colony PCR on colonies transformed pWUR_Cas9sp1_hrfor the detection of deletion mutants. 9 out of the 10 tested colonies (S.C.#1 to S.C.#10) that resulted from the 3-day long editing process in TVMYxgu (TVMY supplemented with xylose, glucose and uracil) medium were deletion mutants. 4 out of the 10 tested colonies (S.C.#1 1 to S.C.#20) that resulted from the 3-day long editing process in TVMYxg (TVMY supplemented with xylose and glucose) medium were deletion mutants. The last 2 lanes are the negative (WT) and positive ( pyrF) controls, respectively. Figure 5. A. Agarose gel electrophoresis showing the resulting products from colony PCR on ET 138 colonies transformed with pWUR_Cas9spR_hr for the detection of hsdR knock-out mutants from the 3-day long editing process in LB2xg medium. The 2.75 kbp PCR fragments that resulted using genome specific primers (Lanes 1 ,3,5,7,9) were digested with the EcoRV restriction enzyme. Each digestion mixture was loaded next to its corresponding PCR- fragment (Lanes 2,4,6,8,10). All the checked colonies were shown to be knock-out mutant cells as the restriction digestion gave 3 bands of 1 .1 , 1.05 and 0.6 kbp. B. Agarose gel electrophoresis showing the resulting products from ET 138 colonies transformed with PCR on pWUR_Cas9spR_hr for the detection of hsdR Knock-out mutants from the 3-day long editing process in TVMYxg medium. The 2.75 kbp PCR-fragments that resulted using genome specific primers (Lanes 1 ,3,5,7,9) were digested with the EcoRV restriction enzyme. Each digestion mixture was loaded next to its corresponding PCR-fragment (Lanes 2,4,6,8,10). For the 2 colonies comprised of knock-out mutant cells (Lanes 1 -2 and 3-4) the restriction digestion gives 3 bands of 1 .1 , 1 .05 and 0.6 kbp. For the 3 non-edited colonies comprised of wild-type cells (Lanes 5-6, 7-8, 9-10), the restriction digestion gives 2 bands of 2.15 kbp and 0.6 kbp. Figure 6. Agarose gel electrophoresis showing the resulting products from colony PCR on the colonies from the 3-day long IdhL knock-in culturing processes in LB2 medium, using ET 138 AsigF AldhL AhsdR cells transformed with either the pWUR_Cas9spKI_hr1 vector (A,B)_or pWUR_Cas9spKI_hr2 vector (C, D). For the colony PCR genome specific primers BG8145 and BG8146 were used with an expected size of the PCR fragment for the knock-in mutation being 3.2 kb, (equal to the size of the PCR fragment for the wild type cells). In contrast, the expected size of the PCR fragment for the knock-out (non-edited) mutants was 2.3 kbp. A. When the 45°C culturing step was performed, the process resulted in 4 out of the 20 tested colonies having the knock-in genotype (20% editing efficiency), 15 colonies with mixed knock- in and wild type genotype and only 1 colony with wild-type genotype. B. When the 45°C culturing step was omitted, the process resulted in only 1 out of the 20 tested colonies having the knock-in genotype, 1 1 colonies with mixed genotype and 8 colonies with the wild-type genotype. C. When the 45°C culturing step was performed, the process resulted in 1 out of the 20 tested colonies having the knock-in genotype, 7 colonies with mixed knock-in and wild- type genotype, and only 1 colony with the wild-type genotype. D. When the culturing step at 45°C was omitted none of the 20 tested colonies had the knock-in genotype, only 6 colonies had the mixed genotype while the remaining 14 colonies had the wild-type genotype.
Figure 7. Sequencing results of the PPfa from pWUR_Cas9sp2. A 7bp-long deletion is located right downstream of the predicted -10 region of the promoter and expands until 8bp upstream of the spacer 2 sequence.
Figure 8. Ο βοο measurements from the B. smithii ET 138 AsigF AldhL spCas9 targeting experiment. The growth of the cells with the spCas9 targeting constructs is greatly affected at 37°C, which is not the case for the cells with the negative control constructs.
Figure 9. Ο βοο measurements from the 7-day long pyrF deletion culturing process. B. smithii ET 138 AldhL AsigF cells transformed with pWUR_Cas9sp1 , pWUR_Cas9sp1_hr, pWUR_Cas9nt or pWUR_Cas9nt_hr were cultured in TVMYXgU medium. After growth at 55°C for 24 hours, the cultures were sequentially transferred every 24 hours to fresh media while gradually lowering the culturing temperature from 55°C to 37°C, with an intermediate transfer at 45°C. After 3 more transfers at 37°C cells were transferred back to 55°C with an intermediate transfer at 45°C. The pWURCas9_sp1 cultures at 37°C showed almost no growth, indicating efficient spCas9 targeting, while the pWUR_Cas9nt and pWUR_Cas9nt_hr control cultures grew at all the temperatures as expected for B. smithii ET 138 cultures. The pWUR_Cas9sp1_hr cultures showed poor growth in the first 2 culturing steps at 37°C, but the growth was reconstituted from the 3rd culturing step at 37°C onwards to control levels, indicating the development of either sgRNA and spCas9 escape mutants, or ApyrF deletion mutants that can avoid the spCas9 targeting.
Figure 10. Phenotypic evaluation of 5-FOA sensitivity and uracil auxotrophy of B. smithii ET 138 pyrF wild-types and mutant. Cells were grown overnight on TVMY medium with the following additions: Plates annotated "Ta" contained 2 g/L 5-FOA and 50 mg/L uracil; Plates annotated "Tb" contained 2 g/L 5-FOA and no uracil; Plates annotated "Td" contained no 5- FOA and no uracil. A. B. smithii ET 138 wild-type. B. B. smithii ET 138 AldhL AsigF. C. B. smithii ET 138 AldhL AsigF ApyrF. DETAILED DESCRIPTION
The following examples illustrate the invention:
EXAMPLES
In experiments Streptococcus pyogenes Cas9 (spCas9) genome editing was applied for the first time to a moderate thermophile i.e. Bacillus smithii, including a gene deletion, a gene knockout via insertion of premature stop codons and a gene insertion. On average it took 1 week from transformation to clean deletion, knock out or knock in - including the plasmid curing step - with an editing efficiency of 90% for the gene deletion and the gene knock out and 20% for the gene insertion.
The major advantage of this system is the limited requirement in genetic parts: only one plasmid and no inducible or well-characterized promoters to drive the spCas9 and sgRNA expression. Since B. smithii grows between 37°C and 65°C, the mesophilic, well-characterized Streptococcus pyogenes Cas9 (spCas9) was used and its expression controlled in ET 138. spCas9 was found to be inactive in ET 138 at temperatures above 42°C and so its activity was tightly controlled by altering the cultivation temperature. The inventors created a clean gene deletion, a gene disruption and a gene insertion by using a system based on one single plasmid. The system employs a plasmid-borne homologous recombination template for introducing the desired modifications to the genome at elevated temperatures, while the non- edited cells are subsequently eliminated by the spCas9 counter-selection tool at 37°C. Moreover, the system could be readily applied for genome editing of other non-model organisms with limited genetic toolbox parts. Example 1 : In vivo expression validation of spCas9 at different temperatures
Inventors designed and constructed the modular pWUR_Cas9nt construct, that encompasses the cas9 gene of S. pyogenes (referred to as cas9sp herein) and an sgRNA-expressing module for which the spacer is predicted not to target any site of the B. smithii genome, (i.e. nt, for non-targeting). The backbone of the pWUR_Cas9nt is the pNW33n vector, which was the only available vector for B. smithii (Bosma, E.F., et al., (2015) Microbial Cell Factories, 14, Art.nr.99; Bosma, E.F., et al., (2015) Applied and Environmental Microbiology, 81 , 1874- 1883). The first basic requirement for the design of the pWUR_Cas9nt was the development of promoters that will drive the expression of the two components of the system: the cas9sp effector and its sgRNA guide module. For many non-model organisms, the number of available promoters and plasmids is very limited. ET 138 is no exception: only two promoters have been evaluated for expression in B. smithii, of which one heterologous and one native (Bosma, E.F., et al., (2015) Microbial Cell Factories, 14, Art.nr.99). The latter is undesired, as the homologous recombination mechanism of ET 138 is very efficient. Desired was to prevent single and double crossover events between the pWUR_Cas9nt construct and the B. smithii genome over the promoter region. Additionally, an inducible system is desirable. For these purposes the xynA promoter (PxynA) from Thermoanaerobacterium saccharolyticum (Currie et al., (2013) Biotechnology for Biofuels, 6, 32) was tested. On the genome of its native host this promoter is induced by xylose and repressed by glucose. To test expression in ET 138, inventors constructed the pWURJacZ vector. pNW33n was used as cloning and expression vector. PxynA was introduced in front of the B. coagulans-der'wed lacZ gene, previously shown to be functional in ET 138 (Bosma, E.F., et al., (2015) Microbial Cell Factories, 14, Art.nr.99). Low expression was observed after overnight culturing in LB2 liquid medium without added carbon source, and strong expression was observed with both xylose and glucose (data not shown). It is probable that the catabolite repression mechanism of ET 138 does not recognize the ere (catabolite repression element) sequence present on PxynA, resulting in lack of transcriptional repression by glucose in B. smithii.ET 138. Although PxynA is not tightly controllable in B. smithii inventors still selected it to drive the expression of cas9 from the pWUR_Cas9nt, maintaining the possibility to explore the effects of different cas9sp expression levels. During all further experiments, xylose and glucose were both added to the media at all the culturing temperatures, to ensure constant induction of cas9sp expression at all times from PxynA. Additionally, the sgRNA-module was placed under the control of the B. coagulans phosphotransacetylase (pfa) promoter Ppfa (Bosma, E.F., et al., (2015) Microbial Cell Factories, 14, Art.nr.99; Kovacs, A.T., et al., (2010) Applied and Environmental Microbiology, 76, 4085-4088) without its RBS. spCas9-expression and toxicity levels in ET 138 were tested by transforming a single batch of competent cells, with the non-targeting pWUR_Cas9nt vector and with the empty pNW33n control vector. The transformed cells were plated on LB2 plates supplemented with chloramphenicol. Plates were incubated overnight only at 55°C, as previous incubation attempts at lower temperatures were not successful (data not shown). One single colony per transformation was used for sequential transfers in LB2xg broth, transferring the cultures from 55°C to 37°C, with two intermediate steps at 45°C and 42°C. Total RNA was isolated from each pWUR_Cas9nt culture after 18 hours of incubation at every temperature and performed semi-quantitative reverse transcription (rt)-PCR using specific primers for the cas9sp gene. Transcription of cas9sp was observed for all temperatures (Figure 1 C). The growth of the cas9Sp-expressing cultures was similar to the pNW33n control cultures at all temperatures (see below), indicating that the expression of cas9sp is not toxic for the ET 138 cells at any of the tested temperatures.
Bacterial strains and growth conditions
Strains used are listed in Table 1. All B. smithii strains were routinely cultured at 55°C unless stated otherwise. TVMY medium and LB2 medium were used as described previously ((Bosma, E.F., et al., (2015) Microbial Cell Factories, 14, Art.nr.99). TVMYxgu is TVMY supplemented with 0,5 g/l xylose, 0,5 g/l glucose and 50 mg/L uracil. TVMYxg is TVMY supplemented with 0,5 g/l xylose and 0,5 g/l glucose. LB2xg is LB2 supplemented with 0,5 g/l xylose and 0,5 g/l glucose. Substrates were added separately as 50% autoclaved solutions after autoclavation of the medium. Uracil was added as 50 mg/ml filter sterilized solution in 1 M NaOH after autoclavation of the medium and addition of the substrates. £ coli strains were grown in LB medium at 37°C. For plates, 30 g of agar (Difco) per liter of medium was used for B. smithii in all experiments; 15 g of agar (Difco) per liter of LB was used for £ coli. If required, chloramphenicol was added in concentrations of 25 μg/mL for £ coli DH5a, 15 μg/mL for £. coli TG90, and 7 μg/mL for B. smithii.
Table 1. Strains used.
Figure imgf000044_0001
E. coli TG90 Cloning host Lopilato, J., ef a/., (1986)
Molecular and General Genetics MGG, 205, 285- 290; Gonzy-Treboul, G., ef a/., (1992) J. Mol. Biol. 224:967-979
B. smithii strains
ET 138 Wild-type, natural isolate (Bosma, E.F., ef a/., (2015)
Applied and Environmental Microbiology, 81 , 1874- 1883)
ET 138 AldhL AsigF ET 138 with clean IdhL and sigF- (Bosma, E.F., ef a/., (2015) deletions Microbial Cell Factories, 14,
Art.nr.99)
ET 138 AldhL AsigF ApyrF ET 138 AldhL AsigF with clean
pyrF-deletion
ET 138 AldhL AsigF AhsdR ET 138 AldhL AsigF with stop
codons and frame shift inserted in
the hsdR gene
ET 138 AsigF AhsdR ET 138 AldhL AsigF AhsdR with the
IdhL gene re-inserted to restore its
original sequence
Genomic DNA isolation, transformations, colony PCR, sequence and phenotypic verification Genomic DNA from B. smithii strains was isolated using the MasterPure™ Gram Positive DNA Purification Kit (Epicentre). Heat shock transformation of E. coli strains was performed according to the NEB supplier's protocol. Transformation of B. smithii strains was performed as described previously in Bosma, E.F., et al., (2015) Microbial Cell Factories, 14, Art.nr.99. Plasmids for transforming B. smithii were extracted from E. coli via maxiprep isolation (Genomed Jetstar 2.0).
After each editing cycle, potential B. smithii ET138 AldhL AsigF ApyrF colonies were randomly selected and subjected to colony PCR using the InstaGene™ Matrix (BIORAD), Taq DNA Polymerase (NEB) and the genome specific primers BG6420+6421 . Purification of PCR products was performed after running them on a 0.8% agarose gel using the Zymoclean™ Gel DNA Recovery Kit. The DNA fragments were subsequently sent for sequencing to GATC. To evaluate the 5-FOA sensitivity and uracil auxotrophy of sequence confirmed strains, B. smithii ET138 AldhL AsigF ApyrF cells were plated on TVMY medium with 30 g/L agar and the following additions: a) 2 g/L 5-FOA and 50 mg/L uracil, b) 2 g/L 5-FOA and no uracil, d) no 5-FOA and no uracil.
After each editing cycle, potential B. smithii ET138 AldhL AsigF AhsdR colonies were randomly selected and subjected to colony PCR using the Phire Plant Direct PCR kit (ThermoFisher Scientific) and the genome specific primers BG7842+7843. Purification of PCR products was performed after running them on a 0.8% agarose gel using the Zymoclean™ Gel DNA Recovery Kit. The DNA fragments were subsequently subjected to EcoRV restriction digestion and sent for sequencing to GATC.
After each editing cycle, potential B. smithii ET138 AsigF AhsdR colonies were randomly selected and subjected to colony PCR using the Phire Plant Direct PCR kit (ThermoFisher Scientific) and the genome specific primers BG8142+8143. Purification of PCR products was performed after running them on a 0.8% agarose gel using the Zymoclean™ Gel DNA Recovery Kit. The DNA fragments were subsequently sent for sequencing to GATC. To evaluate lactate production, cells were grown overnight in TVMY medium containing 10 g/L glucose and subsequently transferred to the same medium and grown for 24 h, after which L- lactate specific measurements were performed using MegaZyme kit K-LATE. Plasmid construction
Plasmids and primers used are shown in Table 2 and 3. Q5 polymerase (NEB) was used for all PCR reactions for cloning purposes. NEB T4 ligase was used for assembling the pWURJacZ, pWUR_Cas9nt, pWUR_Cas9nt_hr, pWUR_Cas 9sp1 , pWUR_Cas9sp1_hr, pWUR_Cas9sp2, pWUR_Cas9sp2_hr, pWUR_Cas9sp3 and pWUR_Cas9sp3_hr vectors. The NEBuilder® HiFi DNA Assembly Master Mix was used for assembling the pWUR_Cas9spR_hr, pWUR_Cas9spKI_hr1 and pWUR_Cas9spKI_hr2 constructs. All the used restriction enzymes were obtained from NEB. Purification of PCR products was performed after running them on a 0.8% agarose gel using the Zymoclean™ Gel DNA Recovery Kit.
To test the PxynA promoter, a DNA fragment comprised of the PxynA and the lacZ gene was synthesized by GeneArt and inserted into pNW33n using digestion with BspHI and Kpnl and subsequent ligation and cloning into E. coli DH5a, creating plasmid pWURJacZ. The PxynA sequence was used exactly as originally described (Currie et al., (2013) Biotechnology for Biofuels, 6, 32), using the sequence until the start codon of the corresponding gene in the original host. For the construction of the basic, modular pWUR_Cas9nt construct, a synthetic gene string was synthesized by GeneArt containing the elements depicted in Figure 1 B except the PxynA promoter. PxynA was amplified from pWURJacZ using primers BG6538 and BG6541. Primer BG6541 replaces the final 6 bp of PxynA by an Xbal site, changing the final -1 to -6 sequence from GTAAGA to TCTAGA and keeping the total length the same as in the original promoter. Primer BG6538 adds a BspHI site on the start of the PxynA. The entire synthesized spCas9 module without promoter for spCas9 was amplified using primers BG6542 and BG6543, keeping the Xbal and Hindi 11 sites already present in the module. Subsequently, vector pNW33n was digested with BspHI and Hind III, the PxynA PCR-product with Xbal and BspHI and the spCas9 module PCR-product with Xbal and Hind III. The three elements were ligated in a 3-point ligation and cloned into E. coli TG90 (TG90 is a derivative of TG1 , carrying the pcnB80 zadvJn W allele; it can be re-constructed using the methods in Gonzy-Treboul, G., et al., (1992) J. Mol. Biol. 224:967-979). Plasmid was extracted and the correct sequence was verified by sequencing, creating plasmid pWUR_Cas9nt (Figure 1A).
To insert the 3 different targeting spacers into pWUR_Cas9nt (which contains a non-targeting spacer), 3 sets of oligos were annealed to create the 3 spacers, after which the annealed spacers were inserted into the construct as follows. Oligo sets were BG6017+6021 for spacer 1 , BG6018+6022 for spacer 2, BG6019+6023 for spacer 3. Each set was annealed by adding 5 μΙ_ 10 mM oligo sets together with 10 μΙ_ NEB buffer 2.1 and 74 μΙ_ MQ water. Mixtures were heated to 94°C for 5 min and gradually cooled down to 37°C at 0.03°C/sec using a PCR machine. Annealed oligos and plasmid pWUR_Cas9nt were digested with BspEI and BsmBI (NEB). First, BspEI digestion was performed at 37°C for 15 min, after which BsmBI was added and the mixture was further incubated at 55°C for 15 min. After gel purification of the digested products, ligation was performed using NEB T4 ligase and mixtures were transformed to £. coli TG90. All constructs were verified by sequencing and all were correct except spacer 2, which is missing 7 nt from the Ppta that is driving spacer expression (Figure 7). As we were not sure whether this would have any influence, we decided to nevertheless use this spacer for future work. Constructs were named pWUR_Cas9sp1 until pWUR_Cas9sp3 according to their corresponding spacer.
To insert the pyrF-flanks into the four different pWUR_Cas9-constructs (nt, sp1 -3), the already fused pyrF-flanks were amplified from a previous plasmid in which the flanks were added as follows: flanks were cloned from genomic DNA of ET 138 using primers BG 5798+5799 (upstream, 958 bp) and BG5800+5801 (downstream, 979 bp), introducing Sail and Xbal restriction sites. The flanks were fused in an overlap extension PCR using primers BG5798+5801 making use of the complementary overhangs in primers BG5799 and BG5800. Subsequently, the flanks and pNW33n were digested with Sail and Xbal, ligated and transformed into E. coli DH5a. To amplify the flanks for insertion into spCas9-editing plasmids, primers BG6850+6849 were used, which both introduce a BspHI site. The five spCas9- plasmids and the amplified pyrF-flanks were digested with BspHI, followed by alkaline phosphatase treatment of the vectors (Thermo Scientific), ligated and transformed in E. coli TG90. Since only one restriction site was used, the flanks could have been inserted in both orientations. For all four constructs, multiple colonies were verified by sequencing and for all constructs the same flank-orientation was selected and used for future experiments, namely with the downstream flank on the PxynA side. The resulting plasmids were named pWUR_Cas9nt_hr and pWUR_Cas9sp1_hr until pWUR_Cas9sp3_hr.
A 4-fragment NEBuilder® HiFi DNA Assembly was designed and executed for the construction of the hsdR-modifying plasmid pWUR_Cas9spR_hr. The backbone of the vector was PCR amplified from pWUR_Cas9sp1 using the Q5 DNA polymerase (NEB) and primers BG7836+7837. The HR fragment upstream of the targeted site in the hsdR gene was PCR amplified from the B. smithii ET 138 genome using the Q5 DNA polymerase (NEB) and primers BG7838+7839. The HR fragment downstream of the targeted site in the hsdR gene was PCR amplified from the B. smithii ET 138 genome using the Q5 DNA polymerase (NEB) and primers BG7840+7841. The cas9sp and sgRNA containing fragment was PCR amplified from the pWUR_Cas9sp1 vector using the Q5 DNA polymerase (NEB) and primers BG7842+7843.
Two 4-fragment NEBuilder® HiFi DNA Assemblies were designed and executed for the construction of the IdhL-restoration plasmids pWUR_Cas9spKI-hr1 and pWUR_Cas9spKI- hr2. The backbone of both vectors was PCR amplified from the pWUR_Cas9sp1 vector using the Q5 DNA polymerase (NEB) and primers BG8134+7837. The HR fragment upstream of and including the IdhL gene was PCR amplified from the B. smithii ET 138 genome using the Q5 DNA polymerase (NEB), primers BG8135+8137 for the pWUR_Cas9spKI-hr1 vector and primers BG8135+8136 for the pWUR_Cas9spKI-hr2 vector. The HR fragment downstream of the IdhL gene was PCR amplified from the B. smithii ET 138 genome using the Q5 DNA polymerase (NEB), primers BG8138+8139 for the pWUR_Cas9spKI-hr1 vector and primers BG8138+8140 for the pWUR_Cas9spKI-hr2 vector. The cas9sp and sgRNA containing fragment of both vectors was PCR amplified from the pWUR_Cas9sp1 vector using the Q5 DNA polymerase (NEB) and primers BG8141 +7842.
Table 2. Plasmids used.
Plasmid Description Reference/origin pNW33n E. coli-Bacillus shuttle vector, cloning vector, CmR. BGSC
(Currie et al., (2013) Biotechnology for Biofuels, 6, 32; Kovacs, A.T., ef al., pWURJacZ pNW33n with B. coagulans lacZ gene under T.
(2010) Applied and saccharolyticum P promoter.
Environmental Microbiology, 76, 4085-4088)
pNW33n with spCas9-module1 containing non- pWUR_Cas9nt
targeting spacer.
pNW33n with spCas9-module1 containing spacer
pWUR_Cas9sp1
targeting the pyrF gene at bp nr 124
pNW33n with spCas9-module1 containing spacer
pWUR_Cas9sp2
targeting the pyrF gene at bp nr 267
pNW33n with spCas9-module1 containing spacer
pWUR_Cas9sp3
targeting the pyrF gene at bp nr 583
pNW33n with spCas9-module1 containing non- pWUR_Cas9nt_hr
targeting spacer and the fused us+ds pyrF-flanks.
pNW33n with spCas9-module1 containing spacer
pWUR_Cas9sp1_hr targeting the pyrF gene at bp nr ... and the fused
us+ds pyrF-flanks.
pNW33n with spCas9-module1 containing spacer
pWUR_Cas9spR_hr targeting the hsdR gene and the fused us+ds hsdR- flanks containing mutations as described in M&M.
pNW33n with spCas9-module1 containing spacer
targeting directly downstream of the IdhL gene and
pWUR_Cas9spKI_hr1 the fused us+ds WhL-flanks (-1000 bp each), in
which the area between the IdhL stop codon and its
terminator is inverted.
pNW33n with spCas9-module1 containing spacer
targeting directly downstream of the IdhL gene and
pWUR_Cas9spKI_hr2 the fused us+ds WhL-flanks (-750 bp each), in
which the area between the IdhL stop codon and its
terminator is inverted.
The spCas9 module contains S. pyogenes Cas9 under the T. saccharolyticum P promoter followed by a B. subtilis-demed Rho-independent terminator, followed by a spacer under the B. coagulans Ppta promoter followed by another B. suM/Zs-derived Rho-independent terminator (Figure 1 A).
Abbreviations: CmR: chloramphenicol resistance gene (chloramphenicol acetyltransferase); BGSC: Bacillus Genetic Stock Centre, USA; us: upstream; ds: downstream; bp: base pairs.
Table 3. Primers used.
Figure imgf000049_0001
6541 GCCTCTAGATTCCTCCCTCAGTAAATTTAATTTATTG [SEQ ID NO: PxynA -Rv, Xbal site 12]
6542 TATGCCTCTAGAATGGATAAGAAATAC [SEQ ID NO: 13] Fw, spCas9 module without
Cas9-promoter
6543 GCCTATCAAGCTTTCTTATAATCAGAAAC [SEQ ID NO: 14] Rv, spCas9 module without
Cas9-promoter
6017 CCGGAAAAATGGATTATTCTTAAAATGGATACCGTTATACACTTT Fw oligo spacer 1
A I M I CAGAATGGAC [SEQ ID NO: 15]
6021 AAACGTCCATTCTGAAAATAAAGTGTATAACGGTATCCATTTTAA Rv oligo spacer 1
GAATAATCCATTTTT [SEQ ID NO: 16]
6018 CCGGAAAAATGGATTATTCTTAAAATGGATACCGTTATACTCAAC Fw oligo spacer 2
GTGCATGCAGCAGGC [SEQ ID NO: 17]
6022 AAACGCCTGCTGCATGCACGTTGAGTATAACGGTATCCATTTTA Rv oligo spacer 2
AGAATAATCCATTTTT [SEQ ID NO: 18]
6020 CCGGAAAAATGGATTATTCTTAAAATGGATACCGTTATACATCTC Fw oligo spacer 3
ATGATCAAAGTCGTG [SEQ ID NO: 19]
6024 AAACCACGACTTTGATCATGAGATGTATAACGGTATCCATTTTAA Rv oligo spacer 3
GAATAATCCATTTTT [SEQ ID NO: 20]
5798 GCCTCTAGATAGGGATAACAGGGTAATATGCAGCGATGGTCCG pyrF-us-Fw, Xbal
GTGTTC [SEQ ID NO: 21 ]
5799 TTTCAGATCTGCTGGTTTACACGCACTTTCCAGCTCCTTC [SEQ pyrF-us-Rv, overhang with ID NO: 22] BG5800
5800 GAAGG AGCTGGAAAGTGCGTGTAAACCAGCAGATCTGAAA [S EQ pyrF-ds-Fw, overhang with ID NO: 23] BG5799
5801 GCCGTCGACTAGGGATAACAGGGTAATGTTCCCATGTTGTGATT pyrF-ds-Rv, Sail
C [SEQ ID NO: 24]
6850 GCCTCATGAATGCAGCGATGGTCCGGTGTTC [SEQ ID NO: 25] pyrF-us-Fw, BspHI site
6849 GCCTCATGAGTTCCCATGTTGTGATTC [SEQ ID NO: 26] pyrF-ds-Rv, BspHI site
6237 ATTGTGATCCCTAGTAACTC [SEQ ID NO: 27] spCas9-specific-Fw, to evaluate expression
6232 GCGCAAAGTATTGTCCATGCC [SEQ ID NO: 28] spCas9-specific-Rv, to evaluate expression
6420 TCGGGGGTTCGTTTCCCTTG [SEQ ID NO: 29] pyrF KO check Fw
6421 CTTACACAG CCAGTG ACG G AAC [SEQ ID NO: 30] pyrF KO check Rv
7836 gaaagacccgtatccaagaaGTTTTAGAGCTAGAAATAGCAAG [SEQ ID FW_pWUR_ori_SpacerR NO: 31]
7837 TCATGACCAAAATCCCTTAAC [SEQ ID NO: 32] RV pWUR ori
7838 tcacgttaagggattttggtcatgaGATATAAGCAATACTGTTTTTCTAATC FW_hsdR_USflank1_ori [SEQ ID NO: 33]
7839 ggaaccgttaggatatcTAATGTTGTACTTTTGCACC [SEQ ID NO: 34] RV_ hsdR _USflank1 7840 aagtacaacattagatatcctaaCGGTTCCTGAACTCGGTGAC [SEQ ID FW_ hsdR _Dsflank1 NO: 35]
7841 attatcctcagctcactagcgccatGGCCGCTGCTTCACGAGT [SEQ ID NO: RV_ hsdR _DSflank1_Cas9 36]
7842 ATGGCGCTAGTGAGCTG [SEQ ID NO: 37] FW_pWUR_cas9
7843 ctaaaacttcttggatacgggtctttcGTATAACGGTATCCATTTTAAGAATAAT RV_pWUR_cas9_SpacerR CC [SEQ ID NO: 38]
7881 AATACCAAACACGCCCTATAGC [SEQ ID NO: 39] FW_ hsdR _check
7882 CTTGCCCTGTTGAAATAATTAATGC [SEQ ID NO: 40] RV_ hsdR _check
8134 ttatgtaaacgaccatacaaGTTTTAGAGCTAGAAATAGCAAG [SEQ ID FW_pNW_ori_SpacerL NO: 41]
8135 tcacgttaagggattttggtcatgaTAAAGCTGGAAGGATGCC [SEQ ID NO: FW LDH DSI ori
42]
8136 tcacgttaagggattttggtcatgaACATCAAAAGGATAGAGACAAAATC FW_LDH_DS2_ori
[SEQ ID NO: 43]
8137 ctttcttaatgctggtatgAAAGGGAGCGTTTTAAGTTAAATG [SEQ ID NO: RV LDH DS
44]
8138 aaacgctccctttcataccagcaTTAAGAAAGTACTTTATTCATCGTTTC FW LDH+US
[SEQ ID NO: 45]
8139 attatcctcagctcactagcgccatATGATGAAATACGCTGGC [SEQ ID NO: RV LDH+US1
46]
8140 attatcctcagctcactagcgccatTTTGGATTTTTGGCAGTATTC [SEQ ID RV LDH+US2
NO: 47]
8141 ctaaaacttgtatggtcgtttacataaGTATAACGGTATCCATTTTAAGAATAAT RV_pNW_spCas9_SpacerL CC [SEQ ID NO: 48]
8142 GCCAACGCTTCATTGTTTCC [SEQ ID NO: 49] FW LDH Check
8143 TCAGCCCATTGACTAGGAAG [SEQ ID NO: 50] RV LDH Check
RNA isolation and rt-PCR
RNA isolation was performed by the phenol extraction based on Van Hijum et al., 2005. Overnight 10 mL cultures were centrifuged at 4°C and 4816 x g for 15 min and immediately used for RNA isolation. After removal of the medium, cells were resuspended in 0.5 mL ice cold TE buffer (pH 8.0) and kept on ice. All samples were divided into two 2 mL screw-capped tubes containing 0.5 g zirconium beads, 30 μΙ_ 10% SDS, 30 μΙ_ 3 M sodium acetate (pH 5.2), and 500 μΙ_ Roti-Phenol (pH 4.5-5.0, Roth). Cells were disrupted using a FastPrep-24 apparatus (MP Biomedicals) at speed 5500 for 45 seconds and centrifuged at 4°C and 10,000 rpm for 5 min. 400 μΙ_ of the water phase from each tube was transferred to a new tube, to which 400 μΙ_ chloroform-isoamyl alcohol (Roth) was added, after which samples were centrifuged at 4°C and 18,400 x g for 3 min. 300 μΙ_ of the aqueous phase was transferred to a new tube and mixed with 300 μΙ_ of the Lysis buffer from the High Pure RNA Isolation Kit (Roche). Subsequently, the rest of the procedure from this kit was performed according to the manufacturer's protocol, except for the DNAse incubation step, which was performed for 45 min. Integrity and concentration of the isolated RNA was checked on Nanodrop-1000.
Reverse Transcriptase PCR was performed using Superscript III Reverse Transcriptase kit (Invitrogen) according to the manufacturer's protocol. For synthesis of the first-strand cDNA, 2 μg of RNA and 200 ng of random primers were used. After cDNA synthesis, the products were used as a template for PCR using spCas9-specific forward and reverse primers BG6237 and BG6232, resulting in a 255 bp product. Products were visualized on a 2% agarose gel ran for 20 min.
Example 2: In vivo validation of spCas9 activity at different temperatures As B. smithii genome encompasses the genes for the basic prokaryotic NHEJ-like system (Bosma, E.F., et al., (2016) Standards in Genomic Sciences, 1 1 ; Shuman, S. and Glickman, M.S. (2007) Nat Rev Micro, 5, 852-861 ). The first approach was to construct a spCas9-based genome editing tool focused on determining the in vivo temperature limits of spCas9 and the capacity of the ET 138 NHEJ-like mechanism to repair the spCas9-induced DSDBs. The pyrF gene encodes the orotidine 5'-phosphate decarboxylase and is part of the operon for pyrimidine biosynthesis. Removal of the gene causes uracil-auxotrophy and resistance to the toxic uracil-analogue 5-fluorootic acid (5-FOA). It is a frequently used selection and counter-selection system in many organisms including thermophiles (Tripathi, S. A et al., (2010) Applied and Environmental Microbiology, 76, 6591 -6599; Kita, A., et al., (2013) Bioscience, Biotechnology and Biochemistry, 77, 301 -306; Chung, D., et al., (2012) PLoS ONE, 7). Initially a clean pyrF deletion mutant was made being ET 138 ldhL sigF pyrF by adding the fused 1 -kb up- and downstream pyrF flanks to pNW33n and applying 5- FOA pressure to select for double cross-over mutants. A total of 9 rounds of subculturing on selective media containing uracil and 5-FOA was required before pure knockouts were obtained with a knockout efficiency of around 50% (data not shown), making the process rather time-consuming.
Then 3 vectors were constructed based on the pWUR_Cas9nt sequence, designated pWUR_Cas9sp1 , pWUR_Cas9sp2, and pWUR_Cas9sp3, each containing a different pyrF- targeting spacer (sp1 -3). A single batch of wild type ET 138 competent cells was transformed with the 3 targeting vectors, the non-targeting control pWUR_Cas9nt and the empty vector control pNW33n. After initial outgrowth at 55°C on LB2 plates without sugar, one confirmed transformant for each construct was subjected to a sequential plating and culturing process in which the temperature was stepwise lowered from 55°C to 37°C to induce spCas9-expression (Figure 2A). Normal growth was observed for all cultures at 55°C, 45°C and 42°C, as well as for the control cultures at 37°C. No growth was observed for any of the cultures with pyrF- targeting sgRNA-modules at 37°C (Figure 2B).
The transformation and culturing process were repeated using the double mutant B. smithii ET138 NdhL AsigF strain (Bosma, E.F., et al., (2015) Microbial Cell Factories, 14, Art.nr.99). This strain is sporulation-deficient, it cannot produce L-lactate and it was constructed as the basic platform strain for further metabolic engineering work. Similar results were obtained as for the wild type B. smithii ET138 strain (Figures 8 and 2B).
In line with the in vitro assay data, the aforementioned results indicate that the designed spCas9 system is active and introducing lethal DSDBs to the ET 138 genome but only at temperatures below 42°C. It also indicates that the NHEJ system in ET 138 is inactive under the tested conditions or not active enough to counteract the spCas9 activity. In addition, the sequencing results of the pWUR_Cas9sp2 construct revealed the deletion of 7 nucleotides near the 3' end of the Ppta (Figure 7). The results of the targeting experiment show that the uncharacterized nature of the Ppta, do not hinder the targeting efficiency of spCas9. Moreover, the truncated Ppta of the pWUR_Cas9sp3 construct clearly expresses enough sgRNA molecules for successful targeting. Example 3: Efficient gene deletion using a HR-CRISPR-Cas9 counter-selection system
A Cas9-based editing system for ET 138 was created, exploiting its efficient homologous recombination mechanism (Bosma, E.F., et al., (2015) Microbial Cell Factories, 14, Art.nr.99) and the temperature-induced spCas9 activity at 37°C. The experimental setup consisted of a single plasmid that combines the recombination template and the spCas9- and sgRNA- expressing modules. Providing the cells with the appropriate plasmid-borne editing template at 55°C and then inducing the expression of active spCas9 at 37°C through a sequential culturing process, is expected to form a powerful homologous recombination and counter- selection system. To generate a pyrF deletion mutant, pWUR_Cas9sp1 was selected as the pyrF-targeting vector for further experiments, which was always compared to the non-targeting control pWUR_Cas9nt. To both vectors, a fusion of the two pyrF-flanks (each 1 kb) was added, creating the pWUR_Cas9nt_hr and pWUR_Cas9sp1_hr vectors (i.e. hr, for homologous recombination) (Figure 3A).
After transforming the 4 constructs (2 with flanks and 2 without flanks) into ET 138 AldhL AsigF at 55°C, one verified transformant per construct was inoculated into TVMY selection medium containing xylose, glucose and supplemented with uracil (TVMYXgU) to complement the auxotrophy in case of successful pyrF deletion. After growth at 55°C for 24 hours, cells were sequentially transferred every 24 hours to fresh media while gradually lowering the culturing temperature from 55°C to 37°C, with an intermediate transfer at 45°C. After 3 more transfers at 37°C to allow for efficient spCas9-counter-selection, cells were transferred back to 55°C with an intermediate transfer at 45°C. As expected, PCR on genomic DNA from the pWUR_Cas9nt and pWURCas9_sp1 cultures showed no pyrF knockout bands at any culturing temperature due to the lack of homologous recombination template in the constructs. In line with the initial in vivo tests, pWURCas9_sp1 cultures at 37°C showed almost no growth while the pWUR_Cas9nt cultures at all the temperatures showed the expected growth for ET 138 culture. PCR on genomic DNA from the liquid cultures containing the vectors with pyrF flanks showed the absence of knockout bands for the pWUR_Cas9nt_hr cultures from the first culturing step at 37°C onwards, but very strong pyrF knockout bands for the pWUR_Cas9sp1_hr cultures for the same culturing steps mentioned above (Figure 4A). The striking difference in the density of the knockout bands between the targeting pWUR_Cas9sp1_hr and the non-targeting pWUR_Cas9nt_hr cultures indicates successful spCas9 activity and pyrF-targeting by the pWUR_Cas9sp1_hr construct. It furthermore indicates that the counter-selection activity of spCas9 is already efficient from the first culturing step at 37°C. Growth of the pWUR_Cas9nt_hr cultures was similar to the control at all temperatures, while the pWUR_Cas9sp1_hr cultures showed poor growth in the first 2 culturing steps at 37°C, but the growth was reconstituted from the 3rd culturing step at 37°C onwards to control levels (Figure 9). After temperature-upshift, colony PCR of the 55°C colonies showed that 5 out of 10 tested pWUR_Cas9sp1_hr colonies were pure pyrF deletion mutants, i.e. an editing efficiency of 50% for the system (data not shown). None of the 10 tested pWUR_Cas9nt_hr colonies (non-targeting control) were pyrF deletion mutants demonstrating functional spCas9-targeting.
To improve the efficiency and speed of the system, the process for the pWUR_Cas9sp1_hr- containing strain was repeated and reduced the number of culturing steps at 37°C from 4 to 1 while keeping the culturing time of each step in a window between 8 and 16 hours. 3 different media were used in order to observe possible medium-dependent variations in the efficiency of the system: TVMY selection medium supplemented with xylose, glucose and uracil (TVMYxgu), TVMY selection medium supplemented with xylose, glucose but not with uracil (TVMYxg) and LB2 medium supplemented with xylose and glucose (LB2xg). After the final culturing step at 55°C, cells were plated on selective agar of the corresponding medium, supplemented with uracil. Colony PCR on 10 randomly selected colonies for each medium and construct showed that from the culturing process on TVMYXgU medium 9 out of 10 colonies were pure pyrF deletion mutants colonies (Figure 4B). From the process with TVMYxg medium, 4 of the checked colonies were pure pyrF deletion mutants, proving the efficiency of the counter-selection tool even in the presence of an auxotrophy barrier (Figure 4B).
To retain spCas9-activity, antibiotics were added in all steps. To allow for subsequent metabolic engineering steps, however, plasmid curing is required. After transferring a sequence-verified pyrF deletion mutant twice in TVMY medium without antibiotics cells were plated on TVMY plates without antibiotics. Colony PCR with plasmid-specific primers showed that all 8 tested colonies had lost the plasmid. Finally, the 5-FOA sensitivity and the uracil auxotrophy of 2 ET 138 AldhL AsigF ApyrF cultures that originated from 2 of the tested colonies ET 138 was verified (Figure 10). Example 4: Using a HR-CRISPR-Cas9 counter-selection system to knockout the Type I restriction modification (RM) system
To increase the potential of ET 138 as platform organism for the production of green chemicals transformation efficiency was improved. ET 138 has a type I Restriction-Modification (R-M) system. Methylation analysis of the PacBio genome sequencing data showed the existence of the single motif "Cm6AGNNNNNNTGT [SEQ ID NO: 51 ]/ACm6AN N N N N N CTG [SEQ ID NO: 52]" with N6-methyladenine (m6A) modifications (unpublished data).
The hsdR gene was knocked out. Between the origin of replication (on) and the cas9sp gene of the pWUR_Cas9nt vector, we introduced a 2 kbp long HR-fragment that expands 289 bp upstream and 1 .65 kb downstream from the start codon of the hsdR gene on the genome of the ET 138. In this HR-fragment we replaced the 25 nt long part between odons 212 and 221 , including the last nucleotide of codon 212, of the hsdR gene with a 8 nt long sequence comprised of 2 stop codons and the EcoRV restriction site, generating a frame shift and facilitating the screening process (Figure 3B). Since the hsdR gene is 2952 nucleotides (984 codons) long, only a fifth of it will be translated due the introduction of the stop codons. We also introduced a spacer in the sgRNA module for spCas9 targeting of the unmodified genomes, completing the construction of the pWUR_Cas9spR_hr editing vector.
B. smithii ET138 AldhL AsigF cells were transformed with the new vector and sequentially cultured as before, gradually lowering the temperature from 55°C to 37°C, with an intermediate transfer at 45°C, and then back up to 55°C. The duration of each culturing step was within a window of 8 to 16 hours. Moreover, we used 2 types of selection media, LB2 and TVMY. Five transformants per medium were subjected to colony PCR, after which the PCR fragments were digested with EcoRV. All colonies from the LB2-culturing process were successfully modified (Figure 5A) giving 100% editing efficiency, whereas 2 of the colonies from the TVMY process were modified giving 40% editing efficiency (Figure 5B). This is in contrast with the result from the pyrF deletion process, where there were no modified colonies resulting from the LB2-culturing process. Plasmid curing was performed as before and the correct mutations were verified by sequencing.
The lack of a functional R-M system in the newly developed ET 138 AldhL AsigF AhsdR strain was confirmed by successfully transforming the plasmid-cured cells with vector pG2K (Reeve, B., et al., (2016) ACS Synth Biol.). In previous attempts we did not succeed in transforming this vector into other (hsdR+) ET 138 strains as it contains the aforementioned methylation motif in its antibiotic resistance marker gene, the kanamycin nucleotidyltransferase (aadA) gene derived from Geobacillus stearothemophilus. This way we added a new antibiotic resistance marker to the toolbox of ET 138, and we confirmed that the ET 138 AldhL AsigF AhsdR strain can be utilized for the expansion of the genetic parts toolbox.
Example 5: Metabolic engineering using spCas9: knock-in of the IdhL gene
Next, the inventors evaluated the applicability of our Cas9-based system in markerless gene chromosomal integrations by knocking into the genome of ET 138 AldhL AsigF AhsdR the 942 bp long genomic fragment between the start and the stop codons of the lactate dehydrogenase (IdhL) gene. The reconstitution of the lactate production in the resulting ET 138 AsigF AhsdR strain would allow for efficient growth under anaerobic conditions, while retaining the advantages of a sporulation- and R-M-deficient strain.
Two versions were constructed of a pWUR_Cas9-based vector that target the ET 138 AldhL AsigF AhsdR genome at the same position between the IdhL stop codon and the beginning of the adjacent rho-independent transcriptional terminator. HR was facilitated with 1 kb flanks (pWUR_Cas9spKI-hr1 ) or 0.75kb flanks (pWUR_Cas9spKI_hr2). For both versions, the region between the IdhL stop codon and its rho-independent transcriptional terminator was inverted, avoiding spCas9 targeting (Figure 3C). The region between the start and stop codon was provided with the wild-type IdhL sequence to allow its knocking in.
ET138 AldhL AsigF AhsdR was transformed with the 2 pWUR_Cas9spKI_hr versions and sequentially cultured the transformants as described before, gradually lowering the temperature from 55 to 37°C, with or without an intermediate transfer at 45°C, and then back up to 55°C. Each culturing step was within a window of 8 to 16 hours. 2 types of selection media, LB2 and TVMY were used. The colony PCR results of the TVMY culturing processes showed that none out of the tested colonies had the knock-in genotype. The colony PCR results of the LB2 culturing processes with the pWUR_Cas9spKI_hr1 transformant showed that with the additional culturing step at 45°C, 4 out of the 20 tested colonies had the knock- in genotype (20% editing efficiency), 15 colonies had a mixed knock-in/wild type genotype and only 1 colony had the wild-type genotype (Figure 6A). When the culturing step at 45°C was omitted, only 1 out of the 20 tested colonies had the knock-in genotype, 1 1 colonies had the mixed genotype and 8 colonies had the wild-type genotype (Figure 6B). The colony PCR results of the LB2 culturing processes with the pWUR_Cas9spKI_hr2 transformant showed that with the additional culturing step at 45°C, 1 out of the 20 tested colonies had the knock- in genotype (5% editing efficiency), 7 colonies had the mixed knock-in and wild-type genotype, and only 1 colony had the wild-type genotype (Figure 6C). When the culturing step at 45°C was omitted none of the 20 tested colonies had the knock-in genotype, only 6 colonies had the mixed genotype while the remaining 14 colonies had the wild-type genotype (Figure 6D). This was the first time that we observed colonies with mixed genotypes using our editing approach. The appearance of such colonies could be explained by relatively inefficient Cas9 targeting when the enzyme is loaded with a suboptimal sgRNA module, as has been described for E. coli (Cui, L. and Bikard, D. (2016) Nucleic Acids Res, 44, 4243-4251 ). Alternative sgRNAs may improve the editing efficiency. Moreover, the results indicate the influence of the HR-template length, as well as the importance of the culturing period before the induction of the counter-selection. The editing efficiency of the tool was higher when we employed the editing construct with the 1 kb HR flanks compared to the editing construct with the 0.75kb HR flanks (20% vs. 5% efficiency, respectively, Figure 6). A culturing period with an additional intermediate step at 45°C allows for efficient homologous recombination and double cross over events to occur, leading to the appearance of the mutants that Cas9 will select for. This is in line with the observations in L. reuterii (Oh & Van Pijkeren, 2014, op. cit). In addition, it may be that the stress of the temperature drop increases the efficiency of the homologous recombination mechanism.
Next, the inventors cured the constructed B. smithii ET138 sigF hsdR strain of the pWUR_Cas9spKI_hr1 plasmid using the sequential transferring approach in LB2 medium without antibiotic at 55°C. However, after the usual 2 transfers none of the tested colonies had lost the plasmid. We repeated the same sequential transferring process but raised the culturing temperature to 65°C, as the pNW33n replicon might be less stable at elevated temperatures (Bosma, E.F., et al., (2015) Microbial Cell Factories, 14, Art.nr.99). After 2 transfers at 65°C, 1 out of 8 tested colonies was confirmed to be plasmid-free by PCR and antibiotic sensitivity. Finally, evaluation of lactate production in the resulting B. smithii ET138 AsigF AhsdR strain under aerobic, microaerobic and anaerobic conditions showed a complete restoration of lactate production to wild-type levels (Table 4).
To evaluate lactate production, cells were grown overnight in TVMY medium containing 10 g/L glucose and subsequently transferred to the same medium and grown for 24 h, after which D- and L-lactate specific measurements were performed using MegaZyme kits K-DATE and K-LATE.
Figure imgf000058_0001
Table 4. Results of the enzymatic assay test for L-lactate production, average results from a technical duplo. Abbreviations: DKO: double knockout B. smithii ET 138 AldhL AsigF; WT: wild-type B. smithii ET 138; DKO_ki: IdhL knock-in strain B. smithii ET 138 AldhL AsigF +ldhL. The results prove the insertion and expression of the IdhL gene in the DKO_ki strain; the lactate production and the growth under anaerobic conditions are reconstituted to the wild type levels.
Example 6: Application of spCas9 in Geobacillus thermoglucosidans spCas9 is used as counter selection tool in Geobacillus. The Geobacillus thermoglucosidans DSM 2542T 1 140-bp gene AOT13_RS16120 (NCBI GenelD: 29239989) was chosen as deletion target for elimination of an internal 943-bp fragment (position 156..1098 of the gene). Upstream and downstream fragments of 0.75 kb were generated by PCR using the following primer combinations at an annealing temperature of 58°C:
2178 (5'-TCACGTTAAGGGATTTTGGTCATGACATGTAAGATAGATAGGGCATC-3' [SEQ ID NO: 53]); and 2179 (5'-CATCTGTTTTCCCAAAGGAGATATGTATTCATTATTATTAG-3' [SEQ ID NO: 54]) and the following primer combinations at an annealing temperature of 66°C:
2180 (5'-ACATATCTCCTTTG G GAAAAC AG ATG CAGG AG G-3' [SEQ ID NO: 55]); and 2181 (5'-ATTATCCTCAGCTCACTAGCGCCATTTTTGCGGATCCCCCTTATTTTTTATC-3' [SEQ ID NO: 56]) and by using chromosomal DNA of G. thermoglucosidans sigF (see WO2016/012296) as a template.
The vector backbone was amplified in two parts using pWUR_Cas9nt as a template, introducing the targeting spacer sequence 5'-AGGAGAATATAGCTAACGTC-3 [SEQ ID NO: 57]. One fragment was generated using the following primer combination using an annealing temperature of 60°C:
2173 (5'-ATGGCGCTAGTGAGCTG-3' [SEQ ID NO: 58]); and
2175 (5'-GACGTTAGCTATATTCTCCTGTATAACGGTATCCATTTTAAGAATAATC-3' [SEQ ID NO: 59]) The other fragment was generated using the following primer combination using an annealing temperature of 63°C:
2171 (5'-GTTATACAGGAGAATATAGCTAACGTCGTTTTAGAGCTAGAAATAGCAAGT TAAAATAAG-3' [SEQ ID NO: 60]); and 2172 (5'-TCATGACCAAAATCCCTTAAC-3' [SEQ ID NO: 61 ])
The four PCR fragments were created with Phusion Flash High-Fidelity PCR master mix (ThermoFisher) according to the manufacturer's instructions. The fragments were separated on a 1 % agarose gel and extracted from gel into 10 μΙ_ h O using a Zymo DNA Clean and concentrator spin column (Zymo Research). The four fragments were assembled into a single plasmid by fusing the 25-bp overlapping regions using the NEBuilder HiFi DNA Assembly Cloning Kit (New England BioLabs). Plasmid DNA was concentrated using a Zymo DNA Clean and Concentrator spin column (Zymo Research) and eluted into and transformed to electrocompetent E. coli TG90 (Gonzy-Treboul, G., Karmzyn-Campelli, C, Stragier, P. 1992. J. Mol. Biol. 224:967-979). Transformants were plated on LB agar plates supplemented with 10 mg/L chloramphenicol and incubated at 37°C. A single colony was selected for plasmid extraction using the ZymoPURE™ Plasmid Midiprep Kit (Zymo Research). Plasmid integrity was confirmed by sequence analysis.
The integration vector was transformed to Geobacillus thermoglucosidans AsigF (see WO2016/012296) by electroporation as described elsewhere (see e.g. WO2016/012296) and plated on TGP plates supplemented with 8 mg/L chloramphenicol. Plates were incubated overnight at 55°C. A single colony was selected and grown overnight at 55°C in TGP broth supplemented with 8 mg/L chloramphenicol. Subsequently, 1 ml transfers were performed to 10 ml fresh prewarmed TGP medium supplemented with 8 mg/L chloramphenicol. Incubations were for 8 h at 68°C, for recombination to occur, for overnight at 55°C, for 8 h at 48°C, transferred and incubated for overnight at 41 °C, for 8 h at 37°C. After these transfer steps the culture was plated at 55°C and colony PCR was performed on 1 1 colonies to check for knockouts using primers 1908 (5'-GTGCCCTGTTTGTTCTAGACG-3' [SEQ ID NO: 62]) and 1909 (5'-GGCAATTGTATTGATTGGTTAAATAGATTGG-3' [SEQ ID NO: 63]). All colonies had the PCR fragment of 1 .7 kb confirming the deletion. This demonstrates the efficiency of the counter-selection tool for gene deletion in Geobacillus.
Figure imgf000060_0001
EXAMPLE 7: Application of spCas9 in Bacillus coagulans spCas9 is evaluated as a counter selection tool in Bacillus coagulans. The Bacillus coagulans DSM 1 759-bp sigF gene (NCBI GenelD: 29812540) was chosen as deletion target. Upstream and downstream fragments of 0.85 kb were generated by PCR using the following primer pair combinations both at an annealing temperature of 58°C and by using chromosomal DNA of B. coagulans DSM 1 as a template:
2561 (5'-TCACGTTAAG G GATTTTGGTCATGAGTGAGTCTG G CTATTGACCTG G-3' [SEQ ID NO: 64]); and 2562 (5'-ATGAAAAAAGCGCACGTCGGCACGACTCCTTAATTG-3' [SEQ ID NO: 65])
2563 (5'-ATTAAGGAGTCGTGCCGACGTGCGCTTTTTTCATTCCC-3' [SEQ ID NO: 66]); and
2564 (5'-ATTATCCTCAGCTCACTAGCGCCATCAGGATATAATGGTCGATGTCCTGTTG-3' [SEQ ID NO: 67]) The vector backbone was amplified in two parts using pWUR_Cas9nt as a template, introducing the targeting spacer sequence 5'-CGATGAGTTAACGAAAAAGC-3' [SEQ ID NO: 68] or the non-targeting spacer sequence 5'-GAAAGACCCGTATCCAAGAA-3' [SEQ ID NO: 69]. For the targeting spacer, one fragment was generated using primer combination: 2173 (5'-ATGGCGCTAGTGAGCTG-3' [SEQ ID NO: 58]); and
2568 (5'-GCTTTTTCGTTAACTCATCGGTATAACGGTATCCATTTTAAGAATAATC-3' [SEQ ID NO: 70])
The other fragment was generated using primer combination:
2567 (5'-GTTATACCGATGAGTTAACGAAAAAGCGTTTTAGAGCTAGAAATAGCA
AGTTAAAATAAG-3' [SEQ ID NO: 71 ]); and 2172 (5'-TCATGACCAAAATCCCTTAAC-3' [SEQ ID NO: 61 ])
For the non-targeting spacer, one fragment was generated using primer combination:
2173 (5'-ATGGCGCTAGTGAGCTG -3' [SEQ ID NO: 58]); and
2678 (5'-TTCTTGGATACGGGTCTTTCGTATAACGGTATCCATTTTAAGAATAATC-3' [SEQ ID NO: 72]) The other fragment was generated using primer combination
2677 (5'-GTTATACGAAAGACCCGTATCCAAGAAGTTTTAGAGCTAGAAATAGCAAGTT AAAATAAG-3' [SEQ ID NO: 73]); and 2172 (5'-TCATGACCAAAATCCCTTAAC-3' [SEQ ID NO: 61 ])
The PCR fragments were created with Phusion Flash High-Fidelity PCR master mix (ThermoFisher) according to the manufacturer's instructions, using an annealing temperature of 58°C for all reactions. For the construction of pMH243, the two PCR products containing the sigF flanks were fused to the two PCR products of the vector containing the targeting spacer. For the construction of pMH249, the two PCR products containing the sigF flanks were fused to the two PCR products of the vector containing the non-targeting spacer. The fragments were assembled into a single plasmid by fusing the 25-bp overlapping regions using the NEBuilder HiFi DNA Assembly Cloning Kit (New England BioLabs). Plasmid DNA was concentrated using a Zymo DNA Clean and Concentrator spin column (Zymo Research) and eluted into 10 μΙ_ hbO and transformed to electrocompetent E. coli TG90 (Gonzy-Treboul, G., Karmzyn-Campelli, C, Stragier, P. 1992. J. Mol. Biol. 224:967-979). Transformants were plated on LB agar plates supplemented with 10 mg/L chloramphenicol and incubated at 37°C. A single colony was selected for plasmid extraction using the ZymoPURE™ Plasmid Midiprep Kit (Zymo Research). Plasmid integrity was confirmed by sequence analysis.
Both integration vectors pMH243 and pMH249 were transformed to Bacillus coagulans by electroporation as described elsewhere (Kovacs, A. T., van Hartskamp, M., Kuipers, O. P., & van Kranenburg, R. 2010. Applied and Environmental Microbiology, 76(12), 4085-4088) and plated on BC plates supplemented with 7 mg/L chloramphenicol (Kovacs, A. T., van Hartskamp, M., Kuipers, O. P., & van Kranenburg, R. 2010. Applied and Environmental Microbiology, 76(12), 4085-4088). Plates were incubated overnight at 45°C. A single colony was selected and grown overnight at 45°C in BC broth supplemented with 7 mg/L chloramphenicol. Subsequently, 0.2 ml transfers were performed to 10 ml fresh, prewarmed BC medium supplemented with 7 mg/L chloramphenicol. Incubations were for 8 h at 65°C, for recombination to occur, and for 24 hours at 45°C. After this, 20 ml was transferred to 10 ml fresh, prewarmed BC medium supplemented with 7 mg/L chloramphenicol and cultures were incubated overnight at 37°C. After these transfer steps, the cultures were plated at 45°C on BC agar plates without chloramphenicol and colony PCR was performed to check for knockouts using primers 351 (5'-CACCATGTCCCGGACAGCAC-3' [SEQ ID NO: 74]) and 352 (5'-GCGATGAAATTGGAACACTGAC-3' [SEQ ID NO: 75]). Out of 31 tested colonies originating from the culture with the targeting spacer, one colony had the PCR fragment of 2.1 kb confirming the deletion, the other 30 had the PCR fragment of the wild type. All of the 40 tested colonies coming from the culture with the non-targeting spacer had the PCR fragment of the wild type. The sigF deletion was confirmed by sequence analysis. This demonstrates the efficiency of the counter-selection tool for gene deletion in Bacillus coagulans.
Figure imgf000063_0001
EXAMPLE 8: Application of spCas9 in Bacillus thermoamylovorans spCas9 is evaluated as counter selection tool in Bacillus thermoamylovorans. The Bacillus thermoamylovorans DSM 23963 762-bp sigF gene was chosen as deletion target.
Upstream and downstream fragments of 0.85 kb were generated by PCR using primer combinations 2553 (5'-TCACGTTAAGGGATTTTGGTCATGAGTGAGTCTGCAAGT
ACATTTTGAATG-3' [SEQ ID NO: 76]) and 2554 (5'-ATTCGTCAAGTGAGAACTACCCCTT AATTG AACACTG CTTTTG-3' [SEQ ID NO: 77]), and 2555 (5'-TGTTCAATTAAGGGGTAG TTCTCACTTGACGAATACTAAACG-3' [SEQ ID NO: 78]) and 2556 (5'-ATTATCCT CAGCTCACTAGCGCCATCTGCATTAATCTCGGAATAATG-3' [SEQ ID NO: 79]) both at an annealing temperature of 58°C and by using chromosomal DNA of B. thermoamylovorans DSM 23963 as a template. The vector backbone was amplified in two parts using
pWUR_Cas9nt as a template, introducing the targeting spacer sequence 5'-ACTAATTA AGAAAAGTCAGG-3' [SEQ ID NO: 80].
One fragment was generated using primer combination 2173 (5'-ATGGCGCTAGTGAGCTG -3' [SEQ ID NO: 58]) and 2558 (5'-CCTGACTTTTCTTAATTAGTGTATAACGGTATCCA
TTTTAAGAATAATC-3' [SEQ ID NO: 81 ]). The other fragment was generated using primer combination 2557 (5'-GTTATACACTAATTAAGAAAAGTCAGGGTTTTAGAGCTAGAAATAG CAAGTTAAAATAAG-3' [SEQ ID NO: 82]) and 2172 (5'-TCATGACCAAAATCCCTTAAC-3' [SEQ ID NO: 61 ]). The PCR fragments were created with Phusion Flash High-Fidelity PCR master mix (ThermoFisher) according to the manufacturer's instructions, using an annealing temperature of 58°C for all reactions. The four fragments were assembled into a single plasmid by fusing the 25-bp overlapping regions using the NEBuilder HiFi DNA Assembly Cloning Kit (New England BioLabs). Plasmid DNA was concentrated using a Zymo DNA Clean and Concentrator spin column (Zymo Research) and eluted into 10 μΙ_ h O and transformed to electrocompetent E. coli TG90 (Gonzy-Treboul, G., Karmzyn-Campelli, C, Stragier, P. 1992. J. Mol. Biol. 224:967-979). Transformants were plated on LB agar plates supplemented with 10 mg/L chloramphenicol and incubated at 37°C.
A single colony was selected for plasmid extraction using the ZymoPURE™ Plasmid Midiprep Kit (Zymo Research). Plasmid integrity was confirmed by sequence analysis. The integration vector was transformed to Bacillus thermoamylovorans by electroporation as described elsewhere (see WO2016/012296), except for the resistance during electroporation being 400 instead of 600 ohm, and plated on TGP plates supplemented with 7 mg/L chloramphenicol. Plates were incubated overnight at 55°C. A single colony was selected and grown overnight at 55°C in TGP broth supplemented with 7 mg/L chloramphenicol. Subsequently, 1 ml transfers were performed to 10 ml fresh prewarmed TGP medium supplemented with 7 mg/L chloramphenicol. Incubations were for 4 h at 65°C, for recombination to occur, for 20 h at 55°C and for 5 h at 50°C, after which 1 .5 ml of the culture was stored at -80°C in 15 % glycerol.
Using an inoculation loop, 10 ml of the frozen glycerol stock was revived in 10 ml fresh prewarmed TGP medium supplemented with 7 mg/L chloramphenicol and grown overnight at 50°C. The glycerol stock storage step is not required and should be considered as optional at any step of the temperature downshift or upshift at 45°C or higher. Next, 1 ml transfers were performed to 10 ml fresh prewarmed TGP medium supplemented with 7 mg/L chloramphenicol. Incubations were for 8 h at 45°C, for overnight at 41 °C and for 8 h 37°C. After this, 0.5 ml was transferred to 10 ml fresh prewarmed TGP medium and the culture was grown overnight at 55°C. After these transfer steps, the culture was plated at 55°C and colony PCR was performed on 82 colonies to check for knockouts using primers 572 (5 - AGCGGTATTGGAGAAATTTG-3' [SEQ ID NO: 83]) and 573 (5'- CGTCACAGCCCATTCATAG-3' [SEQ ID NO: 84]).
Two colonies had the PCR fragment of 1 .8 kb confirming the deletion. 61 colonies showed a mixed genotype of both the wildtype and delta sigF and 29 colonies only had the PCR fragment of the wildtype. The sigF deletion was confirmed by sequence analysis for one of the delta sigF colonies. This demonstrates the efficiency of the counter-selection tool for gene deletion in Bacillus thermoamylovorans.
Figure imgf000065_0001
Example 9: Expanding the tool to mesophiles
Attempts were then made to test if the tool could be expanded to mesophiles. E. coli DH5a transformation efficiency was tested at different temperatures, particularly at 42°C and compared with transformation efficiency at 37°C and 40°C. For this purpose, chemically competent cells (NEB) were transformed from the same batch with 50 pmol of the pUC19 vector, recovered in 1 ml SOB and plated on LB agar plates with 100 μg ml ampicillin for overnight incubation at the corresponding temperature. All the transformations gave approximately the same number of colonies (10000 colonies/nmol of pUC19) regardless of the incubation temperature. The only observed difference was the size of the colonies; the higher the incubation temperature, the smaller the colonies. This result clearly indicates that E. coli, are transformable at near thermophilic temperatures (for which spCas9 is inactive) and therefore the herein described tool for genome editing is applicable to mesophiles. Mesophilic cells can be transformed with the editing constructs, recovered, plated on selective medium and cultured at any temperature that spCas9 is not active (42°C and above) allowing for homologous recombination events to occur. Subsequently, dropping the temperature to mesophilic levels (around 37°C) would induce the spCas9-based counter-selection process. Then by plating the cells again on selective medium at the mesophilic temperature, the colonies can be screened for the surviving mutants that are not targeted by the spCas9. The following are nucleotide sequences of plasmids as described herein:
[SEQ ID NO: 1] pWURJacZ
CCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCGTAATCATGGTCATAGC TGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCA TAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCT C ACTG CCCGCTTTCC AGTCGG G A AACCTGTCGTG CC AG CCCTTC A AACTTCCC AA AGG CG AGCCCTAGTGACATTAGAAAACCGACTGTAAAAAGTACAGTCGGCATTATCTCATATTAT AAAAGCCAGTCATTAGGCCTATCTGACAATTCCTGAATAGAGTTCATAAACAATCCTGCA TGATAACCATCACAAACAGAATGATGTACCTGTAAAGATAGCGGTAAATATATTGAATTA CCTTTATTAATGAATTTTCCTGCTGTAATAATGGGTAGAAGGTAATTACTATTATTATTG ATATTTAAGTTAAACCCAGTAAATGAAGTCCATGGAATAATAGAAAGAGAAAAAGCATTT TCAGGTATAGGTGTTTTGGGAAACAATTTCCCCGAACCATTATATTTCTCTACATCAGAA AGGTATAAATCATAAAACTCTTTGAAGTCATTCTTTACAGGAGTCCAAATACCAGAGAAT GTTTTAGATACACCATCAAAAATTGTATAAAGTGGCTCTAACTTATCCCAATAACCTAAC TCTCCGTCGCTATTGTAACCAGTTCTAAAAGCTGTATTTGAGTTTATCACCCTTGTCACT A AG A AA ATA AATG C AGG GTAA AATTTATATCCTTCTTGTTTTATGTTTCG GTATAA AAC A CTAATATCAATTTCTGTGGTTATACTAAAAGTCGTTTGTTGGTTCAAATAATGATTAAAT ATCTCTTTTCTCTTCCAATTGTCTAAATCAATTTTATTAAAGTTCATTTGATATGCCTCC TAAA I I I I I ATCTAAAGTGAATTTAGGAGGCTTACTTGTCTGCTTTCTTCATTAGAATCA ATCC I I I I I I AAAAGTCAATCCCGTTTGTTGAACTACTCTTTAATAAAATAA I I I I I CCG TTCCCAATTCCACATTGCAATAATAGAAAATCCATCTTCATCGGC I I I I I CGTCATCATC TGTATGAATCAAATCGCCTTCTTCTGTGTCATCAAGGTTTAA I I I I I I ATGTATTTCTTT TAACAAACCACCATAGGAGATTAACCTTTTACGGTGTAAACCTTCCTCCAAATCAGACAA ACGTTTCAAATTCTTTTCTTCATCATCGGTCATAAAATCCGTATCCTTTACAGGATATTT TGCAGTTTCGTCAATTGCCGATTGTATATCCGATTTATATTTA I I I I I CGGTCGAATCAT TTGAACTTTTACATTTGGATCATAGTCTAATTTCATTGCC I I I I I CCAAAATTGAATCCA TTG I I I I I GATTCACGTAGTTTTCTGTATTCTTAAAATAAGTTGGTTCCACACATACCAA TACATGCATGTGCTGATTATAAGAATTATCTTTATTATTTATTGTCACTTCCGTTGCACG CATAAAACCAACAAGA I I I I I ATTAA I I I I I I I ATATTGCATCATTCGGCGAAATCCTTG AGCCATATCTGACAAACTCTTATTTAATTCTTCGCCATCATAAACA I I I I I AACTGTTAA TGTGAGAAACAACCAACGAACTGTTGGCTTTTGTTTAATAACTTCAGCAACAACCTTTTG TG ACTG AATG CC ATGTTTC ATTG CTCTCCTCC AGTTG C AC ATTG G AC AA AGCCTG G ATTT ACAAAACCACACTCGATACAACTTTCTTTCGCCTGTTTCACGATTTTGTTTATACTCTAA TATTTCAGCACAATCTTTTACTCTTTCAGCC I I I I I AAATTCAAGAATATGCAGAAGTTC AAAGTAATCAACATTAGCGATTTTCTTTTCTCTCCATGGTCTCACTTTTCCAC I I I I I GT CTTGTCCACTAAAACCCTTGA I I I I I CATCTGAATAAATGCTACTATTAGGACACATAAT ATTAAAAGAAACCCCCATCTATTTAGTTATTTGTTTGGTCACTTATAACTTTAACAGATG GGG I I I I I CTGTGCAACCAATTTTAAGGGTTTTCAATACTTTAAAACACATACATACCAA CACTTCAACGCACCTTTCAGCAACTAAAATAAAAATGACGTTATTTCTATATGTATCAAG AATAGAAAGAACTCG I I I I I CGCTACGCTCAAAACGCAAAAAAAGCACTCATTCGAGTGC I I I I I CTTATCGCTCCAAATCATGCGA I I I I I I CCTCTTTGCTTTTCTTTGCTCACGAAG TTCTCGATCACGCTGCAAAACATCTTGAAGCGAAAAAGTATTCTTCTTTTCTTCCGATCG CTCATGCTGACGCACGAAAAGCCCTCTAGGCGCATAGGAACAACTCCTAAATGCATGTGA GGGGTTTTCTCGTCCATGTGAACAGTCGCATACGCAATATTTTGTTTCCCATACTGCATT AATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCT CGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAA AGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAA AAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCG I I I I I CCATAGGC TCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGA CAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTC CGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTT CTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCT GTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTG AGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTA GCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCT ACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAA GAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGG I I I I I I I GTTT GCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTA CGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAATGGCG CTAGTGAGCTGAGGATAATATTTTCTATCATATTTCCACTTAGTGTTCCAGTATTAGCGA CAATAACGC I I I I I GTTGCAGTTGGACAATGGAATTCGTGGTTTGATACAATGC I I I I I I GTTCCATGAATCCAAATTTAAGCACATTACAGTATGAATTGCAAAAAGTATTGCAGTCGA CTCAACAGTTTACACAACAAGCGTCTTTTGATATGGGGTTAAGAAATAATTCTAATTCAG TTACCCCGGAAAGCATAAGAGCTGCTATGACGGTAGTGGCAGTGGTTCCTATTATGATGG TTTATCCATTCTTGCAGAAGTATTTTGTAAAGGGTTTAACCATTGGCAGTGTTAAGGGTT AGTAC ATCTTTG G I I I I I I ATAAATGCTGAAAGGGGGGAAATTTAAAAATACTTATTAGA CAAAA I I I I I I AATTGCGAGGTGATAGTAAATAAA I I I I I CAATAAATTAAATTTACTGA GGGAGGAAGTAAGAATGCTAAAAAAACATGAAAAATTTTATTACGGCGGGGATTACAACC CTGAACAATGGGATGAATCTGTATGGAAAGAAGACATGCGGCTGATGAAAAAAGCCGGCG TCAATTATGTAAGCATCAACATTTTCAGCTGGGCAAGGCTGCAGCCGGATGAAGAAACAT ATGA I I I I I CAACGCTCGATAAAATCATGGATATGCTTGCGGAAAACGGCATCGGGGCGG ACCTGGCCACTGCAACTGCGGCACCGCCGGCATGGCTGTCGCGGAAATATCCCGATTCGC TTCCGGTTGACAAGGACGGGTCGCGC I I I I I GCCGGGGTCGCGCCAGCATTATTGCCCGA ACAGCAAAGACTACGCTCGGCTTGCGGCGAAACTGGTCCGCAAAATTGCGGAACGGTACA AAAGCCATCCCGCGCTTGTCATGTGGCATGTGAACAATGAGTATGGCTGCCACATTTCCG AATGCTATTGCGACAACTGCAAAAAGGGTTTTCAAACATGGCTCAAAGAAAAATACGGCA CGATTGAAAACTTGAATAAAAGCTGGTCAACAGACTTCTGGAGCCAGCGTTATTATGAAT GGGAAGAAATCTGTCTGCCAGGGAAAACGCCGACTTTTGCCAATCCGATGCAACAGCTCG ATTACAAAGCCTTTATGGATGATTCACTGCTTGCACTTTATAAAATGGAACGCGATATCC TGAAAACATATACGCCGGATGTGCCGGTAATGACAAACTTAATGGGGCTGCACAAACCGG TGGACGGATTCCATTGGGCGAAAGAAATGGATCTTGTGACATGGGATGCCTACCCAGATC CGTTTGAGGACATCCCGTATGCGCAATTCATGGCCCATGATTTGACGCGCAGTTTGAAAA AACAGCCG I I I I I GCTAATGGAACAGGCAGCCGGCGCGGTGAACTGGCGGGCGCAAAACG CTGTGAAAGCACCGGGTGTGATGCGGCTTTGGAGTTATGAAGCGGCTGCGCACGGGGCGG ACGGCATCATG I I I I I CCAATGGCGCGCATCCCAGGGCGGTGCGGAGAAATTCCATAGCG GGATGGTGCCGCATTCCGGGGATGAAGAAAGCCGGAATTTCCGGGAAGTGGTACAACTCG GCAATGAATTGAAAAATTTGGAAAAGGTAACGGGTTCAGCATATGCATCCGATGTTGCGA TTG I I I I I GACTGGAAAAACTGGTGGGCGTTGGAACTCGATTCAAAACCTTCTTCGCTTG TAACCTATATCAAACAGCTTCTGCCGTTTTATAGGGTGCTTCATACCCAAAACATTGGTG TCGATTTTATTCACCCGGACGAGGCAATGGACCGGTATAAAGTGGTTTTCGCGCCGGCAA GTTACCGGGTGACAAAGACATTTGCGGATAAAGTAAAAGCCTATGTCGAAAACGGCGGTT ATTTTGCCACCAATTTCTTCAGCGGAATTGCGGATGAAAACGAACGTGTGTATCTTGGCG GCTATCCGGGCGCGTACCGGGATATTCTTGGCATTTATGTTGAAGAGTTTGCGCCGATGA AAAAGGGGGCAGTACATCAAATCCGGACCGGGTATGGCGATGCGGCTATCCGCGTGTGGG AGGAAAAGATCCACTTGAAAGGCGCCGAAGCTTTGGCCTGGTTCAAAGACGGCTATTTGG CCGGGTCGCCTGCTGTCACCGCGCATCATTGCGGAAAAGGAAAAGCTTATTATATCGGGA CACAGCCGGATGAACAATATTTGTCTTCTCTTTTAAAAGAAATTTTAAAGGAAGCGGATG TCCGGCCGGCATTGGATGCTCCTCGTGGTGTGGAAGTGGCAGTGCGCAAAAATGGGCATG AAAAA I I I I I GTTCCTGCTGAACCATACGGATCAGGTTCAGTTTGTGGATGCAGGCGGCA
CGTACCCGGAACTGATTTACGGGCGCACTGAAGCAGAAACAGTCCGGCTCTCGCCCCGGG
ATGTGAAAATTTTGCAGGTAATTGAAAAATAGGGTAC [SEQ ID NO: 2] pWUR_Ca9nt
AGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACAC AACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACAT TAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCCCTTCAAACTT CCCAAAGGCGAGCCCTAGTGACATTAGAAAACCGACTGTAAAAAGTACAGTCGGCATTATCTCAT ATTATAAAAGCCAGTCATTAGGCCTATCTGACAATTCCTGAATAGAGTTCATAAACAATCCTGCAT GATAACCATCACAAACAGAATGATGTACCTGTAAAGATAGCGGTAAATATATTGAATTACCTTTAT TAATGAATTTTCCTGCTGTAATAATGGGTAGAAGGTAATTACTATTATTATTGATATTTAAGTTAAA CCCAGTAAATGAAGTCCATGGAATAATAGAAAGAGAAAAAGCATTTTCAGGTATAGGTGTTTTGG GAAACAATTTCCCCGAACCATTATATTTCTCTACATCAGAAAGGTATAAATCATAAAACTCTTTGA AGTCATTCTTTACAGGAGTCCAAATACCAGAGAATGTTTTAGATACACCATCAAAAATTGTATAAA GTGG CTCTA ACTTATCCC AATAACCTAACTCTCCGTCG CTATTGTA ACC AGTTCTA AA AG CTGTAT TTGAGTTTATCACCCTTGTCACTAAGAAAATAAATGCAGGGTAAAATTTATATCCTTCTTGTTTTAT GTTTCGGTATAAAACACTAATATCAATTTCTGTGGTTATACTAAAAGTCGTTTGTTGGTTCAAATA ATGATTAAATATCTCTTTTCTCTTCCAATTGTCTAAATCAATTTTATTAAAGTTCATTTGATATGCCT CCTAAA I I I I I ATCTAAAGTGAATTTAGGAGGCTTACTTGTCTGCTTTCTTCATTAGAATCAATCCT I I I I I AAAAGTCAATCCCGTTTGTTGAACTACTCTTTAATAAAATAA I I I I I CCGTTCCCAATTCCA CATTGCAATAATAGAAAATCCATCTTCATCGGC I I I I I CGTCATCATCTGTATGAATCAAATCGCC TTCTTCTGTGTCATCAAGGTTTAA I I I I I I ATGTATTTCTTTTA AC A AACC ACC ATAG G AG ATTAAC CTTTTACG GTGTAA ACCTTCCTCC AA ATC AG AC AA ACGTTTC AA ATTCTTTTCTTC ATC ATCG GTC A TAAAATCCGTATCCTTTACAGGATATTTTGCAGTTTCGTCAATTGCCGATTGTATATCCGATTTATA TTTA I I I I I CGGTCGAATCATTTGAACTTTTACATTTGGATCATAGTCTAATTTCATTGCC I I I I I CC AAAATTGAATCCATTG I I I I I GATTCACGTAGTTTTCTGTATTCTTAAAATAAGTTGGTTCCACACA TACCAATACATGCATGTGCTGATTATAAGAATTATCTTTATTATTTATTGTCACTTCCGTTGCACGC ATAAAACCAACAAGA I I I I I ATTAA I I I I I I I ATATTGCATCATTCGGCGAAATCCTTGAGCCATAT CTGACAAACTCTTATTTAATTCTTCGCCATCATAAACA I I I I I AACTGTTAATGTGAGAAACAACCA ACGAACTGTTGGCTTTTGTTTAATAACTTCAGCAACAACCTTTTGTGACTGAATGCCATGTTTCAT TGCTCTCCTCCAGTTGCACATTGGACAAAGCCTGGATTTACAAAACCACACTCGATACAACTTTCT TTCGCCTGTTTCACGATTTTGTTTATACTCTAATATTTCAGCACAATCTTTTACTCTTTCAGCCTTTT TAAATTCAAGAATATGCAGAAGTTCAAAGTAATCAACATTAGCGATTTTCTTTTCTCTCCATGGTC TCACTTTTCCAC I I I I I GTCTTGTCCACTAAAACCCTTGA I I I I I CATCTGAATAAATGCTACTATT AGGACACATAATATTAAAAGAAACCCCCATCTATTTAGTTATTTGTTTGGTCACTTATAACTTTAAC AGATGGGG I I I I I CTGTGCAACCAATTTTAAGGGTTTTCAATACTTTAAAACACATACATACCAAC ACTTCAACGCACCTTTCAGCAACTAAAATAAAAATGACGTTATTTCTATATGTATCAAGAATAGAA AGAACTCG I I I I I CGCTACGCTCAAAACGCAAAAAAAGCACTCATTCGAGTGC I I I I I CTTATCGC TCCAAATCATGCGA I I I I I I CCTCTTTGCTTTTCTTTGCTCACGAAGTTCTCGATCACGCTGCAAA ACATCTTGAAGCGAAAAAGTATTCTTCTTTTCTTCCGATCGCTCATGCTGACGCACGAAAAGCCCT CTAGGCGCATAGGAACAACTCCTAAATGCATGTGAGGGGTTTTCTCGTCCATGTGAACAGTCGC ATACGCAATATTTTGTTTCCCATACTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTT GCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGG CGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAG GAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGG CG I I I I I CCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTG GCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCT CCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGC TTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGT GTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCA ACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAG GTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACA GTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATC CGGCAAACAAACCACCGCTGGTAGCGGTGG I I I I I I I GTTTGCAAGCAGCAGATTACGCGCAGA AAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAA CTCACGTTAAGGGATTTTGGTCATGAATGGCGCTAGTGAGCTGAGGATAATATTTTCTATCATATT TCCACTTAGTGTTCCAGTATTAGCGACAATAACGC I I I I I GTTGCAGTTGGACAATGGAATTCGTG GTTTGATACAATGC I I I I I I GTTCCATGAATCCAAATTTAAGCACATTACAGTATGAATTGCAAAA AGTATTGCAGTCGACTCAACAGTTTACACAACAAGCGTCTTTTGATATGGGGTTAAGAAATAATTC TAATTCAGTTACCCCGGAAAGCATAAGAGCTGCTATGACGGTAGTGGCAGTGGTTCCTATTATGA TGGTTTATCCATTCTTGCAGAAGTATTTTGTAAAGGGTTTAACCATTGGCAGTGTTAAGGGTTAGT ACATCTTTGG I I I I I I ATAAATGCTGAAAGGGGGGAAATTTAAAAATACTTATTAGACAAAATTTT TTAATTGCGAGGTGATAGTAAATAAA I I I I I CAATAAATTAAATTTACTGAGGGAGGAATCTAGAA TGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCAC TG ATG A ATATA AG GTTCCGTCTA AA AAGTTC A AG GTTCTGG G A AATAC AG ACCG CC AC AGTATC A AAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGACAGCGGAAGCGACTCGTCTAAAA CGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGGAGA M I M I C AAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTC I I I I I I GGTGGAAG A AG AC AAG A AG C ATG A ACGTC ATCCTA I I I I I GGAAATATAGTAGATGAAGTTGCTTATCACGAG AAATATCCAACTATCTATCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGATTTGCGC TTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCA I I I I I I GATTGAGGGAGATTTA AATCCTGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGTACAAACCTACAATCAATTATTT GAAGAAAACCCTATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAA ATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGA ATCTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATG CTAAATTACAGCTTTCAAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAG ATCAATATGCTGATTTG I I I I I GGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCT AAGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAACGCTACGATGAAC ATCATCAAGACTTGACTCTTTTAAAAGCATTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAA TC I I I I I I GATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAA TTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTA AATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCA CTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCA I I I I I AAAAGACAATC GTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGC AATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGA AGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCT TCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATT GACAAAGGTCAAATATGTTACTGAAGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGA AAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATT ATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTT CATTAGGTACCTACCATGATTTGCTAAAAATTATTAAAGATAAAGA I I I I I I GGATAATGAAGAAA ATGAAGATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGAGATGATTGAG GAAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCG TTATACTGGTTGGGGACGTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCA AAACAATATTAGA I I I I I I GAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATG ATGATAGTTTGACATTTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTA CATGAACATATTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAA GTTGTTGATGAATTGGTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGC ACGTGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCGAA GAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCA AAATGAAAAGCTCTATCTCTATTATCTCCAAAATGGAAGAGACATGTATGTGGACCAAGAATTAG ATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGATT CAATAGACAATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGT GAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCA ACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTT TTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGT CGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAA TCTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTAC CATCATGCCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAA ACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTC TGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATAATGAACTTCTTCAA AACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAA CTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCC CCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTAC CAAAAAGAAATTCGGACAAACTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGT TTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAA GAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAA ATCCGATTGAC I I I I I AGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTAC CTAAATATAGTC I I I I I GAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTA CAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAA I I I I I I ATATTTAGCTAGTCATTAT GAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGC ATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATGCCA ATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAA AATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAA CAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCA TCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTAGGAGGTGACTGAATCAGACAAAAT GGCCTGCTTATGAAGCGGGCCA I I I I I GTTTAATCCTGTCGACTTGCCGGAATTCTTTCACAGCC GCATCCCTTTCTTCCGCAGGCAGCGCCTTCCAGCCCGCCCAGTCGAGAGAACGGAAATCATGCA GTGAGTACCAGCCATCTAGTGTTTCTGCTGCTTCGCTCA I I I I I CTCCCTCCAAATG I I I I I CTCTT CCCTTACTATAACACAGTTTAGAAGAGGGAGAAAATATTACGGCTGCCGCGAAATTGTCTAAAAG AAGCCTTCTTTTGCCTTTGATTTTCCGGAAAAATGGATTATTCTTAAAATGGATACCGTTATACGA GGAGTACCGGTTGAGACGAGTCTCGGAAGCTCAAACGTCTCTGTTTTAGAGCTAGAAATAGCAA GTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTACGATAG ACATATAAAGAGAGCGGCCATACAGGCCGCCTCTTTTCTGTTCTTGGCGACTCCCCGTCTAGTGG ATCACTCAGCAACTCCTCAGCTGCAGCAGCTGCCTCGAGTGCAAAAACAGCCCGCAGATCAACA TCCGCGGGCTGTTTCTGATTATAAGAA [SEQ ID NO: 3] pWUR_Ca9spl
AGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACAC AACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACAT TAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCCCTTCAAACTT CCCAAAGGCGAGCCCTAGTGACATTAGAAAACCGACTGTAAAAAGTACAGTCGGCATTATCTCAT ATTATAAAAGCCAGTCATTAGGCCTATCTGACAATTCCTGAATAGAGTTCATAAACAATCCTGCAT GATAACCATCACAAACAGAATGATGTACCTGTAAAGATAGCGGTAAATATATTGAATTACCTTTAT TAATGAATTTTCCTGCTGTAATAATGGGTAGAAGGTAATTACTATTATTATTGATATTTAAGTTAAA CCCAGTAAATGAAGTCCATGGAATAATAGAAAGAGAAAAAGCATTTTCAGGTATAGGTGTTTTGG GAAACAATTTCCCCGAACCATTATATTTCTCTACATCAGAAAGGTATAAATCATAAAACTCTTTGA AGTCATTCTTTACAGGAGTCCAAATACCAGAGAATGTTTTAGATACACCATCAAAAATTGTATAAA GTGG CTCTA ACTTATCCC AATAACCTAACTCTCCGTCG CTATTGTA ACC AGTTCTA AA AG CTGTAT TTGAGTTTATCACCCTTGTCACTAAGAAAATAAATGCAGGGTAAAATTTATATCCTTCTTGTTTTAT GTTTCGGTATAAAACACTAATATCAATTTCTGTGGTTATACTAAAAGTCGTTTGTTGGTTCAAATA ATGATTAAATATCTCTTTTCTCTTCCAATTGTCTAAATCAATTTTATTAAAGTTCATTTGATATGCCT CCTAAA I I I I I ATCTAAAGTGAATTTAGGAGGCTTACTTGTCTGCTTTCTTCATTAGAATCAATCCT I I I I I AAAAGTCAATCCCGTTTGTTGAACTACTCTTTAATAAAATAA I I I I I CCGTTCCCAATTCCA CATTGCAATAATAGAAAATCCATCTTCATCGGC I I I I I CGTCATCATCTGTATGAATCAAATCGCC TTCTTCTGTGTCATCAAGGTTTAA I I I I I I ATGTATTTCTTTTA AC A AACC ACC ATAG G AG ATTAAC CTTTTACG GTGTAA ACCTTCCTCC AA ATC AG AC AA ACGTTTC AA ATTCTTTTCTTC ATC ATCG GTC A TAAAATCCGTATCCTTTACAGGATATTTTGCAGTTTCGTCAATTGCCGATTGTATATCCGATTTATA TTTA I I I I I CGGTCGAATCATTTGAACTTTTACATTTGGATCATAGTCTAATTTCATTGCC I I I I I CC AAAATTGAATCCATTG I I I I I GATTCACGTAGTTTTCTGTATTCTTAAAATAAGTTGGTTCCACACA TACCAATACATGCATGTGCTGATTATAAGAATTATCTTTATTATTTATTGTCACTTCCGTTGCACGC ATAAAACCAACAAGA I I I I I ATTAA I I I I I I I ATATTGCATCATTCGGCGAAATCCTTGAGCCATAT CTGACAAACTCTTATTTAATTCTTCGCCATCATAAACA I I I I I AACTGTTAATGTGAGAAACAACCA ACGAACTGTTGGCTTTTGTTTAATAACTTCAGCAACAACCTTTTGTGACTGAATGCCATGTTTCAT TGCTCTCCTCCAGTTGCACATTGGACAAAGCCTGGATTTACAAAACCACACTCGATACAACTTTCT TTCGCCTGTTTCACGATTTTGTTTATACTCTAATATTTCAGCACAATCTTTTACTCTTTCAGCCTTTT TAAATTCAAGAATATGCAGAAGTTCAAAGTAATCAACATTAGCGATTTTCTTTTCTCTCCATGGTC TCACTTTTCCAC I I I I I GTCTTGTCCACTAAAACCCTTGA I I I I I CATCTGAATAAATGCTACTATT AGGACACATAATATTAAAAGAAACCCCCATCTATTTAGTTATTTGTTTGGTCACTTATAACTTTAAC AGATGGGG I I I I I CTGTGCAACCAATTTTAAGGGTTTTCAATACTTTAAAACACATACATACCAAC ACTTCAACGCACCTTTCAGCAACTAAAATAAAAATGACGTTATTTCTATATGTATCAAGAATAGAA AGAACTCG I I I I I CGCTACGCTCAAAACGCAAAAAAAGCACTCATTCGAGTGC I I I I I CTTATCGC TCCAAATCATGCGA I I I I I I CCTCTTTG CTTTTCTTTG CTC ACG AAGTTCTCG ATC ACG CTG C AA A ACATCTTGAAGCGAAAAAGTATTCTTCTTTTCTTCCGATCGCTCATGCTGACGCACGAAAAGCCCT CTAGGCGCATAGGAACAACTCCTAAATGCATGTGAGGGGTTTTCTCGTCCATGTGAACAGTCGC ATACGCAATATTTTGTTTCCCATACTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTT GCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGG CGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAG GAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGG CG I I I I I CCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTG GCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCT CCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGC TTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGT GTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCA ACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAG GTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACA GTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATC CGGCAAACAAACCACCGCTGGTAGCGGTGG I I I I I I I GTTTGCAAGCAGCAGATTACGCGCAGA AAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAA CTCACGTTAAGGGATTTTGGTCATGAATGGCGCTAGTGAGCTGAGGATAATATTTTCTATCATATT TCCACTTAGTGTTCCAGTATTAGCGACAATAACGC I I I I I GTTGCAGTTGGACAATGGAATTCGTG GTTTGATACAATGC I I I I I I GTTCCATGAATCCAAATTTAAGCACATTACAGTATGAATTGCAAAA AGTATTGCAGTCGACTCAACAGTTTACACAACAAGCGTCTTTTGATATGGGGTTAAGAAATAATTC TAATTCAGTTACCCCGGAAAGCATAAGAGCTGCTATGACGGTAGTGGCAGTGGTTCCTATTATGA TGGTTTATCCATTCTTGCAGAAGTATTTTGTAAAGGGTTTAACCATTGGCAGTGTTAAGGGTTAGT ACATCTTTGG I I I I I I ATAAATGCTGAAAGGGGGGAAATTTAAAAATACTTATTAGACAAAATTTT TTAATTGCGAGGTGATAGTAAATAAA I I I I I CAATAAATTAAATTTACTGAGGGAGGAATCTAGAA TGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCAC TG ATG A ATATA AG GTTCCGTCTA AA AAGTTC A AG GTTCTGG G A AATAC AG ACCG CC AC AGTATC A AAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGACAGCGGAAGCGACTCGTCTAAAA CGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGGAGA M I M I C AAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTC I I I I I I GGTGGAAG A AG AC AAG A AG C ATG A ACGTC ATCCTA I I I I I GGAAATATAGTAGATGAAGTTGCTTATCACGAG AAATATCCAACTATCTATCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGATTTGCGC TTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCA I I I I I I GATTGAGGGAGATTTA AATCCTGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGTACAAACCTACAATCAATTATTT GAAGAAAACCCTATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAA ATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGA ATCTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATG CTAAATTACAGCTTTCAAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAG ATCAATATGCTGATTTG I I I I I GGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCT AAGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAACGCTACGATGAAC ATCATCAAGACTTGACTCTTTTAAAAGCATTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAA TC I I I I I I GATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAA TTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTA AATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCA CTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCA I I I I I AAAAGACAATC GTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGC AATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGA AGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCT TCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATT GACAAAGGTCAAATATGTTACTGAAGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGA AAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATT ATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTT CATTAGGTACCTACCATGATTTGCTAAAAATTATTAAAGATAAAGA I I I I I I GGATAATGAAGAAA ATGAAGATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGAGATGATTGAG GAAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCG TTATACTGGTTGGGGACGTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCA AAACAATATTAGA I I I I I I GAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATG ATGATAGTTTGACATTTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTA CATGAACATATTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAA GTTGTTGATGAATTGGTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGC ACGTGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCGAA GAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCA AAATGAAAAGCTCTATCTCTATTATCTCCAAAATGGAAGAGACATGTATGTGGACCAAGAATTAG ATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGATT CAATAGACAATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGT GAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCA ACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTT TTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGT CGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAA TCTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTAC CATCATGCCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAA ACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTC TGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATAATGAACTTCTTCAA AACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAA CTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCC CCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTAC CAAAAAGAAATTCGGACAAACTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGT TTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAA GAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAA ATCCGATTGAC I I I I I AGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTAC CTAAATATAGTC I I I I I GAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTA CAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAA I I I I I I ATATTTAGCTAGTCATTAT GAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGC ATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATGCCA ATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAA AATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAA CAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCA TCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTAGGAGGTGACTGAATCAGACAAAAT GGCCTGCTTATGAAGCGGGCCA I I I I I GTTTA ATCCTGTCG ACTTGCCG G AATTCTTTC AC AG CC GCATCCCTTTCTTCCGCAGGCAGCGCCTTCCAGCCCGCCCAGTCGAGAGAACGGAAATCATGCA GTG AGTACC AGCC ATCTAGTGTTTCTG CTG CTTCG CTC A I I I I I CTCCCTCCAAATG I I I I I CTCTT CCCTTACTATAACACAGTTTAGAAGAGGGAGAAAATATTACGGCTGCCGCGAAATTGTCTAAAAG AAGCCTTCTTTTGCCTTTGATTTTCCGGAAAAATGGATTATTCTTAAAATGGATACCGTTATACAC TTTATTTTCAGAATGGACGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCA ACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTACGATAGACATATAAAGAGAGCGGCCATACAG GCCGCCTCTTTTCTGTTCTTGGCGACTCCCCGTCTAGTGGATCACTCAGCAACTCCTCAGCTGCA GCAGCTGCCTCGAGTGCAAAAACAGCCCGCAGATCAACATCCGCGGGCTGTTTCTGATTATAAG AA
[SEQ ID NO: 4] pWUR_Ca9sp2
AGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACAC
AACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACAT
TAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCCCTTCAAACTT CCCAAAGGCGAGCCCTAGTGACATTAGAAAACCGACTGTAAAAAGTACAGTCGGCATTATCTCAT ATTATAAAAGCCAGTCATTAGGCCTATCTGACAATTCCTGAATAGAGTTCATAAACAATCCTGCAT GATAACCATCACAAACAGAATGATGTACCTGTAAAGATAGCGGTAAATATATTGAATTACCTTTAT TAATGAATTTTCCTGCTGTAATAATGGGTAGAAGGTAATTACTATTATTATTGATATTTAAGTTAAA CCCAGTAAATGAAGTCCATGGAATAATAGAAAGAGAAAAAGCATTTTCAGGTATAGGTGTTTTGG GAAACAATTTCCCCGAACCATTATATTTCTCTACATCAGAAAGGTATAAATCATAAAACTCTTTGA AGTCATTCTTTACAGGAGTCCAAATACCAGAGAATGTTTTAGATACACCATCAAAAATTGTATAAA GTGGCTCTAACTTATCCCAATAACCTAACTCTCCGTCGCTATTGTAACCAGTTCTAAAAGCTGTAT TTGAGTTTATCACCCTTGTCACTAAGAAAATAAATGCAGGGTAAAATTTATATCCTTCTTGTTTTAT GTTTCGGTATAAAACACTAATATCAATTTCTGTGGTTATACTAAAAGTCGTTTGTTGGTTCAAATA ATGATTAAATATCTCTTTTCTCTTCCAATTGTCTAAATCAATTTTATTAAAGTTCATTTGATATGCCT CCTAAA I I I I I ATCTAAAGTGAATTTAGGAGGCTTACTTGTCTGCTTTCTTCATTAGAATCAATCCT I I I I I AAAAGTCAATCCCGTTTGTTGAACTACTCTTTAATAAAATAA I I I I I CCGTTCCCAATTCCA CATTGCAATAATAGAAAATCCATCTTCATCGGC I I I I I CGTCATCATCTGTATGAATCAAATCGCC TTCTTCTGTGTCATCAAGGTTTAA I I I I I I ATGTATTTCTTTTAACAAACCACCATAGGAGATTAAC CTTTTACG GTGTAA ACCTTCCTCC AA ATC AG AC AA ACGTTTC AA ATTCTTTTCTTC ATC ATCG GTC A TAAAATCCGTATCCTTTACAGGATATTTTGCAGTTTCGTCAATTGCCGATTGTATATCCGATTTATA TTTA I I I I I CGGTCGAATCATTTGAACTTTTACATTTGGATCATAGTCTAATTTCATTGCC I I I I I CC AAAATTGAATCCATTG I I I I I GATTCACGTAGTTTTCTGTATTCTTAAAATAAGTTGGTTCCACACA TACCAATACATGCATGTGCTGATTATAAGAATTATCTTTATTATTTATTGTCACTTCCGTTGCACGC ATAAAACCAACAAGA I I I I I ATTAA I I I I I I I ATATTGCATCATTCGGCGAAATCCTTGAGCCATAT CTGACAAACTCTTATTTAATTCTTCGCCATCATAAACA I I I I I AACTGTTAATGTGAGAAACAACCA ACGAACTGTTGGCTTTTGTTTAATAACTTCAGCAACAACCTTTTGTGACTGAATGCCATGTTTCAT TGCTCTCCTCCAGTTGCACATTGGACAAAGCCTGGATTTACAAAACCACACTCGATACAACTTTCT TTCGCCTGTTTCACGATTTTGTTTATACTCTAATATTTCAGCACAATCTTTTACTCTTTCAGCCTTT^ TAAATTCAAGAATATGCAGAAGTTCAAAGTAATCAACATTAGCGATTTTCTTTTCTCTCCATGGTC TCACTTTTCCAC I I I I I GTCTTGTCCACTAAAACCCTTGA I I I I I CATCTGAATAAATGCTACTATT AGGACACATAATATTAAAAGAAACCCCCATCTATTTAGTTATTTGTTTGGTCACTTATAACTTTAAC AGATGGGG I I I I I CTGTGCAACCAATTTTAAGGGTTTTCAATACTTTAAAACACATACATACCAAC ACTTCAACGCACCTTTCAGCAACTAAAATAAAAATGACGTTATTTCTATATGTATCAAGAATAGAA AGAACTCG I I I I I CGCTACGCTCAAAACGCAAAAAAAGCACTCATTCGAGTGC I I I I I CTTATCGC TCCAAATCATGCGA I I I I I I CCTCTTTG CTTTTCTTTG CTC ACG AAGTTCTCG ATC ACG CTG C AA A ACATCTTGAAGCGAAAAAGTATTCTTCTTTTCTTCCGATCGCTCATGCTGACGCACGAAAAGCCCT CTAGGCGCATAGGAACAACTCCTAAATGCATGTGAGGGGTTTTCTCGTCCATGTGAACAGTCGC ATACGCAATATTTTGTTTCCCATACTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTT GCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGG CGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAG GAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGG CG I I I I I CCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTG GCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCT CCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGC TTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGT GTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCA ACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAG GTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACA GTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATC CGGCAAACAAACCACCGCTGGTAGCGGTGG I I I I I I I GTTTGCAAGCAGCAGATTACGCGCAGA AAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAA CTCACGTTAAGGGATTTTGGTCATGAATGGCGCTAGTGAGCTGAGGATAATATTTTCTATCATATT TCCACTTAGTGTTCCAGTATTAGCGACAATAACGC I I I I I GTTGCAGTTGGACAATGGAATTCGTG GTTTGATACAATGC I I I I I I GTTCCATGAATCCAAATTTAAGCACATTACAGTATGAATTGCAAAA AGTATTGCAGTCGACTCAACAGTTTACACAACAAGCGTCTTTTGATATGGGGTTAAGAAATAATTC TAATTCAGTTACCCCGGAAAGCATAAGAGCTGCTATGACGGTAGTGGCAGTGGTTCCTATTATGA TGGTTTATCCATTCTTGCAGAAGTATTTTGTAAAGGGTTTAACCATTGGCAGTGTTAAGGGTTAGT ACATCTTTGG I I I I I I ATAAATGCTGAAAGGGGGGAAATTTAAAAATACTTATTAGACAAAATTTT TTAATTGCGAGGTGATAGTAAATAAA I I I I I CAATAAATTAAATTTACTGAGGGAGGAATCTAGAA TGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCAC TGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCA AAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGACAGCGGAAGCGACTCGTCTAAAA CGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGGAGA M I M I C AAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTC I I I I I I GGTGGAAG A AG AC AAG A AG C ATG A ACGTC ATCCTA I I I I I GGAAATATAGTAGATGAAGTTGCTTATCACGAG AAATATCCAACTATCTATCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGATTTGCGC TTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCA I I I I I I GATTGAGGGAGATTTA AATCCTGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGTACAAACCTACAATCAATTATTT GAAGAAAACCCTATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAA ATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGA ATCTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATG CTAAATTACAGCTTTCAAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAG ATCAATATGCTGATTTG I I I I I GGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCT AAGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAACGCTACGATGAAC ATCATCAAGACTTGACTCTTTTAAAAGCATTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAA TC I I I I I I GATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAA TTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTA AATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCA CTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCA I I I I I AAAAGACAATC GTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGC AATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGA AGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCT TCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATT GACAAAGGTCAAATATGTTACTGAAGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGA AAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATT ATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTT CATTAGGTACCTACCATGATTTGCTAAAAATTATTAAAGATAAAGA I I I I I I GGATAATGAAGAAA ATGAAGATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGAGATGATTGAG GAAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCG TTATACTGGTTGGGGACGTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCA AAACAATATTAGA I I I I I I GAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATG ATGATAGTTTGACATTTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTA CATGAACATATTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAA GTTGTTGATGAATTGGTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGC ACGTGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCGAA GAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCA AAATGAAAAGCTCTATCTCTATTATCTCCAAAATGGAAGAGACATGTATGTGGACCAAGAATTAG ATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGATT CAATAGACAATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGT GAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCA ACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTT TTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGT CGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAA TCTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTAC CATCATGCCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAA ACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTC TGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATAATGAACTTCTTCAA AACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAA CTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCC CCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTAC CAAAAAGAAATTCGGACAAACTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGT TTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAA GAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAA ATCCGATTGAC I I I I I AGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTAC CTAAATATAGTC I I I I I GAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTA CAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAA I I I I I I ATATTTAGCTAGTCATTAT GAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGC ATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATGCCA ATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAA AATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAA CAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCA TCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTAGGAGGTGACTGAATCAGACAAAAT GGCCTGCTTATGAAGCGGGCCA I I I I I GTTTAATCCTGTCGACTTGCCGGAATTCTTTCACAGCC GCATCCCTTTCTTCCGCAGGCAGCGCCTTCCAGCCCGCCCAGTCGAGAGAACGGAAATCATGCA GTG AGTACC AGCC ATCTAGTGTTTCTG CTG CTTCG CTC A I I I I I CTCCCTCCAAATG I I I I I CTCTT CCCTTACTATAACACAGTTTAGAAGAGGGAGAAAATATTACGGCTGCCGCGAAATTGTCTAAAAG AAGCCTTCTTTTGCCTTTGATTTTCCGGAAAAATGGATTATTCTTAAAACGTTATACTCAACGTGC ATG C AG C AG GCGTTTTAG AG CTAG AA ATAGC AAGTTAA AATA AG GCTAGTCCGTTATC A ACTTG A AAAAGTGGCACCGAGTCGGTGCTTTTACGATAGACATATAAAGAGAGCGGCCATACAGGCCGCC TCTTTTCTGTTCTTGGCGACTCCCCGTCTAGTGGATCACTCAGCAACTCCTCAGCTGCAGCAGCT GCCTCGAGTGCAAAAACAGCCCGCAGATCAACATCCGCGGGCTGTTTCTGATTATAAGAA [SEQ ID NO: 5] pWUR_Ca9sp3
AGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACAC AACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACAT TAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCCCTTCAAACTT CCCAAAGGCGAGCCCTAGTGACATTAGAAAACCGACTGTAAAAAGTACAGTCGGCATTATCTCAT ATTATAAAAGCCAGTCATTAGGCCTATCTGACAATTCCTGAATAGAGTTCATAAACAATCCTGCAT GATAACCATCACAAACAGAATGATGTACCTGTAAAGATAGCGGTAAATATATTGAATTACCTTTAT TAATGAATTTTCCTGCTGTAATAATGGGTAGAAGGTAATTACTATTATTATTGATATTTAAGTTAAA CCCAGTAAATGAAGTCCATGGAATAATAGAAAGAGAAAAAGCATTTTCAGGTATAGGTGTTTTGG GAAACAATTTCCCCGAACCATTATATTTCTCTACATCAGAAAGGTATAAATCATAAAACTCTTTGA AGTCATTCTTTACAGGAGTCCAAATACCAGAGAATGTTTTAGATACACCATCAAAAATTGTATAAA GTGG CTCTA ACTTATCCC AATAACCTAACTCTCCGTCG CTATTGTA ACC AGTTCTA AA AG CTGTAT TTGAGTTTATCACCCTTGTCACTAAGAAAATAAATGCAGGGTAAAATTTATATCCTTCTTGTTTTAT GTTTCGGTATAAAACACTAATATCAATTTCTGTGGTTATACTAAAAGTCGTTTGTTGGTTCAAATA ATGATTAAATATCTCTTTTCTCTTCCAATTGTCTAAATCAATTTTATTAAAGTTCATTTGATATGCCT CCTAAA I I I I I ATCTAAAGTGAATTTAGGAGGCTTACTTGTCTGCTTTCTTCATTAGAATCAATCCT I I I I I AAAAGTCAATCCCGTTTGTTGAACTACTCTTTAATAAAATAA I I I I I CCGTTCCCAATTCCA CATTGCAATAATAGAAAATCCATCTTCATCGGC I I I I I CGTCATCATCTGTATGAATCAAATCGCC TTCTTCTGTGTCATCAAGGTTTAA I I I I I I ATGTATTTCTTTTA AC A AACC ACC ATAG G AG ATTAAC CTTTTACG GTGTAA ACCTTCCTCC AA ATC AG AC AA ACGTTTC AA ATTCTTTTCTTC ATC ATCG GTC A TAAAATCCGTATCCTTTACAGGATATTTTGCAGTTTCGTCAATTGCCGATTGTATATCCGATTTATA TTTA I I I I I CGGTCGAATCATTTGAACTTTTACATTTGGATCATAGTCTAATTTCATTGCC I I I I I CC AAAATTGAATCCATTG I I I I I GATTCACGTAGTTTTCTGTATTCTTAAAATAAGTTGGTTCCACACA TACCAATACATGCATGTGCTGATTATAAGAATTATCTTTATTATTTATTGTCACTTCCGTTGCACGC ATAAAACCAACAAGA I I I I I ATTAA I I I I I I I ATATTGCATCATTCGGCGAAATCCTTGAGCCATAT CTGACAAACTCTTATTTAATTCTTCGCCATCATAAACA I I I I I AACTGTTAATGTGAGAAACAACCA ACGAACTGTTGGCTTTTGTTTAATAACTTCAGCAACAACCTTTTGTGACTGAATGCCATGTTTCAT TGCTCTCCTCCAGTTGCACATTGGACAAAGCCTGGATTTACAAAACCACACTCGATACAACTTTCT TTCGCCTGTTTCACGATTTTGTTTATACTCTAATATTTCAGCACAATCTTTTACTCTTTCAGCCTTTT TAAATTCAAGAATATGCAGAAGTTCAAAGTAATCAACATTAGCGATTTTCTTTTCTCTCCATGGTC TCACTTTTCCAC I I I I I GTCTTGTCCACTAAAACCCTTGA I I I I I CATCTGAATAAATGCTACTATT AGGACACATAATATTAAAAGAAACCCCCATCTATTTAGTTATTTGTTTGGTCACTTATAACTTTAAC AGATGGGG I I I I I CTGTGCAACCAATTTTAAGGGTTTTCAATACTTTAAAACACATACATACCAAC ACTTCAACGCACCTTTCAGCAACTAAAATAAAAATGACGTTATTTCTATATGTATCAAGAATAGAA AGAACTCG I I I I I CGCTACGCTCAAAACGCAAAAAAAGCACTCATTCGAGTGC I I I I I CTTATCGC TCCAAATCATGCGA I I I I I I CCTCTTTGCTTTTCTTTGCTCACGAAGTTCTCGATCACGCTGCAAA ACATCTTGAAGCGAAAAAGTATTCTTCTTTTCTTCCGATCGCTCATGCTGACGCACGAAAAGCCCT CTAGGCGCATAGGAACAACTCCTAAATGCATGTGAGGGGTTTTCTCGTCCATGTGAACAGTCGC ATACGCAATATTTTGTTTCCCATACTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTT GCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGG CGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAG GAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGG CG I I I I I CCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTG GCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCT CCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGC TTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGT GTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCA ACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAG GTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACA GTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATC CGGCAAACAAACCACCGCTGGTAGCGGTGG I I I I I I I GTTTGCAAGCAGCAGATTACGCGCAGA AAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAA CTCACGTTAAGGGATTTTGGTCATGAATGGCGCTAGTGAGCTGAGGATAATATTTTCTATCATATT TCCACTTAGTGTTCCAGTATTAGCGACAATAACGC I I I I I GTTGCAGTTGGACAATGGAATTCGTG GTTTGATACAATGC I I I I I I GTTCCATGAATCCAAATTTAAGCACATTACAGTATGAATTGCAAAA AGTATTGCAGTCGACTCAACAGTTTACACAACAAGCGTCTTTTGATATGGGGTTAAGAAATAATTC TAATTCAGTTACCCCGGAAAGCATAAGAGCTGCTATGACGGTAGTGGCAGTGGTTCCTATTATGA TGGTTTATCCATTCTTGCAGAAGTATTTTGTAAAGGGTTTAACCATTGGCAGTGTTAAGGGTTAGT ACATCTTTGG I I I I I I ATAAATGCTGAAAGGGGGGAAATTTAAAAATACTTATTAGACAAAATTTT TTAATTGCGAGGTGATAGTAAATAAA I I I I I CAATAAATTAAATTTACTGAGGGAGGAATCTAGAA TGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCAC TG ATG A ATATA AG GTTCCGTCTA AA AAGTTC A AG GTTCTGG G A AATAC AG ACCG CC AC AGTATC A AAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGACAGCGGAAGCGACTCGTCTAAAA CGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGGAGA M I M I C AAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTC I I I I I I GGTGGAAG A AG AC AAG A AG C ATG A ACGTC ATCCTA I I I I I GGAAATATAGTAGATGAAGTTGCTTATCACGAG AAATATCCAACTATCTATCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGATTTGCGC TTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCA I I I I I I GATTGAGGGAGATTTA AATCCTGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGTACAAACCTACAATCAATTATTT GAAGAAAACCCTATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAA ATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGA ATCTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATG CTAAATTACAGCTTTCAAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAG ATCAATATGCTGATTTG I I I I I GGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCT AAGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAACGCTACGATGAAC ATCATCAAGACTTGACTCTTTTAAAAGCATTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAA TC I I I I I I GATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAA TTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTA AATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCA CTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCA I I I I I AAAAGACAATC GTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGC AATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGA AGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCT TCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATT GACAAAGGTCAAATATGTTACTGAAGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGA AAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATT ATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTT CATTAGGTACCTACCATGATTTGCTAAAAATTATTAAAGATAAAGA I I I I I I GGATAATGAAGAAA ATGAAGATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGAGATGATTGAG GAAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCG TTATACTGGTTGGGGACGTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCA AAACAATATTAGA I I I I I I GAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATG ATGATAGTTTGACATTTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTA CATGAACATATTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAA GTTGTTGATGAATTGGTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGC ACGTGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCGAA GAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCA AAATGAAAAGCTCTATCTCTATTATCTCCAAAATGGAAGAGACATGTATGTGGACCAAGAATTAG ATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGATT CAATAGACAATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGT GAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCA ACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTT TTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGT CGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAA TCTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTAC CATCATGCCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAA ACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTC TGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATAATGAACTTCTTCAA AACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAA CTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCC CCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTAC CAAAAAGAAATTCGGACAAACTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGT TTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAA GAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAA ATCCGATTGAC I I I I I AGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTAC CTAAATATAGTC I I I I I GAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTA CAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAA I I I I I I ATATTTAGCTAGTCATTAT GAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGC ATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATGCCA ATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAA AATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAA CAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCA TCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTAGGAGGTGACTGAATCAGACAAAAT GGCCTGCTTATGAAGCGGGCCA I I I I I GTTTAATCCTGTCGACTTGCCGGAATTCTTTCACAGCC GCATCCCTTTCTTCCGCAGGCAGCGCCTTCCAGCCCGCCCAGTCGAGAGAACGGAAATCATGCA GTGAGTACCAGCCATCTAGTGTTTCTGCTGCTTCGCTCA I I I I I CTCCCTCCAAATG I I I I I CTCTT CCCTTACTATAACACAGTTTAGAAGAGGGAGAAAATATTACGGCTGCCGCGAAATTGTCTAAAAG AAGCCTTCTTTTGCCTTTGATTTTCCGGAAAAATGGATTATTCTTAAAATGGATACCGTTATACAT CTCATGATCAAAGTCGTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATC AACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTACGATAGACATATAAAGAGAGCGGCCATACA GGCCGCCTCTTTTCTGTTCTTGGCGACTCCCCGTCTAGTGGATCACTCAGCAACTCCTCAGCTGC AGCAGCTGCCTCGAGTGCAAAAACAGCCCGCAGATCAACATCCGCGGGCTGTTTCTGATTATAA GAA
[SEQ ID NO: 6] pWUR_Cas9nt_hr
AGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATT CCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGC TAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGC CAGCCCTTCAAACTTCCCAAAGGCGAGCCCTAGTGACATTAGAAAACCGACTGTAAAAAG TACAGTCGGCATTATCTCATATTATAAAAGCCAGTCATTAGGCCTATCTGACAATTCCTG AATAGAGTTCATAAACAATCCTGCATGATAACCATCACAAACAGAATGATGTACCTGTAA AGATAGCGGTAAATATATTGAATTACCTTTATTAATGAATTTTCCTGCTGTAATAATGGG TAGAAGGTAATTACTATTATTATTGATATTTAAGTTAAACCCAGTAAATGAAGTCCATGG AATAATAGAAAGAGAAAAAGCATTTTCAGGTATAGGTGTTTTGGGAAACAATTTCCCCGA ACCATTATATTTCTCTACATCAGAAAGGTATAAATCATAAAACTCTTTGAAGTCATTCTT TACAGGAGTCCAAATACCAGAGAATGTTTTAGATACACCATCAAAAATTGTATAAAGTGG CTCTAACTTATCCC AATA ACCTA ACTCTCCGTCG CTATTGTA ACC AGTTCTA AA AG CTGT ATTTGAGTTTATCACCCTTGTCACTAAGAAAATAAATGCAGGGTAAAATTTATATCCTTC TTGTTTTATGTTTCGGTATAAAACACTAATATCAATTTCTGTGGTTATACTAAAAGTCGT TTGTTGGTTCAAATAATGATTAAATATCTCTTTTCTCTTCCAATTGTCTAAATCAATTTT ATTAA AGTTC ATTTG ATATG CCTCCTA AA I I I I I ATCTAAAGTGAATTTAGGAGGCTTAC TTGTCTGCTTTCTTCATTAGAATCAATCC I I I I I I AAAAGTCAATCCCGTTTGTTGAACT ACTCTTTAATAAAATAA I I I I I CCGTTCCCAATTCCACATTGCAATAATAGAAAATCCAT CTTCATCGGC I I I I I CGTCATCATCTGTATGAATCAAATCGCCTTCTTCTGTGTCATCAA GGTTTAA I I I I I I ATGTATTTCTTTTAACAAACCACCATAGGAGATTAACCTTTTACGGT GTAAACCTTCCTCCAAATCAGACAAACGTTTCAAATTCTTTTCTTCATCATCGGTCATAA AATCCGTATCCTTTACAGGATATTTTGCAGTTTCGTCAATTGCCGATTGTATATCCGATT TATATTTA I I I I I CGGTCGAATCATTTGAACTTTTACATTTGGATCATAGTCTAATTTCA TTGCC I I I I I CCAAAATTGAATCCATTG I I I I I GATTCACGTAGTTTTCTGTATTCTTAA AATAAGTTGGTTCCACACATACCAATACATGCATGTGCTGATTATAAGAATTATCTTTAT TATTTATTGTCACTTCCGTTGCACGCATAAAACCAACAAGA I I I I I ATTAA I I I I I I I AT ATTGCATCATTCGGCGAAATCCTTGAGCCATATCTGACAAACTCTTATTTAATTCTTCGC CATCATAAACA I I I I I AACTGTTAATGTGAGAAACAACCAACGAACTGTTGGCTTTTGTT TAATAACTTCAGCAACAACCTTTTGTGACTGAATGCCATGTTTCATTGCTCTCCTCCAGT TGCACATTGGACAAAGCCTGGATTTACAAAACCACACTCGATACAACTTTCTTTCGCCTG TTTCACGATTTTGTTTATACTCTAATATTTCAGCACAATCTTTTACTCTTTCAGCCTTTT TAAATTCAAGAATATGCAGAAGTTCAAAGTAATCAACATTAGCGATTTTCTTTTCTCTCC ATG GTCTC ACTTTTCC AC I I I I I GTCTTGTCCACTAAAACCCTTGA I I I I I CATCTGAAT AAATGCTACTATTAGGACACATAATATTAAAAGAAACCCCCATCTATTTAGTTATTTGTT TGGTCACTTATAACTTTAACAGATGGGG I I I I I CTGTGCAACCAATTTTAAGGGTTTTCA ATACTTTAAAACACATACATACCAACACTTCAACGCACCTTTCAGCAACTAAAATAAAAA TGACGTTATTTCTATATGTATCAAGAATAGAAAGAACTCG I I I I I CGCTACGCTCAAAAC GCAAAAAAAGCACTCATTCGAGTGC I I I I I CTTATCGCTCCAAATCATGCGA I I I I I I CC TCTTTGCTTTTCTTTGCTCACGAAGTTCTCGATCACGCTGCAAAACATCTTGAAGCGAAA AAGTATTCTTCTTTTCTTCCGATCGCTCATGCTGACGCACGAAAAGCCCTCTAGGCGCAT AGGAACAACTCCTAAATGCATGTGAGGGGTTTTCTCGTCCATGTGAACAGTCGCATACGC AATATTTTGTTTCCCATACTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTG CGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTG CGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGAT AACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCC GCGTTGCTGGCG I I I I I CCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGC TCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGA AGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTT CTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTG TAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGC GCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTG GCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTC TTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTG CTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACC GCTGGTAGCGGTGG I I I I I I I GTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCT CAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGT TAAGGGATTTTGGTCATGAATGCAGCGATGGTCCGGTGTTCCCAATTGGGGAGGTTGTAT TGTGAATCCATTGCATGTCCAATTGCCAGGTTTAGAGTTGAAAAATCCAGTGATGCCGGC CTCAGGCTGCTTCGGATTTGGAAGAGAGTTCAGCCAGTTATATGATTTGAATCAGCTGGG AGCGATTATGATCAAAGCGACCACACCGGAACCGAGGTTTGGAAATCCGACTCCGCGAGT AGCAGAAACACCTTCGGGAATGTTGAATGCCATTGGTCTGCAAAATCCAGGATTAAAGAA AGTGATCAGTGACGAGCTTCCATGGCTCTCACAATTTGATGTACCGATTATCGCCAATAT CGCCGGATCGGAAATGGAAGACTATGTAACGGTTGCTGAAGCCATCTCACAAGTGGACAA CGTGAAAGCGTTGGAACTGAATATTTCTTGTCCGAATGTTAAAACAGGCGGGATTGCTTT TGGGACGGTACCGGAAATTGCAAAAGAACTGACGAAAAAAGTGAAAGAAGTATCCGAAGT TCCGGTATATGTGAAATTGTCTCCTAATGTCACCAATATTGTGGAAATGGCAAAAGCAGT AGAAGAAGGCGGAGCGGACGGTATTACGATGATTAATACGCTTTTAGGCATGAAAATAGA CATCCATACTGGAAGGGCTCTATTGGCAAACGGCACCGGAGGTTTGTCAGGTCCCGCTGT CAAACCGATTGCGGTGCGGATGATCTATGAAGTGAGCCAGCATGTCCATATTCCGATCAT TGGAATGGGCGGTATTTCCTCGCTCGATGATATTCTCGAA I I I I I I I ATGCGGGGGCCAG CGCAGTGGCCATCGGTACAGCGAATTTTGTTGATCCGTTTATCTGTCAAAAGCTTATCGC TCAATTGCAGGACTACGTTGAGGCAAATGGAATTGATCATATTAGCGAACTGACAGGAAG GAGCTGGAAAGTGCGTGTAAACCAGCAGATCTGAAAAAAAGCTGTAAAAATGGTGAGAGG AGAACAATCACATGCAGAACGATCATATACAAAGTAAAAGAATAGCAGAACAGCTTCTCA ATATTGAAGCCATCTATTTGCAACCAAATGATCCATTTACATGGTCCTCAGGAATAAAAT CGCCCATATATTGCGACAATCGTCTTACCCTTTCTTATCCGGCGGTGCGAAAAGAAGTTG CGAAAGGACTCGTTTCTTTGATTAACGAACA I I I I I C AG A AGTAC AATG C ATTG CCGG G A CTGCGACAGCGGGCATTCCTCATGCGGCTTGGGTAAGCGATCTACTCGATTTGCCCATGT GCTATGTTCGTTCAAAAGCGAAAAGCCATGGAAAAGGGAAACAAATAGAGGGAAAAGTAC AGCCGGGCGTTAAAACGGTGGTCGTAGAAGACTTAATTTCGACAGGAGGAAGCGCTATTC AAGCAGCTAACGTCTTGCGAGAATCAGGATGCGAGGTGCTTGGAGTCGTGTCGA I I I I I A CTTATGAACTTGATATAGGAAAGCAGCTGCTTGATGAAGCCAATTTAAAAGCTTATAGTT TAAGCAATTATTCAGCGCTAATGGAAGTTGCTTTAGAAAAAGGAGCGATTCAACCCAAGG ATTTAGAGACTCTTCGTCAATGGCGGGAAAATCCTGAGCATTGGCCGATGGTACAATCGT AATACATAGATGAAAAAATAAGGAAGAGAGCGTCTCTTCCTTA I I I I I I CAATTTCACCA CTAAATCAGGATCCGGTGTAACATAAATGGTTTGCTGATTGTCATAGATGACAAATCCAG GTTTTGAACCGTTCGG I I I I I I GACATAACGAATTTTCGTGTAATCTACCGGTACTGAAC TGGAATGTTTTGCTTTGCTGAAATAAGCGGCCAGGTTGGCGGCTTCCAACAGCGTTTCTT CGGATGGGCTATTATGGCGAATCACAACATGGGAACTCATGAATGGCGCTAGTGAGCTGA GGATAATATTTTCTATCATATTTCCACTTAGTGTTCCAGTATTAGCGACAATAACGCTTT TTGTTGCAGTTGGACAATGGAATTCGTGGTTTGATACAATGC I I I I I I GTTCCATGAATC CAAATTTAAGCACATTACAGTATGAATTGCAAAAAGTATTGCAGTCGACTCAACAGTTTA CACAACAAGCGTCTTTTGATATGGGGTTAAGAAATAATTCTAATTCAGTTACCCCGGAAA GCATAAGAGCTGCTATGACGGTAGTGGCAGTGGTTCCTATTATGATGGTTTATCCATTCT TGCAGAAGTATTTTGTAAAGGGTTTAACCATTGGCAGTGTTAAGGGTTAGTACATCTTTG G I I I I I I ATAAATGCTGAAAGGGGGGAAATTTAAAAATACTTATTAGACAAAA I I I I I I A ATTGCGAGGTGATAGTAAATAAA I I I I I CAATAAATTAAATTTACTGAGGGAGGAATCTA GAATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGG TGATCACTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACC GCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGACAGCGG AAGCGACTCGTCTAAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTT GTTATCTACAGGAGA I I I I I I CAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATC GACTTGAAGAGTC I I I I I I GGTGGAAGAAGACAAGAAGCATGAACGTCATCCTA M I N G GAAATATAGTAGATGAAGTTGCTTATCACGAGAAATATCCAACTATCTATCATCTGCGAA AAAAATTGGTAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGC ATATGATTAAGTTTCGTGGTCA I I I I I I GATTGAGGGAGATTTAAATCCTGATAATAGTG ATGTGGACAAACTATTTATCCAGTTGGTACAAACCTACAATCAATTATTTGAAGAAAACC CTATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAA GACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGA ATCTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAG AAGATGCTAAATTACAGCTTTCAAAAGATACTTACGATGATGATTTAGATAATTTATTGG CGCAAATTGGAGATCAATATGCTGATTTG I I I I I GGCAGCTAAGAATTTATCAGATGCTA TTTTACTTTCAGATATCCTAAGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTT CAATGATTAAACGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCATTAGTTC GACAACAACTTCCAGAAAAGTATAAAGAAATC I I I I I I GATCAATCAAAAAACGGATATG CAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTT TAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGC GCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGC ATGCTATTTTGAGAAGACAAGAAGACTTTTATCCA I I I I I AAAAGACAATCGTGAGAAGA TTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAATA GTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAG AAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATA AAAATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGG TTTATAACGAATTGACAAAGGTCAAATATGTTACTGAAGGAATGCGAAAACCAGCATTTC TTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAA CCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAA TTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGTACCTACCATGATTTGCTAAAAA TTATTAAAGATAAAGA I I I I I I GGATAATGAAGAAAATGAAGATATCTTAGAGGATATTG TTTTAACATTGACCTTATTTGAAGATAGGGAGATGATTGAGGAAAGACTTAAAACATATG CTCACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGG GACGTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATAT TAGA I I I I I I GAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATG ATAGTTTGACATTTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTT TACATGAACATATTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGA CTGTAAAAGTTGTTGATGAATTGGTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATCG TTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGC GTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATC CTGTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTCCAAAATGGAA GAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAAGTGATTATGATGTCGATC ACATTGTTCCACAAAGTTTCCTTAAAGACGATTCAATAGACAATAAGGTCTTAACGCGTT CTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGA AAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATT TAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCC AATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGA ATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAAT CTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACA
ATTACCATCATGCCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGA AATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTA AAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACT CTAATATAATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAAC GCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATT TTG CC AC AGTGCG C AA AGTATTGTCC ATG CCCC A AGTC A ATATTGTC AAG A AA AC AG A AG TACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAACTTA TTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAG CTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCG TTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTG AC I I I I I AGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTA AATATAGTC I I I I I GAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAAT TACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAA I I I I I I ATATTTAGCTA GTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGG AGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTG TTATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACA AACCAATACGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAATCTTGGAG CTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAACGATATACGTCTACAA A AG A AGTTTTAG ATG CC ACTCTTATCC ATC A ATCC ATC ACTG GTCTTTATG A AAC ACG C A TTGATTTGAGTCAGCTAGGAGGTGACTGAATCAGACAAAATGGCCTGCTTATGAAGCGGG CCA I I I I I GTTTAATCCTGTCGACTTGCCGGAATTCTTTCACAGCCGCATCCCTTTCTTC CGCAGGCAGCGCCTTCCAGCCCGCCCAGTCGAGAGAACGGAAATCATGCAGTGAGTACCA GCCATCTAGTGTTTCTGCTGCTTCGCTCA I I I I I CTCCCTCCAAATG I I I I I CTCTTCCC TTACTATAACACAGTTTAGAAGAGGGAGAAAATATTACGGCTGCCGCGAAATTGTCTAAA AG AAGCCTTCTTTTG CCTTTG ATTTTCCG G A AA AATG G ATTATTCTTAA AATG G ATACCG TTATACGAGGAGTACCGGTTGAGACGAGTCTCGGAAGCTCAAACGTCTCTGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCTTTTACGATAGACATATAAAGAGAGCGGCCATACAGGCCGCCTCTTTTCTGTTC TTGGCGACTCCCCGTCTAGTGGATCACTCAGCAACTCCTCAGCTGCAGCAGCTGCCTCGA GTGCAAAAACAGCCCGCAGATCAACATCCGCGGGCTGTTTCTGATTATAAGAA
[SEQ ID NO: 7] pWUR_Cas9spl_hr
GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCAC CGAGTCGGTGCTTTTACGATAGACATATAAAGAGAGCGGCCATACAGGCCGCCTCTTTTCTGTTC TTGGCGACTCCCCGTCTAGTGGATCACTCAGCAACTCCTCAGCTGCAGCAGCTGCCTCGAGTGC AAAAACAGCCCGCAGATCAACATCCGCGGGCTGTTTCTGATTATAAGAAAGCTTGGCGTAATCAT GGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGA AGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTC ACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCCCTTCAAACTTCCCAAAGGCGAGCCC TAGTGACATTAGAAAACCGACTGTAAAAAGTACAGTCGGCATTATCTCATATTATAAAAGCCAGT CATTAGGCCTATCTGACAATTCCTGAATAGAGTTCATAAACAATCCTGCATGATAACCATCACAAA CAGAATGATGTACCTGTAAAGATAGCGGTAAATATATTGAATTACCTTTATTAATGAATTTTCCTG CTGTAATAATGGGTAGAAGGTAATTACTATTATTATTGATATTTAAGTTAAACCCAGTAAATGAAG TCCATGGAATAATAGAAAGAGAAAAAGCATTTTCAGGTATAGGTGTTTTGGGAAACAATTTCCCC GAACCATTATATTTCTCTACATCAGAAAGGTATAAATCATAAAACTCTTTGAAGTCATTCTTTACAG GAGTCCAAATACCAGAGAATGTTTTAGATACACCATCAAAAATTGTATAAAGTGGCTCTAACTTAT CCCAATAACCTAACTCTCCGTCGCTATTGTAACCAGTTCTAAAAGCTGTATTTGAGTTTATCACCC TTGTCACTAAGAAAATAAATGCAGGGTAAAATTTATATCCTTCTTGTTTTATGTTTCGGTATAAAAC ACTAATATCAATTTCTGTGGTTATACTAAAAGTCGTTTGTTGGTTCAAATAATGATTAAATATCTCT TTTCTCTTCCAATTGTCTAAATCAATTTTATTAAAGTTCATTTGATATGCCTCCTAAA I I I I I ATCTA AAGTGAATTTAGGAGGCTTACTTGTCTGCTTTCTTCATTAGAATCAATCC I I I I I I AAAAGTCAATC CCGTTTGTTGAACTACTCTTTAATAAAATAA I I I I I CCGTTCCCAATTCCACATTGCAATAATAGAA AATCCATCTTCATCGGC I I I I I CGTCATCATCTGTATGAATCAAATCGCCTTCTTCTGTGTCATCAA GGTTTAA I I I I I I ATGTATTTCTTTTAACAAACCACCATAGGAGATTAACCTTTTACGGTGTAAACC TTCCTCCAAATCAGACAAACGTTTCAAATTCTTTTCTTCATCATCGGTCATAAAATCCGTATCCTTT ACAGGATATTTTGCAGTTTCGTCAATTGCCGATTGTATATCCGATTTATATTTA I I I I I CGGTCGAA TCATTTGAACTTTTACATTTGGATCATAGTCTAATTTCATTGCC I I I I I CCAAAATTGAATCCATTG I I I I I GATTCACGTAGTTTTCTGTATTCTTAAAATAAGTTGGTTCCACACATACCAATACATGCATG TGCTGATTATAAGAATTATCTTTATTATTTATTGTCACTTCCGTTGCACGCATAAAACCAACAAGAT TTTTATTAA I I I I I I I ATATTGCATCATTCGGCGAAATCCTTGAGCCATATCTGACAAACTCTTATT TAATTCTTCG CC ATC ATA AAC A I I I I I AACTGTTAATGTGAGAAACAACCAACGAACTGTTGGCTT TTGTTTAATAACTTCAGCAACAACCTTTTGTGACTGAATGCCATGTTTCATTGCTCTCCTCCAGTT GCACATTGGACAAAGCCTGGATTTACAAAACCACACTCGATACAACTTTCTTTCGCCTGTTTCACG ATTTTGTTTATACTCTAATATTTCAGCACAATCTTTTACTCTTTCAGCC I I I I I AAATTCAAGAATAT GCAGAAGTTCAAAGTAATCAACATTAGCGATTTTCTTTTCTCTCCATGGTCTCACTTTTCCACTTTT TGTCTTGTCCACTAAAACCCTTGA I I I I I CATCTGAATAAATGCTACTATTAGGACACATAATATTA AAAGAAACCCCCATCTATTTAGTTATTTGTTTGGTCACTTATAACTTTAACAGATGGGG I I I I I CT GTGCAACCAATTTTAAGGGTTTTCAATACTTTAAAACACATACATACCAACACTTCAACGCACCTT TCAGCAACTAAAATAAAAATGACGTTATTTCTATATGTATCAAGAATAGAAAGAACTCG I I I I I CG CTACGCTCAAAACGCAAAAAAAGCACTCATTCGAGTGC I I I I I CTTATCGCTCCAAATCATGCGAT I I I I I CCTCTTTGCTTTTCTTTGCTCACGAAGTTCTCGATCACGCTGCAAAACATCTTGAAGCGAA AAAGTATTCTTCTTTTCTTCCGATCGCTCATGCTGACGCACGAAAAGCCCTCTAGGCGCATAGGA ACAACTCCTAAATGCATGTGAGGGGTTTTCTCGTCCATGTGAACAGTCGCATACGCAATATTTTG TTTCCCATACTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCT CTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGC TCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGA GCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCG I I I I I CCATAGGC TCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGG ACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGC CGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGC TGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCG TTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGAC TTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTA CAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCT CTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCG CTGGTAGCGGTGG I I I I I I I GTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAA GATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTT GGTCATGAATGCAGCGATGGTCCGGTGTTCCCAATTGGGGAGGTTGTATTGTGAATCCATTGCA TGTCCAATTGCCAGGTTTAGAGTTGAAAAATCCAGTGATGCCGGCCTCAGGCTGCTTCGGATTTG GAAGAGAGTTCAGCCAGTTATATGATTTGAATCAGCTGGGAGCGATTATGATCAAAGCGACCAC ACCGGAACCGAGGTTTGGAAATCCGACTCCGCGAGTAGCAGAAACACCTTCGGGAATGTTGAAT GCCATTGGTCTGCAAAATCCAGGATTAAAGAAAGTGATCAGTGACGAGCTTCCATGGCTCTCACA ATTTGATGTACCGATTATCGCCAATATCGCCGGATCGGAAATGGAAGACTATGTAACGGTTGCTG AAGCCATCTCACAAGTGGACAACGTGAAAGCGTTGGAACTGAATATTTCTTGTCCGAATGTTAAA ACAGGCGGGATTGCTTTTGGGACGGTACCGGAAATTGCAAAAGAACTGACGAAAAAAGTGAAAG AAGTATCCGAAGTTCCGGTATATGTGAAATTGTCTCCTAATGTCACCAATATTGTGGAAATGGCA AAAGCAGTAGAAGAAGGCGGAGCGGACGGTATTACGATGATTAATACGCTTTTAGGCATGAAAA TAGACATCCATACTGGAAGGGCTCTATTGGCAAACGGCACCGGAGGTTTGTCAGGTCCCGCTGT CAAACCGATTGCGGTGCGGATGATCTATGAAGTGAGCCAGCATGTCCATATTCCGATCATTGGAA TGGGCGGTATTTCCTCGCTCGATGATATTCTCGAA I I I I I I I ATGCGGGGGCCAGCGCAGTGGCC ATCGGTACAGCGAATTTTGTTGATCCGTTTATCTGTCAAAAGCTTATCGCTCAATTGCAGGACTAC GTTG AG GC A AATG G A ATTG ATC ATATTAG CG A ACTG AC AG G A AGG AG CTG G AA AGTG CGTGTAA ACCAGCAGATCTGAAAAAAAGCTGTAAAAATGGTGAGAGGAGAACAATCACATGCAGAACGATC ATATACAAAGTAAAAGAATAGCAGAACAGCTTCTCAATATTGAAGCCATCTATTTGCAACCAAATG ATCCATTTACATGGTCCTCAGGAATAAAATCGCCCATATATTGCGACAATCGTCTTACCCTTTCTT ATCCGGCGGTGCGAAAAGAAGTTGCGAAAGGACTCGTTTCTTTGATTAACGAACA I I I I I CAGAA GTACAATGCATTGCCGGGACTGCGACAGCGGGCATTCCTCATGCGGCTTGGGTAAGCGATCTAC TCGATTTGCCCATGTGCTATGTTCGTTCAAAAGCGAAAAGCCATGGAAAAGGGAAACAAATAGAG GGAAAAGTACAGCCGGGCGTTAAAACGGTGGTCGTAGAAGACTTAATTTCGACAGGAGGAAGC GCTATTCAAGCAGCTAACGTCTTGCGAGAATCAGGATGCGAGGTGCTTGGAGTCGTGTCGATTT TTACTTATGAACTTGATATAGGAAAGCAGCTGCTTGATGAAGCCAATTTAAAAGCTTATAGTTTAA GCAATTATTCAGCGCTAATGGAAGTTGCTTTAGAAAAAGGAGCGATTCAACCCAAGGATTTAGAG ACTCTTCGTCAATGGCGGGAAAATCCTGAGCATTGGCCGATGGTACAATCGTAATACATAGATGA A AA AATA AG G A AG AG AGCGTCTCTTCCTTATTTTTTC AATTTC ACC ACTAA ATC AG G ATCCG GTGT AACATAAATGGTTTGCTGATTGTCATAGATGACAAATCCAGGTTTTGAACCGTTCGG I I I I I I GAC ATAACGAATTTTCGTGTAATCTACCGGTACTGAACTGGAATGTTTTGCTTTGCTGAAATAAGCGG CCAGGTTGGCGGCTTCCAACAGCGTTTCTTCGGATGGGCTATTATGGCGAATCACAACATGGGA ACTCATGAATGGCGCTAGTGAGCTGAGGATAATATTTTCTATCATATTTCCACTTAGTGTTCCAGT ATT AG CG AC AATA ACG C I I I I I GTTGCAGTTGGACAATGGAATTCGTGGTTTGATACAATGCTTTT TTGTTCCATGAATCCAAATTTAAGCACATTACAGTATGAATTGCAAAAAGTATTGCAGTCGACTCA ACAGTTTACACAACAAGCGTCTTTTGATATGGGGTTAAGAAATAATTCTAATTCAGTTACCCCGGA AAGCATAAGAGCTGCTATGACGGTAGTGGCAGTGGTTCCTATTATGATGGTTTATCCATTCTTGC AGAAGTATTTTGTAAAGGGTTTAACCATTGGCAGTGTTAAGGGTTAGTACATCTTTGG I I I I I I AT AAATGCTGAAAGGGGGGAAATTTAAAAATACTTATTAGACAAAA I I I I I I AATTGCGAGGTGATA GTAAATAAA I I I I I CAATAAATTAAATTTACTGAGGGAGGAATCTAGAATGGATAAGAAATACTCA ATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGAATATAAGGTTC CGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGG GCTCTTTTATTTGACAGTGGAGAGACAGCGGAAGCGACTCGTCTAAAACGGACAGCTCGTAGAA GGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGGAGA I I I I I I CAAATGAGATGGCGAAA GTAGATGATAGTTTCTTTCATCGACTTGAAGAGTC I I I I I I GGTGGAAGAAGACAAGAAGCATGA ACGTCATCCTA I I I I I GGAAATATAGTAGATGAAGTTGCTTATCACGAGAAATATCCAACTATCTA TCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTT AGCGCATATGATTAAGTTTCGTGGTCA I I I I I I GATTGAGGGAGATTTAAATCCTGATAATAGTGA TGTGGACAAACTATTTATCCAGTTGGTACAAACCTACAATCAATTATTTGAAGAAAACCCTATTAA CGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAA ATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGAATCTCATTGCTTTGTCAT TGGGTTTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAA AAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGT TTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAATACTGAAA TAACTAAGGCTCCCCTATCAGCTTCAATGATTAAACGCTACGATGAACATCATCAAGACTTGACTC TTTTAAAAGCATTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAATC I I I I I I GATCAATCAA AAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAA CCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCT GCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATG CTATTTTGAGAAGACAAGAAGACTTTTATCCA I I I I I AAAAGACAATCGTGAGAAGATTGAAAAAA TCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGA TGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCT TCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTA CCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTT ACTGAAGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACT CTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATG TTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGTACCTACCATGA TTTGCTAAAAATTATTAAAGATAAAGA I I I I I I GGATAATGAAGAAAATGAAGATATCTTAGAGGA TATTGTTTTAACATTGACCTTATTTGAAGATAGGGAGATGATTGAGGAAAGACTTAAAACATATGC TCACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTT TGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGA I I I I I I GA AATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGATAGTTTGACATTTAAAG AAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTACATGAACATATTGCAAATTTA GCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAGTTGTTGATGAATTGGTCAA AGTAATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAGACAACT CAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAG GAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCT ATTATCTCCAAAATGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAAGTGATT ATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGATTCAATAGACAATAAGGTCTTAA CGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATG AAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACG AAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGA AACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACTAAATACGATG AAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCC GAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCGTATC TAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTATG GTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCA ACCGCAAAATATTTCTTTTACTCTAATATAATGAACTTCTTCAAAACAGAAATTACACTTGCAAATG GAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAA AGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAA CAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAACTT ATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTA TTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGT TACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTGAC I I I I I AGAAGCTA AAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAATATAGTC I I I I I GAGTTAG AAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAGCTGGCTCT GCCAAGCAAATATGTGAA I I I I I I ATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGA AGATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGATGAGATTATTGAGC AAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCAT ATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGA CGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAACGATATACGT CTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCA TTGATTTGAGTCAGCTAGGAGGTGACTGAATCAGACAAAATGGCCTGCTTATGAAGCGGGCCAT TTTTGTTTAATCCTGTCGACTTGCCGGAATTCTTTCACAGCCGCATCCCTTTCTTCCGCAGGCAGC GCCTTCCAGCCCGCCCAGTCGAGAGAACGGAAATCATGCAGTGAGTACCAGCCATCTAGTGTTT CTGCTGCTTCGCTCA I I I I I CTCCCTCCAAATG I I I I I CTCTTCCCTTACTATAACACAGTTTAGAA GAGGGAGAAAATATTACGGCTGCCGCGAAATTGTCTAAAAGAAGCCTTCTTTTGCCTTTGATTTT CCGGAAAAATGGATTATTCTTAAAATGGATACCGTTATACACTTTATTTTCAGAATGGAC
[SEQ ID NO: 8] pWUR_Cas9spR_hr
GAAAGACCCGTATCCAAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTACGATAGACATATAAAGAGA GCGGCCATACAGGCCGCCTCTTTTCTGTTCTTGGCGACTCCCCGTCTAGTGGATCACTCA GC A ACTCCTC AG CTG C AG C AG CTG CCTCG AGTG C AA AA AC AGCCCGC AG ATC AAC ATCCG CGGGCTGTTTCTGATTATAAGAAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGT GAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAG CCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTT TCCAGTCGGGAAACCTGTCGTGCCAGCCCTTCAAACTTCCCAAAGGCGAGCCCTAGTGAC ATTAGAAAACCGACTGTAAAAAGTACAGTCGGCATTATCTCATATTATAAAAGCCAGTCA TTAGGCCTATCTGACAATTCCTGAATAGAGTTCATAAACAATCCTGCATGATAACCATCA CAAACAGAATGATGTACCTGTAAAGATAGCGGTAAATATATTGAATTACCTTTATTAATG AATTTTCCTGCTGTAATAATGGGTAGAAGGTAATTACTATTATTATTGATATTTAAGTTA AACCCAGTAAATGAAGTCCATGGAATAATAGAAAGAGAAAAAGCATTTTCAGGTATAGGT GTTTTGGGAAACAATTTCCCCGAACCATTATATTTCTCTACATCAGAAAGGTATAAATCA TAAAACTCTTTGAAGTCATTCTTTACAGGAGTCCAAATACCAGAGAATGTTTTAGATACA CCATCAAAAATTGTATAAAGTGGCTCTAACTTATCCCAATAACCTAACTCTCCGTCGCTA TTGTAACCAGTTCTAAAAGCTGTATTTGAGTTTATCACCCTTGTCACTAAGAAAATAAAT GCAGGGTAAAATTTATATCCTTCTTGTTTTATGTTTCGGTATAAAACACTAATATCAATT TCTGTGGTTATACTAAAAGTCGTTTGTTGGTTCAAATAATGATTAAATATCTCTTTTCTC TTCCAATTGTCTAAATCAATTTTATTAAAGTTCATTTGATATGCCTCCTAAA I I I I I ATC TAAAGTGAATTTAGGAGGCTTACTTGTCTGCTTTCTTCATTAGAATCAATCC I I I I I I AA AAGTCAATCCCGTTTGTTGAACTACTCTTTAATAAAATAA I I I I I CCGTTCCCAATTCCA CATTGCAATAATAGAAAATCCATCTTCATCGGC I I I I I CGTCATCATCTGTATGAATCAA ATCGCCTTCTTCTGTGTCATCAAGGTTTAA I I I I I I ATGTATTTCTTTTAACAAACCACC ATAGGAGATTAACCTTTTACGGTGTAAACCTTCCTCCAAATCAGACAAACGTTTCAAATT CTTTTCTTCATCATCGGTCATAAAATCCGTATCCTTTACAGGATATTTTGCAGTTTCGTC AATTGCCGATTGTATATCCGATTTATATTTA I I I I I CGGTCGAATCATTTGAACTTTTAC ATTTGGATCATAGTCTAATTTCATTGCC I I I I I CCAAAATTGAATCCATTG I I I I I GATT CACGTAGTTTTCTGTATTCTTAAAATAAGTTGGTTCCACACATACCAATACATGCATGTG CTGATTATAAGAATTATCTTTATTATTTATTGTCACTTCCGTTGCACGCATAAAACCAAC AAGA I I I I I ATTAA I I I I I I I ATATTGCATCATTCGGCGAAATCCTTGAGCCATATCTGA CAAACTCTTATTTAATTCTTCGCCATCATAAACA I I I I I AACTGTTAATGTGAGAAACAA CCAACGAACTGTTGGCTTTTGTTTAATAACTTCAGCAACAACCTTTTGTGACTGAATGCC ATGTTTCATTGCTCTCCTCCAGTTGCACATTGGACAAAGCCTGGATTTACAAAACCACAC TCGATACAACTTTCTTTCGCCTGTTTCACGATTTTGTTTATACTCTAATATTTCAGCACA ATCTTTTACTCTTTCAGCC I I I I I AAATTCAAGAATATGCAGAAGTTCAAAGTAATCAAC ATTAGCGATTTTCTTTTCTCTCCATGGTCTCACTTTTCCAC I I I I I GTCTTGTCCACTAA AACCCTTGA I I I I I CATCTGAATAAATGCTACTATTAGGACACATAATATTAAAAGAAAC CCCCATCTATTTAGTTATTTGTTTGGTCACTTATAACTTTAACAGATGGGG I I I I I CTGT GCAACCAATTTTAAGGGTTTTCAATACTTTAAAACACATACATACCAACACTTCAACGCA CCTTTCAGCAACTAAAATAAAAATGACGTTATTTCTATATGTATCAAGAATAGAAAGAAC TCG I I I I I CG CTACGCTC A AA ACG C AA AA AA AG C ACTC ATTCG AGTGC I I I I I CTTATCG CTCCAAATCATGCGA I I I I I I CCTCTTTGCTTTTCTTTGCTCACGAAGTTCTCGATCACG CTGCAAAACATCTTGAAGCGAAAAAGTATTCTTCTTTTCTTCCGATCGCTCATGCTGACG CACGAAAAGCCCTCTAGGCGCATAGGAACAACTCCTAAATGCATGTGAGGGGTTTTCTCG TCCATGTGAACAGTCGCATACGCAATATTTTGTTTCCCATACTGCATTAATGAATCGGCC AACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACT CGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATAC GGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAA AGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCG I I I I I CCATAGGCTCCGCCCCCCTG ACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAA GATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGC TTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCAC GCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAAC CCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGG TAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGT ATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAA CAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCT CTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGG I I I I I I I GTTTGCAAGCAGCAGA TTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACG CTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATATAAGCAATACTGTT TTTCTAATCCATTAGTAAGAAAACAATTTATTAAAATTGCTACTGGTGCTACCGTTTACG GTATTTCGAAAACTAGTATAAAACAAATTAAGATTCCTGTGCCTCCAATTGAAGAACAAA GAACAATTGCAAAGACATTAAAGGGATTGGACAACAAAATTCAGAATGAAAAAGAGAAGT TAGAACCTTTAATTTATATAAAGAAGGGCCTCATGCAAATCCTTCTTACAGGCAAAGTCC GTGTCAAAGTCGAGGATGAGGTGATGTCACAGTGACCGTCGCAAGTGAATGGAATGAGTT AAATCTCGTGGAAAAACGTTTAATCCATCAGCTTCAAGAGTTGGGCTATGAACACGTCAC AGGCTCAGCTCTTGAGTCTGAACGTGATTCGTTGAGCGAAGTCATTTTGAAGGAGCGGTT AAAGAAGGCGATTCAGCGGTTAAATCCATGGATTGATGAGAACAACCTTGCCCGCATTAT TTACAACCTTACTCACATTGAAGCAACAGGCCTCATGCAAGCGAATGAAAAGTTTCATGA GATGCTTGTTAACTATATTTCAATTCAACAAGATTTAGGAAAAGGGAAAAAGAATCAAAC AGTTAAACTGATTGATTTTGATAACCCTTATAACAATGAATTTCTTGTTGTGGACCAGTT CCGCATTCAAGATGCAAGAGGCACGATTCGCCCTGACGTTGTGA I I I I I GTGAATGGATT GCCTTTAGTCGTCGTCGAATGCAAAAGCCCAACGTTGCAGGAAGATACGCAAATTTCAAA AGCAGTCAATCAATTGCGCCGCTATCAGGAAACTCATGAGCAACTTTTCTACTATAACCA GTTTATGATTGCGACAAGCAATTATCGTGCAAAAGCGGGAACGATTGGTGCAAAAGTACA ACATTAGATATCCTAACGGTTCCTGAACTCGGTGACTCACCTACGAAACAAGACATCTTA GTTGCGGGGATGTTAGCGAAAGAAAACTTGCTTGACTTGGTTCAAAACTTCATTGTCTAT GAAGTCGACGGCGGACGGGTGATTAAAAAGATTGCTCGTTATCAACAATTCCGTGCCGTT AATAAAGCGATTCACCGCATTTTACATGCAAAAACGCCGAAGGACCGCGGCGGGGTGATT TGGCATACACAAGGTTCAGGTAAGTCTTTAACGATGTTATATTTAGCTGTAAAACTTAGA AGAATCAAAGAGTTGCAAAACCCAACGATTGTCGTTGTGACCGACCGTAAAGACTTGGAT GACCAAATTACAAGAACATTCCGTAAGTGTGGTTTCCCGAATCCGCAACAAGCGGAAAGT GTCAAAGATCTGAAAAAGCTGTTACAACAAGGTCCTGGTTCTACCATTATGACCCTTGTG CAAAAGTTTCAGCCAGACAAGGAACAAGATGAGTATCCAGAACTTTCAAGGTCAGAAAAT A I I I I I GTATTAGTCGACGAATCCCACCGCACGCAATATAAAGGTCTAGCGATGAACATG CGGAAAGGATTGCCGAATGCATGTTACATCGGTTTTACTGGAACGCCAATCGATAAAGAA GATAAAAGTACAACAAGAACGTTCGGCTCTTATATTGACAAATATACGATTGAACAAGCG GTAGATGACGGGGCAACGGTTCCTA I I I I I I ATGAAGCGCGAATGGTTAACCTGCATTTG CACGGCGATTCTTTAGACCGATTATTTGCCCGTAAATTTAGTGAGTATAGCGAGGAAGAC CGTGAGCGGATTAAAAAGAAATATGCAAAGGAAGAAGCGATTTTAGCCGCTCCAAGTCGG ATTGAACAAATTGCCTTAGATATTGTGGAACACTTTGAAAATTATATTTTGCCAAATGGT TTTAAAGCACAAGTAGTGGCAGTTACTCGTGAAGCAGCGGCCATGGCGCTAGTGAGCTGA GGATAATATTTTCTATCATATTTCCACTTAGTGTTCCAGTATTAGCGACAATAACGCTTT TTGTTGCAGTTGGACAATGGAATTCGTGGTTTGATACAATGC I I I I I I GTTCCATGAATC CAAATTTAAGCACATTACAGTATGAATTGCAAAAAGTATTGCAGTCGACTCAACAGTTTA CACAACAAGCGTCTTTTGATATGGGGTTAAGAAATAATTCTAATTCAGTTACCCCGGAAA GCATAAGAGCTGCTATGACGGTAGTGGCAGTGGTTCCTATTATGATGGTTTATCCATTCT TGCAGAAGTATTTTGTAAAGGGTTTAACCATTGGCAGTGTTAAGGGTTAGTACATCTTTG G I I I I I I ATAAATGCTGAAAGGGGGGAAATTTAAAAATACTTATTAGACAAAA I I I I I I A ATTGCGAGGTGATAGTAAATAAA I I I I I CAATAAATTAAATTTACTGAGGGAGGAATCTA GAATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGG TGATCACTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACC GCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGACAGCGG AAGCGACTCGTCTAAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTT GTTATCTACAGGAGA I I I I I I CAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATC GACTTGAAGAGTC I I I I I I GGTGGAAGAAGACAAGAAGCATGAACGTCATCCTA M I N G GAAATATAGTAGATGAAGTTGCTTATCACGAGAAATATCCAACTATCTATCATCTGCGAA AAAAATTGGTAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGC ATATGATTAAGTTTCGTGGTCA I I I I I I GATTGAGGGAGATTTAAATCCTGATAATAGTG ATGTGGACAAACTATTTATCCAGTTGGTACAAACCTACAATCAATTATTTGAAGAAAACC CTATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAA GACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGA ATCTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAG AAGATGCTAAATTACAGCTTTCAAAAGATACTTACGATGATGATTTAGATAATTTATTGG CGCAAATTGGAGATCAATATGCTGATTTG I I I I I GGCAGCTAAGAATTTATCAGATGCTA TTTTACTTTCAGATATCCTAAGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTT CAATGATTAAACGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCATTAGTTC GACAACAACTTCCAGAAAAGTATAAAGAAATC I I I I I I G ATC A ATC A AA AA ACG G ATATG CAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTT TAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGC GCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGC ATGCTATTTTGAGAAGACAAGAAGACTTTTATCCA I I I I I AAAAGACAATCGTGAGAAGA TTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAATA GTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAG AAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATA AAAATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGG TTTATAACGAATTGACAAAGGTCAAATATGTTACTGAAGGAATGCGAAAACCAGCATTTC TTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAA CCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAA TTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGTACCTACCATGATTTGCTAAAAA TTATTAAAGATAAAGA I I I I I I GGATAATGAAGAAAATGAAGATATCTTAGAGGATATTG TTTTAACATTGACCTTATTTGAAGATAGGGAGATGATTGAGGAAAGACTTAAAACATATG CTCACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGG GACGTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATAT TAGA I I I I I I GAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATG ATAGTTTGACATTTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTT TACATGAACATATTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGA CTGTAAAAGTTGTTGATGAATTGGTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATCG TTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGC GTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATC CTGTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTCCAAAATGGAA GAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAAGTGATTATGATGTCGATC ACATTGTTCCACAAAGTTTCCTTAAAGACGATTCAATAGACAATAAGGTCTTAACGCGTT CTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGA AAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATT TAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCC AATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGA ATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAAT CTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACA ATTACCATCATGCCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGA AATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTA AAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACT CTAATATAATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAAC GCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATT TTG CC AC AGTGCG C AA AGTATTGTCC ATG CCCC AAGTC AATATTGTC A AG A AA AC AG A AG TACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAACTTA TTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAG CTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCG TTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTG AC I I I I I AGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTA AATATAGTC I I I I I GAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAAT TACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAA I I I I I I ATATTTAGCTA GTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGG AGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTG TTATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACA AACCAATACGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAATCTTGGAG CTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAACGATATACGTCTACAA AAGAAGTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCA TTGATTTGAGTCAGCTAGGAGGTGACTGAATCAGACAAAATGGCCTGCTTATGAAGCGGG CCA I I I I I GTTTAATCCTGTCGACTTGCCGGAATTCTTTCACAGCCGCATCCCTTTCTTC CGCAGGCAGCGCCTTCCAGCCCGCCCAGTCGAGAGAACGGAAATCATGCAGTGAGTACCA GCCATCTAGTGTTTCTGCTGCTTCGCTCA I I I I I CTCCCTCCAAATG I I I I I CTCTTCCC TTACTATAACACAGTTTAGAAGAGGGAGAAAATATTACGGCTGCCGCGAAATTGTCTAAA AG AAGCCTTCTTTTG CCTTTG ATTTTCCG G A AA AATG G ATTATTCTTAA AATG G ATACCG TTATAC
[SEQ ID NO: 9] pWUR_Cas9spKI_hrl
TTATGTAAACGACCATACAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTACGATAGACATATAAAGAGA GCGGCCATACAGGCCGCCTCTTTTCTGTTCTTGGCGACTCCCCGTCTAGTGGATCACTCA GC A ACTCCTC AG CTG C AG C AG CTG CCTCG AGTG C AA AA AC AGCCCGC AG ATC AAC ATCCG CGGGCTGTTTCTGATTATAAGAAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGT GAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAG CCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTT TCCAGTCGGGAAACCTGTCGTGCCAGCCCTTCAAACTTCCCAAAGGCGAGCCCTAGTGAC ATTAGAAAACCGACTGTAAAAAGTACAGTCGGCATTATCTCATATTATAAAAGCCAGTCA TTAGGCCTATCTGACAATTCCTGAATAGAGTTCATAAACAATCCTGCATGATAACCATCA CAAACAGAATGATGTACCTGTAAAGATAGCGGTAAATATATTGAATTACCTTTATTAATG AATTTTCCTGCTGTAATAATGGGTAGAAGGTAATTACTATTATTATTGATATTTAAGTTA AACCCAGTAAATGAAGTCCATGGAATAATAGAAAGAGAAAAAGCATTTTCAGGTATAGGT GTTTTGGGAAACAATTTCCCCGAACCATTATATTTCTCTACATCAGAAAGGTATAAATCA TAAAACTCTTTGAAGTCATTCTTTACAGGAGTCCAAATACCAGAGAATGTTTTAGATACA CCATCAAAAATTGTATAAAGTGGCTCTAACTTATCCCAATAACCTAACTCTCCGTCGCTA TTGTAACCAGTTCTAAAAGCTGTATTTGAGTTTATCACCCTTGTCACTAAGAAAATAAAT GCAGGGTAAAATTTATATCCTTCTTGTTTTATGTTTCGGTATAAAACACTAATATCAATT TCTGTGGTTATACTAAAAGTCGTTTGTTGGTTCAAATAATGATTAAATATCTCTTTTCTC TTCCAATTGTCTAAATCAATTTTATTAAAGTTCATTTGATATGCCTCCTAAA I I I I I ATC TAAAGTGAATTTAGGAGGCTTACTTGTCTGCTTTCTTCATTAGAATCAATCC I I I I I I AA AAGTCAATCCCGTTTGTTGAACTACTCTTTAATAAAATAA I I I I I CCGTTCCCAATTCCA CATTGCAATAATAGAAAATCCATCTTCATCGGC I I I I I CGTCATCATCTGTATGAATCAA ATCGCCTTCTTCTGTGTCATCAAGGTTTAA I I I I I I ATGTATTTCTTTTAACAAACCACC ATAGGAGATTAACCTTTTACGGTGTAAACCTTCCTCCAAATCAGACAAACGTTTCAAATT CTTTTCTTC ATC ATCG GTC ATAA AATCCGTATCCTTTAC AG G ATATTTTG C AGTTTCGTC AATTGCCGATTGTATATCCGATTTATATTTA I I I I I CGGTCGAATCATTTGAACTTTTAC ATTTGGATCATAGTCTAATTTCATTGCC I I I I I CCAAAATTGAATCCATTG I I I I I GATT CACGTAGTTTTCTGTATTCTTAAAATAAGTTGGTTCCACACATACCAATACATGCATGTG CTGATTATAAGAATTATCTTTATTATTTATTGTCACTTCCGTTGCACGCATAAAACCAAC AAGA I I I I I ATTAA I I I I I I I ATATTGCATCATTCGGCGAAATCCTTGAGCCATATCTGA CAAACTCTTATTTAATTCTTCGCCATCATAAACA I I I I I AACTGTTAATGTGAGAAACAA CCAACGAACTGTTGGCTTTTGTTTAATAACTTCAGCAACAACCTTTTGTGACTGAATGCC ATGTTTCATTGCTCTCCTCCAGTTGCACATTGGACAAAGCCTGGATTTACAAAACCACAC TCGATACAACTTTCTTTCGCCTGTTTCACGATTTTGTTTATACTCTAATATTTCAGCACA ATCTTTTACTCTTTCAGCC I I I I I AAATTCAAGAATATGCAGAAGTTCAAAGTAATCAAC ATTAGCGATTTTCTTTTCTCTCCATGGTCTCACTTTTCCAC I I I I I GTCTTGTCCACTAA AACCCTTGA I I I I I CATCTGAATAAATGCTACTATTAGGACACATAATATTAAAAGAAAC CCCCATCTATTTAGTTATTTGTTTGGTCACTTATAACTTTAACAGATGGGG I I I I I CTGT GCAACCAATTTTAAGGGTTTTCAATACTTTAAAACACATACATACCAACACTTCAACGCA CCTTTCAGCAACTAAAATAAAAATGACGTTATTTCTATATGTATCAAGAATAGAAAGAAC TCG I I I I I CG CTACGCTC A AA ACG C AA AA AA AG C ACTC ATTCG AGTGC I I I I I CTTATCG CTCC AA ATC ATG CG A I I I I I I CCTCTTTGCTTTTCTTTGCTCACGAAGTTCTCGATCACG CTGCAAAACATCTTGAAGCGAAAAAGTATTCTTCTTTTCTTCCGATCGCTCATGCTGACG CACGAAAAGCCCTCTAGGCGCATAGGAACAACTCCTAAATGCATGTGAGGGGTTTTCTCG TCCATGTGAACAGTCGCATACGCAATATTTTGTTTCCCATACTGCATTAATGAATCGGCC AACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACT CGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATAC GGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAA AGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCG I I I I I CCATAGGCTCCGCCCCCCTG ACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAA GATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGC TTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCAC GCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAAC CCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGG TAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGT ATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAA CAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCT CTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGG I I I I I I I GTTTGCAAGCAGCAGA TTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACG CTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGATAAAGCTGGAAGGATGCC GAAATAAGTTAAGATATCGAAGAAGGCAACAATAAAAATAATCGGCATTAATACGCTCAT GAAAAAGGACGGTGTACCATTTAAAGATACTAGGTCACCAAAGACGAAATTGATACCGGA CATAGAGCAATGGATAAGCCATAAGAAAAACTTTGAAACGATTTGGATGAATTGTCCACC TATTTTAGTGGTTAACATAAACCAAGTAATAACCAATTGAATAACAAACATCAAAAGGAT AGAGACAAAATCTATGCCTTTACGATTGATAGAGCATAAATAGGCAATAGCAAAAACAAC GATAATTCCAATGATGTTAATGATAAAAGACAATGAAATTCCTCCCTGAACCTACGCACA TTTGATTAATTAAGCGTTCTTATGTTGTCCGGTC I I I I I I I CGTTTATACTTTTCGAGCG TATTAGTGAATCGAATTTAAAAACATACAAAAATCGACGTCAAAATCCCCCGCAACGACG AAATAAAATTCTAAAAATAATAAATAGTCGTTTCAAATTAAATTTTAAATGCAGGGTGGG GAATATGCAAGATCAAATAAGTGTAAAAAATGCGCATTTTCAAGGGGATTATTGACAGTT CAAAAAGAAG I I I I I AA I I I I I AAAATTTTAAAATTATAGTGAACATAGAAAAAAACTAA AGTAGAATCTATATAATGTCAAAAAATTCCGGTTTCATTCATTCAATCAACATATTATTT
CTTCATACATGGAACCATTTCGGCGTCCAGAAAAACGATGCGGAAATGCAACTAAAATTC GAATAATAACGATCGTTTTGATTAAATCGAGTTTGAATACCCCTTGCCCTCTCAAATGAG I I I I I GCATGGTTTTGTCTTCTCAGAATCATGGTTTATGATAGAACGAAACTGGAAATAA ACTTCTGCAGCTCGCCAAAAGACAGGGCATATATAAAAAGGGAGCGGAATAAGCATTTAA CTTAAAACGCTCCCTTTCATACCAGCATTAAGAAAGTACTTTATTCATCGTTTCTTTCAA AACAGCAGCTGAGTGGTTGAA I I I I I C I I I I I CTTCTTCATTTAATTTCATTTCGATGAT TTCACGAACCCCATTTCGGTTAACGACAGCAGGTGCACCGATGTAAAGATCTTCTTGACC GTATTCTCCTTCAAGCAGAACGGATACAGTCAGAATGCTGTTTTCATTATGAAGGATCGC TTTAGTGATGCGGACAAGAGACATTCCGATTCCGTAATAGGTGGCTCCTTTTCTTTCGAT GATATGGTATGCAGCATCCCGTACGTTTACAAAGATCTCATCCAGATCTGCTCTAGTGAT TTCACCC I I I I I GATGAAGTAATCGAGTGGTTCAATACCAATAGTAGCATGGCTCCAAAC CGGAAGTTCTGTATCGCCGTGTTCACCAATAACAGCTGCGTGAACATTTCTTGGATCGAT ATTTAAACGTTCTCCAAGTTGAAAGCGCAGCCGGGCTGAGTCGAGTACAGTGCCGGAACC GATGACACGTTCTTTAGGAAGTCCTGATTCTTTCCAGGTCACATAAGTAAGGATGTCGAC TGGATTTGTAGCTACTAGGAAAATTCCGTCAAATCCGCTGTTCATAATATTTCGAATTAT CTCTTTGAAAATCTTAGTG I I I I I I I CGACTAATTGCAGACGGGTTTCTCCCGG M I N G TGCTAATCCGGCTGTTATGACAACTAAATCAGCTTGTCTACAATCTTTATAATCTCCATT CCAAA I I I I I GCAGGAGAAGGGGCGAACGGTATTCCGTGATTAAGGTCCATGGCTTCGCC TTCCGCTTTTGCTTTGTTGACATCTATCAATACTATTTCTTCAGCTACACCTTGATTGAT TAGGGAGTAAGCATAGCTGCAACCTACTGCACCAGTTCCAACGACTACAACACGATTTAC I I I I I C I I I I I I CATAATAAGAAACTCCTTTCGTCATTTCTGGTTGATTGCAAAATGTGT GAGAATATAACCTAACCCATATGTAAAAAATGGTTTACATAGGGTAAGTCTACAAATTTA TGTACTATTAGAATATAATTTCCTGTTAGCAGTAAATGTTATGCCAACATTCATTAAAAG ATGCTGCAGCCAAAGACACACGTGAAAGGGTACGGTTCTTCATAAAACGTTGATAATTTA GAAGGAAAAGGAATGGCTGCAGCGATTTACAAGTTCATTATACAACACTAAATGTGAATT TATTCACAATATAAATGAATAATTTGTGAAC I I I I I CACACACTGATTTATTTTA I I I I I GGATGAAACCA I I I I I I I ATAGAAACAGGATGAATCGGCGTTTTCTTGCAAAAAAACGGG CGAATGGGAAATAGGGCCAACATGTTTTAAAGTCCATACAAAAACCCCCTATAGTCATTT AGGTATCATTCGTTTCTTTCATTTCCATTGCCTATAAGGGGATCTAATCAAATGACCAAT TTATTC I I I I I I ATCGTTAAAATCATGATCTTCCGAAAACTCGGCGTTATATGCCAGTTT ATGATTGGCTCTCA I I I I I GTCCGCCTGTTCGTTTGAACGTCCGGAAAACGATTCAGAAT AAATTGATGAATAAACGCTTCTGCTATTGTGATGATAACGGCTGAAATAATGCTTCCCCA AGCAATCTGCATATAGTTATTAAGAAGAATACTGCCAAAAATCCAAACGCTCGTATAGGC AAGAAGAAAATCAACTATTGATGCAGACGTATTTCCAAAATAGGGAAGAATCAACCGATC TCCTATTAAAAAAGACATAATCGTCGTCAGTAAGCTGAAAGAAAAAATATCCAAATAGGT AGCATCAAAAAATAGATCGAGACCTACTGCAAATGCGATTAGGCAAGCAATAAATTTTAT AAGTAAAATGCCAGCGTATTTCATCATATGGCGCTAGTGAGCTGAGGATAATATTTTCTA TCATATTTCCACTTAGTGTTCCAGTATTAGCGACAATAACGC I I I I I GTTGCAGTTGGAC AATGGAATTCGTGGTTTGATACAATGC I I I I I I GTTCC ATG AATCC AA ATTTA AG C AC AT TACAGTATGAATTGCAAAAAGTATTGCAGTCGACTCAACAGTTTACACAACAAGCGTCTT TTGATATGGGGTTAAGAAATAATTCTAATTCAGTTACCCCGGAAAGCATAAGAGCTGCTA TGACGGTAGTGGCAGTGGTTCCTATTATGATGGTTTATCCATTCTTGCAGAAGTATTTTG TAA AG GGTTTA ACC ATTG GC AGTGTTA AG GGTTAGTAC ATCTTTG G I I I I I I ATA A ATG C TGAAAGGGGGGAAATTTAAAAATACTTATTAGACAAAA I I I I I I AATTGCGAGGTGATAG TAAATAAA I I I I I CAATAAATTAAATTTACTGAGGGAGGAATCTAGAATGGATAAGAAAT ACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGAAT ATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAA AAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGACAGCGGAAGCGACTCGTCTAA AACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGGAGA I I I I I I CAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTT TTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCCTA I I I I I GGAAATATAGTAGATG AAGTTGCTTATCACGAGAAATATCCAACTATCTATCATCTGCGAAAAAAATTGGTAGATT CTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTC GTGGTCA I I I I I I GATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTAT TTATCCAGTTGGTACAAACCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTG GAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATC TCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGAATCTCATTGCTTTGT CATTGGGTTTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTAC AGCTTTCAAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATC AATATGCTGATTTG I I I I I GGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATA TCCTAAGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAACGCT ACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCATTAGTTCGACAACAACTTCCAG AAAAGTATAAAGAAATC I I I I I I GATCAATCAAAAAACGGATATGCAGGTTATATTGATG GGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGATG GTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCT TTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAA GACAAGAAGACTTTTATCCA I I I I I AAAAGACAATCGTGAGAAGATTGAAAAAATCTTGA CTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGA TGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGATAAAG GTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATG AAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGA CAAAGGTCAAATATGTTACTGAAGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGA AGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAA AAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAG ATAGATTTAATGCTTCATTAGGTACCTACCATGATTTGCTAAAAATTATTAAAGATAAAG A I I I I I I GGATAATGAAGAAAATGAAGATATCTTAGAGGATATTGTTTTAACATTGACCT TATTTGAAGATAGGGAGATGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGATG ATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAA AATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGA I I I I I I GAAAT CAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGATAGTTTGACATTTA AAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTACATGAACATATTG CAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAGTTGTTG ATGAATTGGTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGCAC GTGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCG AAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTC A ATTG C AA A ATG A AA AG CTCTATCTCTATTATCTCC AA AATG G AAG AG AC ATGTATGTGG ACCAAGAATTAGATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAA GTTTCCTTAAAGACGATTCAATAGACAATAAGGTCTTAACGCGTTCTGATAAAAATCGTG GTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGAC AACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAAC GTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTC GCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACTAAATACGATG AAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTG ACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCC ATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTG AATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGT CTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATAATGAACT TCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAA CTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCA AAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGAT TCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAACTTATTGCTCGTAAAAAAG ACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAG TGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAG GGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTGAC I I I I I AGAAGCTA AAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAATATAGTC M I N G AGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATG AGCTGGCTCTGCCAAGCAAATATGTGAA I I I I I I ATATTTAGCTAGTCATTATGAAAAGT TGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATT ATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATG CCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAAC AAGCAGAAAATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTA AATATTTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGATG CCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGC TAGGAGGTGACTGAATCAGACAAAATGGCCTGCTTATGAAGCGGGCCA I I I I I GTTTAAT CCTGTCGACTTGCCGGAATTCTTTCACAGCCGCATCCCTTTCTTCCGCAGGCAGCGCCTT CCAGCCCGCCCAGTCGAGAGAACGGAAATCATGCAGTGAGTACCAGCCATCTAGTGTTTC TGCTGCTTCGCTCA I I I I I CTCCCTCCAAATG I I I I I CTCTTCCCTTACTATAACACAGT TTAGAAGAGGGAGAAAATATTACGGCTGCCGCGAAATTGTCTAAAAGAAGCCTTCTTTTG CCTTTGATTTTCCGGAAAAATGGATTATTCTTAAAATGGATACCGTTATAC
[SEQ ID NO: 10] pWUR_Cas9spKI_hr2 TTATGTAAACGACCATACAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTACGATAGACATATAAAGAGA GCGGCCATACAGGCCGCCTCTTTTCTGTTCTTGGCGACTCCCCGTCTAGTGGATCACTCA GC A ACTCCTC AG CTG C AG C AG CTG CCTCG AGTG C AA AA AC AGCCCGC AG ATC AAC ATCCG CGGGCTGTTTCTGATTATAAGAAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGT GAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAG CCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTT TCCAGTCGGGAAACCTGTCGTGCCAGCCCTTCAAACTTCCCAAAGGCGAGCCCTAGTGAC ATTAGAAAACCGACTGTAAAAAGTACAGTCGGCATTATCTCATATTATAAAAGCCAGTCA TTAGGCCTATCTGACAATTCCTGAATAGAGTTCATAAACAATCCTGCATGATAACCATCA CAAACAGAATGATGTACCTGTAAAGATAGCGGTAAATATATTGAATTACCTTTATTAATG AATTTTCCTGCTGTAATAATGGGTAGAAGGTAATTACTATTATTATTGATATTTAAGTTA AACCCAGTAAATGAAGTCCATGGAATAATAGAAAGAGAAAAAGCATTTTCAGGTATAGGT GTTTTGGGAAACAATTTCCCCGAACCATTATATTTCTCTACATCAGAAAGGTATAAATCA TAAAACTCTTTGAAGTCATTCTTTACAGGAGTCCAAATACCAGAGAATGTTTTAGATACA CCATCAAAAATTGTATAAAGTGGCTCTAACTTATCCCAATAACCTAACTCTCCGTCGCTA TTGTAACCAGTTCTAAAAGCTGTATTTGAGTTTATCACCCTTGTCACTAAGAAAATAAAT GCAGGGTAAAATTTATATCCTTCTTGTTTTATGTTTCGGTATAAAACACTAATATCAATT TCTGTGGTTATACTAAAAGTCGTTTGTTGGTTCAAATAATGATTAAATATCTCTTTTCTC TTCCAATTGTCTAAATCAATTTTATTAAAGTTCATTTGATATGCCTCCTAAA I I I I I ATC TAAAGTGAATTTAGGAGGCTTACTTGTCTGCTTTCTTCATTAGAATCAATCC I I I I I I AA AAGTCAATCCCGTTTGTTGAACTACTCTTTAATAAAATAA I I I I I CCGTTCCCAATTCCA CATTGCAATAATAGAAAATCCATCTTCATCGGC I I I I I CGTCATCATCTGTATGAATCAA ATCGCCTTCTTCTGTGTCATCAAGGTTTAA I I I I I I ATGTATTTCTTTTAACAAACCACC ATAGGAGATTAACCTTTTACGGTGTAAACCTTCCTCCAAATCAGACAAACGTTTCAAATT CTTTTCTTCATCATCGGTCATAAAATCCGTATCCTTTACAGGATATTTTGCAGTTTCGTC AATTGCCGATTGTATATCCGATTTATATTTA I I I I I CGGTCGAATCATTTGAACTTTTAC ATTTGGATCATAGTCTAATTTCATTGCC I I I I I CCAAAATTGAATCCATTG I I I I I GATT CACGTAGTTTTCTGTATTCTTAAAATAAGTTGGTTCCACACATACCAATACATGCATGTG CTGATTATAAGAATTATCTTTATTATTTATTGTCACTTCCGTTGCACGCATAAAACCAAC AAGA I I I I I ATTAA I I I I I I I ATATTGCATCATTCGGCGAAATCCTTGAGCCATATCTGA C A AACTCTTATTTA ATTCTTCG CC ATC ATA AAC A I I I I I AACTGTTAATGTGAGAAACAA CCAACGAACTGTTGGCTTTTGTTTAATAACTTCAGCAACAACCTTTTGTGACTGAATGCC ATGTTTCATTGCTCTCCTCCAGTTGCACATTGGACAAAGCCTGGATTTACAAAACCACAC TCGATACAACTTTCTTTCGCCTGTTTCACGATTTTGTTTATACTCTAATATTTCAGCACA ATCTTTTACTCTTTCAGCC I I I I I AAATTCAAGAATATGCAGAAGTTCAAAGTAATCAAC ATTAGCGATTTTCTTTTCTCTCCATGGTCTCACTTTTCCAC I I I I I GTCTTGTCCACTAA AACCCTTGA I I I I I CATCTGAATAAATGCTACTATTAGGACACATAATATTAAAAGAAAC CCCCATCTATTTAGTTATTTGTTTGGTCACTTATAACTTTAACAGATGGGG I I I I I CTGT GCAACCAATTTTAAGGGTTTTCAATACTTTAAAACACATACATACCAACACTTCAACGCA CCTTTCAGCAACTAAAATAAAAATGACGTTATTTCTATATGTATCAAGAATAGAAAGAAC TCG I I I I I CG CTACGCTC A AA ACG C AA AA AA AG C ACTC ATTCG AGTGC I I I I I CTTATCG CTCC AA ATC ATG CG A I I I I I I CCTCTTTGCTTTTCTTTGCTCACGAAGTTCTCGATCACG CTGCAAAACATCTTGAAGCGAAAAAGTATTCTTCTTTTCTTCCGATCGCTCATGCTGACG CACGAAAAGCCCTCTAGGCGCATAGGAACAACTCCTAAATGCATGTGAGGGGTTTTCTCG TCCATGTGAACAGTCGCATACGCAATATTTTGTTTCCCATACTGCATTAATGAATCGGCC AACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACT CGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATAC GGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAA AGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCG I I I I I CCATAGGCTCCGCCCCCCTG ACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAA GATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGC TTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCAC GCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAAC CCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGG TAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGT ATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAA CAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCT CTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGG I I I I I I I GTTTGCAAGCAGCAGA TTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACG CTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAACATCAAAAGGATAGAGA CAAAATCTATGCCTTTACGATTGATAGAGCATAAATAGGCAATAGCAAAAACAACGATAA TTCCAATGATGTTAATGATAAAAGACAATGAAATTCCTCCCTGAACCTACGCACATTTGA TTAATTAAGCGTTCTTATGTTGTCCGGTC I I I I I I I CGTTTATACTTTTCGAGCGTATTA GTGAATCGAATTTAAAAACATACAAAAATCGACGTCAAAATCCCCCGCAACGACGAAATA AAATTCTAAAAATAATAAATAGTCGTTTCAAATTAAATTTTAAATGCAGGGTGGGGAATA TGCAAGATCAAATAAGTGTAAAAAATGCGCATTTTCAAGGGGATTATTGACAGTTCAAAA AGAAG I I I I I AA I I I I I AAAATTTTAAAATTATAGTGAACATAGAAAAAAACTAAAGTAG AATCTATATAATGTCAAAAAATTCCGGTTTCATTCATTCAATCAACATATTATTTCTTCA TACATGGAACCATTTCGGCGTCCAGAAAAACGATGCGGAAATGCAACTAAAATTCGAATA ATAACGATCGTTTTGATTAAATCGAGTTTGAATACCCCTTGCCCTCTCAAATGAG I I I I I GCATGGTTTTGTCTTCTCAGAATCATGGTTTATGATAGAACGAAACTGGAAATAAACTTC TGCAGCTCGCCAAAAGACAGGGCATATATAAAAAGGGAGCGGAATAAGCATTTAACTTAA AACGCTCCCTTTCATACCAGCATTAAGAAAGTACTTTATTCATCGTTTCTTTCAAAACAG CAGCTGAGTGGTTGAA I I I I I C I I I I I CTTCTTCATTTAATTTCATTTCGATGATTTCAC GAACCCCATTTCGGTTAACGACAGCAGGTGCACCGATGTAAAGATCTTCTTGACCGTATT CTCCTTCAAGCAGAACGGATACAGTCAGAATGCTGTTTTCATTATGAAGGATCGCTTTAG TGATGCGGACAAGAGACATTCCGATTCCGTAATAGGTGGCTCCTTTTCTTTCGATGATAT GGTATGCAGCATCCCGTACGTTTACAAAGATCTCATCCAGATCTGCTCTAGTGATTTCAC CC I I I I I GATGAAGTAATCGAGTGGTTCAATACCAATAGTAGCATGGCTCCAAACCGGAA GTTCTGTATCGCCGTGTTCACCAATAACAGCTGCGTGAACATTTCTTGGATCGATATTTA AACGTTCTCCAAGTTGAAAGCGCAGCCGGGCTGAGTCGAGTACAGTGCCGGAACCGATGA CACGTTCTTTAGGAAGTCCTGATTCTTTCCAGGTCACATAAGTAAGGATGTCGACTGGAT TTGTAGCTACTAGGAAAATTCCGTCAAATCCGCTGTTCATAATATTTCGAATTATCTCTT TGAAAATCTTAGTG I I I I I I I CGACTAATTGCAGACGGGTTTCTCCCGG I I I I I GTGCTA ATCCGGCTGTTATGACAACTAAATCAGCTTGTCTACAATCTTTATAATCTCCATTCCAAA
I I I I I GCAGGAGAAGGGGCGAACGGTATTCCGTGATTAAGGTCCATGGCTTCGCCTTCCG CTTTTGCTTTGTTGACATCTATCAATACTATTTCTTCAGCTACACCTTGATTGATTAGGG AGTAAGCATAGCTGCAACCTACTGCACCAGTTCCAACGACTACAACACGATTTAC I I I I I C I I I I I I CATAATAAGAAACTCCTTTCGTCATTTCTGGTTGATTGCAAAATGTGTGAGAA TATAACCTAACCCATATGTAAAAAATGGTTTACATAGGGTAAGTCTACAAATTTATGTAC TATTAGAATATAATTTCCTGTTAGCAGTAAATGTTATGCCAACATTCATTAAAAGATGCT GCAGCCAAAGACACACGTGAAAGGGTACGGTTCTTCATAAAACGTTGATAATTTAGAAGG AAAAGGAATGGCTGCAGCGATTTACAAGTTCATTATACAACACTAAATGTGAATTTATTC ACAATATAAATGAATAATTTGTGAAC I I I I I CACACACTGATTTATTTTA I I I I I GGATG AAACCA I I I I I I I ATAGAAACAGGATGAATCGGCGTTTTCTTGCAAAAAAACGGGCGAAT GGGAAATAGGGCCAACATGTTTTAAAGTCCATACAAAAACCCCCTATAGTCATTTAGGTA TCATTCGTTTCTTTCATTTCCATTGCCTATAAGGGGATCTAATCAAATGACCAATTTATT C I I I I I I ATCGTTAAAATCATGATCTTCCGAAAACTCGGCGTTATATGCCAGTTTATGAT TGGCTCTCA I I I I I GTCCGCCTGTTCGTTTGAACGTCCGGAAAACGATTCAGAATAAATT GATGAATAAACGCTTCTGCTATTGTGATGATAACGGCTGAAATAATGCTTCCCCAAGCAA TCTGCATATAGTTATTAAGAAGAATACTGCCAAAAATCCAAAATGGCGCTAGTGAGCTGA GGATAATATTTTCTATCATATTTCCACTTAGTGTTCCAGTATTAGCGACAATAACGCTTT TTGTTGCAGTTGGACAATGGAATTCGTGGTTTGATACAATGC I I I I I I GTTCCATGAATC CAAATTTAAGCACATTACAGTATGAATTGCAAAAAGTATTGCAGTCGACTCAACAGTTTA CACAACAAGCGTCTTTTGATATGGGGTTAAGAAATAATTCTAATTCAGTTACCCCGGAAA GCATAAGAGCTGCTATGACGGTAGTGGCAGTGGTTCCTATTATGATGGTTTATCCATTCT TGCAGAAGTATTTTGTAAAGGGTTTAACCATTGGCAGTGTTAAGGGTTAGTACATCTTTG G I I I I I I ATAAATGCTGAAAGGGGGGAAATTTAAAAATACTTATTAGACAAAA I I I I I I A ATTGCGAGGTGATAGTAAATAAA I I I I I CAATAAATTAAATTTACTGAGGGAGGAATCTA GAATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGG TGATCACTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACC GCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGACAGCGG AAGCGACTCGTCTAAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTT GTTATCTACAGGAGA I I I I I I CAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATC GACTTGAAGAGTC I I I I I I GGTGGAAGAAGACAAGAAGCATGAACGTCATCCTA M I N G GAAATATAGTAGATGAAGTTGCTTATCACGAGAAATATCCAACTATCTATCATCTGCGAA AAAAATTGGTAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGC ATATGATTAAGTTTCGTGGTCA I I I I I I GATTGAGGGAGATTTAAATCCTGATAATAGTG ATGTGGACAAACTATTTATCCAGTTGGTACAAACCTACAATCAATTATTTGAAGAAAACC CTATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAA GACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGA ATCTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAG AAGATGCTAAATTACAGCTTTCAAAAGATACTTACGATGATGATTTAGATAATTTATTGG CGCAAATTGGAGATCAATATGCTGATTTG I I I I I GGCAGCTAAGAATTTATCAGATGCTA TTTTACTTTCAGATATCCTAAGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTT CAATGATTAAACGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCATTAGTTC GACAACAACTTCCAGAAAAGTATAAAGAAATC I I I I I I GATCAATCAAAAAACGGATATG CAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTT TAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGC GCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGC ATGCTATTTTGAGAAGACAAGAAGACTTTTATCCA I I I I I AAAAGACAATCGTGAGAAGA TTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAATA GTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAG AAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATA AAAATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGG TTTATAACGAATTGACAAAGGTCAAATATGTTACTGAAGGAATGCGAAAACCAGCATTTC TTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAA CCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAA TTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGTACCTACCATGATTTGCTAAAAA TTATTAAAGATAAAGA I I I I I I GGATAATGAAGAAAATGAAGATATCTTAGAGGATATTG TTTTAACATTGACCTTATTTGAAGATAGGGAGATGATTGAGGAAAGACTTAAAACATATG CTCACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGG GACGTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATAT TAGA I I I I I I GAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATG ATAGTTTGACATTTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTT TACATGAACATATTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGA CTGTAAAAGTTGTTGATGAATTGGTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATCG TTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGC GTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATC CTGTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTCCAAAATGGAA GAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAAGTGATTATGATGTCGATC ACATTGTTCCACAAAGTTTCCTTAAAGACGATTCAATAGACAATAAGGTCTTAACGCGTT CTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGA AAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATT TAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCC AATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGA ATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAAT CTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACA ATTACCATCATGCCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGA AATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTA AAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACT CTAATATAATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAAC GCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATT TTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAG TACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAACTTA TTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAG CTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCG TTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTG AC I I I I I AGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTA AATATAGTC I I I I I GAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAAT TACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAA I I I I I I ATATTTAGCTA GTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGG AGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTG TTATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACA AACCAATACGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAATCTTGGAG CTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAACGATATACGTCTACAA A AG A AGTTTTAG ATG CC ACTCTTATCC ATC A ATCC ATC ACTG GTCTTTATG A AAC ACG C A TTGATTTGAGTCAGCTAGGAGGTGACTGAATCAGACAAAATGGCCTGCTTATGAAGCGGG CCA I I I I I GTTTAATCCTGTCGACTTGCCGGAATTCTTTCACAGCCGCATCCCTTTCTTC CGCAGGCAGCGCCTTCCAGCCCGCCCAGTCGAGAGAACGGAAATCATGCAGTGAGTACCA GCCATCTAGTGTTTCTGCTGCTTCGCTCA I I I I I CTCCCTCCAAATG I I I I I CTCTTCCC
TTACTATAACACAGTTTAGAAGAGGGAGAAAATATTACGGCTGCCGCGAAATTGTCTAAA AG AAGCCTTCTTTTG CCTTTG ATTTTCCG G A AA AATG G ATTATTCTTAA AATG G ATACCG TTATAC

Claims

1 . A method of microbial genome editing, comprising the steps of:
(a) introducing into cells:
(i) at least one guide RNA or at least one polynucleotide comprising a sequence encoding a guide RNA; wherein the or each guide RNA is substantially complementary to a target polynucleotide sequence(s) in a microbial genome;
(ii) a nuclease, or a polynucleotide comprising a sequence encoding the nuclease; wherein the nuclease forms a ribonuclease complex with the guide RNA, and wherein the ribonuclease complex makes site-specific double stranded DNA breaks (DSDB) in the microbial genome;
(iii) a homologous recombination polynucleotide or a polynucleotide comprising a sequence encoding an homologous recombination polynucleotide having a sequence substantially complementary to a target region containing the target in the microbial genome, and having regions upstream and downstream that flank the target;
(b) incubating the cells at a first temperature whereby homologous recombination occurs between the homologous recombination polynucleotide and the target region, wherein the modified microbial genome is resistant to site-specific DSDB by the ribonuclease complex; and
(c) incubating the cells at a second temperature whereby ribonuclease complex mediated site-specific DSDB occurs in unmodified cells;
wherein the first incubation temperature is greater than the second incubation temperature.
2. A method as claimed in claim 1 , further comprising incubating the cells at a third temperature; wherein the third temperature is greater than the second temperature; optionally wherein the third temperature is the same as the first temperature.
3. A method as claimed in claim 1 or claim 2, wherein one or more of (i), (ii) or (iii) are introduced into the cells as part of a polynucleotide vector, e.g. a plasmid.
4. A method as claimed in any of claims 1 to 3, wherein (i), (ii) or (iii) are introduced into the cells substantially separately, simultaneously or sequentially.
5. A method as claimed in any preceding claim, wherein the sequence of the homologous recombination polynucleotide has at least one mis-match with the guide RNA.
6. A method as claimed in any preceding claim, wherein the sequence of the homologous recombination polynucleotide does not include a PAM sequence recognised by the ribonuclease complex.
7. A method as claimed in any preceding claim, wherein the microbes have substantially no endogenous non-homologous end joining repair mechanism.
8. A method as claimed in any preceding claim, wherein the homologous recombination polynucleotide is a single stranded DNA.
9. A method as claimed in claims 1 to 7, wherein the homologous recombination polynucleotide is a double stranded DNA.
10. A method as claimed in claims 1 to 7, wherein the homologous recombination polynucleotide is a plasmid-borne double stranded DNA.
1 1 . A method as claimed in any preceding claim, wherein the homologous recombination polynucleotide further comprises a polynucleotide sequence between the upstream and downstream flanking regions.
12. A method as claimed in any preceding claim, wherein the homologous recombination polynucleotide has a sequence fully complementary to a target region containing the target in the microbial genome, and having regions upstream and downstream that flank the target.
13. A method as claimed in any preceding claim, further comprising at least one homologous recombination polynucleotide or a polynucleotide comprising a sequence encoding a homologous recombination polynucleotide having a sequence substantially complementary to a second target region containing the target in the microbial genome.
14. A method as claimed in any preceding claim, which removes a polynucleotide sequence from the microbial genome.
15. A method as claimed in claims 1 to 13, which inserts one or more gene(s), or fragment thereof, in to the microbial genome.
16. A method as claimed in any claims 1 to 13, which modifies or replaces at least one nucleotide in the microbial genome.
17. A method as claimed in any preceding claim, further comprising at least a second guide RNA or at least one polynucleotide comprising a sequence encoding a guide RNA; wherein the second guide RNA is substantially complementary to a second target polynucleotide sequence(s) in a microbial genome.
18. A method as claimed in any preceding claim, wherein the guide RNA comprises a crRNA and tracrRNA.
19. A method as claimed in claim 18, wherein the guide RNA comprises a single chimeric guide RNA.
20. A method as claimed in claims 1 to 17, wherein the guide RNA comprises a crRNA.
21 . A method as claimed in claims 1 to 19, wherein the nuclease is Cas9, preferably Streptococcus pyogenes Cas9, or C2C1.
22. A method as claimed in claims 1 to 17 or 19 to 20, wherein the nuclease is Cpfl .
23. A method as claimed in claims 1 to 20, wherein the nuclease is C2C3 or Argonaute.
24. A method as claimed in any preceding claim, wherein at the first incubation temperature the ribonuclease complex has substantially no nuclease activity.
25. A method as claimed in any preceding claim, further comprising incubation at one or more intervening temperatures between the first incubation temperature and the second incubation temperature, wherein the or each intervening temperature is lower than the first incubation temperature and higher than the second incubation temperature.
26. A method as claimed in any preceding claim, wherein the first temperature is gradually lowered to the second temperature via a linear gradient.
27. A method as claimed in any preceding claim, wherein the first incubation temperature is between 20 °C and 70 °C, preferably 45 °C to 55 °C.
28. A method as claimed in any preceding claim, wherein the second incubation temperature is between 20 °C and 50 °C, preferably 35 °C to 45 °C.
29. A method as claimed in any preceding claim, wherein the first incubation temperature is higher than 39 °C, preferably higher than 40 °C, more preferably higher than 41 °C, and even more preferably higher than 42 °C.
30. A method as claimed in any preceding claim, wherein the second incubation temperature is lower than 45 °C, preferably lower than 44 °C, more preferably lower than 43 °C, and even more preferably lower than 42 °C.
31 . A method as claimed in any preceding claim, wherein step (c) includes changing the culture medium for fresh culture medium at least once; optionally two or more times.
32. A method as claimed in any preceding claim, wherein the microbes are grown in media comprising TVMY medium, optionally further comprising any carbon source selected from: xylose, uracil and glucose, or any combination thereof.
33. A method as claimed in claims 1 to 31 , wherein the microbes are grown in media comprising LB2 medium, optionally further comprising any carbon source selected from: xylose, uracil and glucose, or any combination thereof.
34. A method as claimed in any preceding claim, wherein the microbes are selected from: bacteria, archaea, fungi and yeast.
35. A method as claimed in any preceding claim, wherein the microbes are mesophilic or thermophilic.
36. A method as claimed in claim 35, wherein the thermophilic microbes are selected from: Thermophilic Bacilli, including Aeribacillus, Alicyclobacillus, Anoxybacillus, Bacillus, Geobacillus, Paenibacillus species. Thermophilic Clostridia, including Anaerobacter, Anaerobacterium, Caldicellulosiruptor, Clostridium, Moorella, Thermoanaerobacter, Thermoanaerobacterium, Thermobrachium, Thermohalobacter species and Thermophilic Lactobacillus species.
37. A method as claimed in claim 35, wherein the mesophilic microbes are selected from:
Bacillus species, Escherichia coli and Lactobacillus species.
38. A selectable replicable plasmid comprising a guide RNA or a polynucleotide comprising a sequence encoding a guide RNA under the control of a first heterologous promoter and with a terminator; a non-codon optimised polynucleotide comprising a sequence encoding a nuclease under the control of a second heterologous promoter with a terminator; and homologous recombination polynucleotide or a polynucleotide comprising a sequence encoding a homologous recombination polynucleotide.
39. A selectable replicable plasmid as claimed in claim 38, wherein the promoters are inducible.
40. A selectable replicable plasmid, as claimed in claim 38 or claim 39, wherein the first heterologous promoter is Bacillus coagulans phosphotransacetylase (pfa) promoter Ppta without its ribosome binding sequence and the terminator is a Rho-independent terminator; and wherein the second promoter is the xynA promoter from
Thermoanaerobacterium saccharolyticum (PxynA) and the terminator is a Rho- independent terminator.
41 . A kit for performing the method as claimed in claims 1 to 37, wherein the kit comprises a plasmid as claimed in claims 38 to 40 and instructions for use.
42. A clonal library obtainable by the method as claimed in any preceding method, wherein the clonal library comprises a plurality of clones harbouring the modified microbial genome that is resistant to site-specific DSDB by the ribonuclease complex.
43. A microbial host cell modified by the methods as claimed in claims 1 to 37.
44. A method for producing a modified microbial host cell with an edited genome, wherein the genome is edited in a method according to any one of claims 1 to 37.
PCT/EP2017/077975 2016-11-02 2017-11-01 Microbial genome editing WO2018083128A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB1618507.6A GB201618507D0 (en) 2016-11-02 2016-11-02 Microbial genome editing
GB1618507.6 2016-11-02

Publications (2)

Publication Number Publication Date
WO2018083128A2 true WO2018083128A2 (en) 2018-05-11
WO2018083128A3 WO2018083128A3 (en) 2019-02-14

Family

ID=57963637

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2017/077975 WO2018083128A2 (en) 2016-11-02 2017-11-01 Microbial genome editing

Country Status (2)

Country Link
GB (1) GB201618507D0 (en)
WO (1) WO2018083128A2 (en)

Cited By (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109337904A (en) * 2018-11-02 2019-02-15 中国科学院动物研究所 Genome editing system and method based on C2c1 nuclease
US10323236B2 (en) 2011-07-22 2019-06-18 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US10465176B2 (en) 2013-12-12 2019-11-05 President And Fellows Of Harvard College Cas variants for gene editing
US10508298B2 (en) 2013-08-09 2019-12-17 President And Fellows Of Harvard College Methods for identifying a target site of a CAS9 nuclease
US10597679B2 (en) 2013-09-06 2020-03-24 President And Fellows Of Harvard College Switchable Cas9 nucleases and uses thereof
CN111235130A (en) * 2019-11-15 2020-06-05 武汉大学 II-type V-type CRISPR protein CeCas12a and application thereof in gene editing
US10682410B2 (en) 2013-09-06 2020-06-16 President And Fellows Of Harvard College Delivery system for functional nucleases
US10704062B2 (en) 2014-07-30 2020-07-07 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
US10858639B2 (en) 2013-09-06 2020-12-08 President And Fellows Of Harvard College CAS9 variants and uses thereof
CN112410234A (en) * 2019-08-21 2021-02-26 江南大学 Visual screening method for multi-target editing recombinant aspergillus strains
US10947530B2 (en) 2016-08-03 2021-03-16 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US11046948B2 (en) 2013-08-22 2021-06-29 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
CN113403294A (en) * 2021-06-04 2021-09-17 广州大学 Fusion protein, base editing tool and application thereof
CN113710802A (en) * 2019-03-08 2021-11-26 齐默尔根公司 Convergent genome editing in microorganisms
US11214780B2 (en) 2015-10-23 2022-01-04 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US11268082B2 (en) 2017-03-23 2022-03-08 President And Fellows Of Harvard College Nucleobase editors comprising nucleic acid programmable DNA binding proteins
WO2022074113A1 (en) 2020-10-08 2022-04-14 Wageningen Universiteit Universal riboswitch for inducible gene expression
US11306324B2 (en) 2016-10-14 2022-04-19 President And Fellows Of Harvard College AAV delivery of nucleobase editors
US11319532B2 (en) 2017-08-30 2022-05-03 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11447770B1 (en) 2019-03-19 2022-09-20 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
WO2022217086A1 (en) 2021-04-09 2022-10-13 Vor Biopharma Inc. Photocleavable guide rnas and methods of use thereof
CN115279898A (en) * 2019-10-23 2022-11-01 成对植物服务股份有限公司 Compositions and methods for RNA templated editing in plants
US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
US11542496B2 (en) 2017-03-10 2023-01-03 President And Fellows Of Harvard College Cytosine to guanine base editor
WO2023283585A2 (en) 2021-07-06 2023-01-12 Vor Biopharma Inc. Inhibitor oligonucleotides and methods of use thereof
US11560566B2 (en) 2017-05-12 2023-01-24 President And Fellows Of Harvard College Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation
WO2023015182A1 (en) 2021-08-02 2023-02-09 Vor Biopharma Inc. Compositions and methods for gene modification
WO2023049926A2 (en) 2021-09-27 2023-03-30 Vor Biopharma Inc. Fusion polypeptides for genetic editing and methods of use thereof
US11661590B2 (en) 2016-08-09 2023-05-30 President And Fellows Of Harvard College Programmable CAS9-recombinase fusion proteins and uses thereof
WO2023102393A1 (en) * 2021-11-30 2023-06-08 Pioneer Hi-Bred International, Inc. High efficiency large scale chromosomal genome manipulation
US11732274B2 (en) 2017-07-28 2023-08-22 President And Fellows Of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE)
CN116656528A (en) * 2023-03-15 2023-08-29 天津科技大学 Pseudomonas and method for preparing deodorizing nitrogen-fixing bacteria agent by utilizing same
US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
US11898179B2 (en) 2017-03-09 2024-02-13 President And Fellows Of Harvard College Suppression of pain by gene editing
US11912985B2 (en) 2020-05-08 2024-02-27 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence
WO2024073751A1 (en) 2022-09-29 2024-04-04 Vor Biopharma Inc. Methods and compositions for gene modification and enrichment

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014093694A1 (en) * 2012-12-12 2014-06-19 The Broad Institute, Inc. Crispr-cas nickase systems, methods and compositions for sequence manipulation in eukaryotes
WO2014143381A1 (en) * 2013-03-09 2014-09-18 Agilent Technologies, Inc. Methods of in vivo engineering of large sequences using multiple crispr/cas selections of recombineering events
CN103981216B (en) * 2014-05-23 2016-06-22 安徽省农业科学院水稻研究所 A kind of key plasmid vector and application
US11339399B2 (en) * 2014-12-31 2022-05-24 Viridos, Inc. Compositions and methods for high efficiency in vivo genome editing
CN105177038B (en) * 2015-09-29 2018-08-24 中国科学院遗传与发育生物学研究所 A kind of CRISPR/Cas9 systems of efficient fixed point editor Plant Genome

Cited By (57)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10323236B2 (en) 2011-07-22 2019-06-18 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US12006520B2 (en) 2011-07-22 2024-06-11 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US10954548B2 (en) 2013-08-09 2021-03-23 President And Fellows Of Harvard College Nuclease profiling system
US11920181B2 (en) 2013-08-09 2024-03-05 President And Fellows Of Harvard College Nuclease profiling system
US10508298B2 (en) 2013-08-09 2019-12-17 President And Fellows Of Harvard College Methods for identifying a target site of a CAS9 nuclease
US11046948B2 (en) 2013-08-22 2021-06-29 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
US10858639B2 (en) 2013-09-06 2020-12-08 President And Fellows Of Harvard College CAS9 variants and uses thereof
US11299755B2 (en) 2013-09-06 2022-04-12 President And Fellows Of Harvard College Switchable CAS9 nucleases and uses thereof
US10597679B2 (en) 2013-09-06 2020-03-24 President And Fellows Of Harvard College Switchable Cas9 nucleases and uses thereof
US10682410B2 (en) 2013-09-06 2020-06-16 President And Fellows Of Harvard College Delivery system for functional nucleases
US10912833B2 (en) 2013-09-06 2021-02-09 President And Fellows Of Harvard College Delivery of negatively charged proteins using cationic lipids
US11124782B2 (en) 2013-12-12 2021-09-21 President And Fellows Of Harvard College Cas variants for gene editing
US10465176B2 (en) 2013-12-12 2019-11-05 President And Fellows Of Harvard College Cas variants for gene editing
US11053481B2 (en) 2013-12-12 2021-07-06 President And Fellows Of Harvard College Fusions of Cas9 domains and nucleic acid-editing domains
US10704062B2 (en) 2014-07-30 2020-07-07 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US11578343B2 (en) 2014-07-30 2023-02-14 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US12043852B2 (en) 2015-10-23 2024-07-23 President And Fellows Of Harvard College Evolved Cas9 proteins for gene editing
US11214780B2 (en) 2015-10-23 2022-01-04 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US11999947B2 (en) 2016-08-03 2024-06-04 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US11702651B2 (en) 2016-08-03 2023-07-18 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US10947530B2 (en) 2016-08-03 2021-03-16 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US11661590B2 (en) 2016-08-09 2023-05-30 President And Fellows Of Harvard College Programmable CAS9-recombinase fusion proteins and uses thereof
US12084663B2 (en) 2016-08-24 2024-09-10 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
US11306324B2 (en) 2016-10-14 2022-04-19 President And Fellows Of Harvard College AAV delivery of nucleobase editors
US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
US11820969B2 (en) 2016-12-23 2023-11-21 President And Fellows Of Harvard College Editing of CCR2 receptor gene to protect against HIV infection
US11898179B2 (en) 2017-03-09 2024-02-13 President And Fellows Of Harvard College Suppression of pain by gene editing
US11542496B2 (en) 2017-03-10 2023-01-03 President And Fellows Of Harvard College Cytosine to guanine base editor
US11268082B2 (en) 2017-03-23 2022-03-08 President And Fellows Of Harvard College Nucleobase editors comprising nucleic acid programmable DNA binding proteins
US11560566B2 (en) 2017-05-12 2023-01-24 President And Fellows Of Harvard College Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation
US11732274B2 (en) 2017-07-28 2023-08-22 President And Fellows Of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE)
US11932884B2 (en) 2017-08-30 2024-03-19 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11319532B2 (en) 2017-08-30 2022-05-03 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
CN109337904A (en) * 2018-11-02 2019-02-15 中国科学院动物研究所 Genome editing system and method based on C2c1 nuclease
CN112961853A (en) * 2018-11-02 2021-06-15 中国科学院动物研究所 Genome editing system and method based on C2C1 nuclease
CN113710802A (en) * 2019-03-08 2021-11-26 齐默尔根公司 Convergent genome editing in microorganisms
US11795452B2 (en) 2019-03-19 2023-10-24 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11643652B2 (en) 2019-03-19 2023-05-09 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11447770B1 (en) 2019-03-19 2022-09-20 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
CN112410234A (en) * 2019-08-21 2021-02-26 江南大学 Visual screening method for multi-target editing recombinant aspergillus strains
CN115279898A (en) * 2019-10-23 2022-11-01 成对植物服务股份有限公司 Compositions and methods for RNA templated editing in plants
CN111235130A (en) * 2019-11-15 2020-06-05 武汉大学 II-type V-type CRISPR protein CeCas12a and application thereof in gene editing
US11912985B2 (en) 2020-05-08 2024-02-27 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence
US12031126B2 (en) 2020-05-08 2024-07-09 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence
WO2022074113A1 (en) 2020-10-08 2022-04-14 Wageningen Universiteit Universal riboswitch for inducible gene expression
WO2022217086A1 (en) 2021-04-09 2022-10-13 Vor Biopharma Inc. Photocleavable guide rnas and methods of use thereof
CN113403294B (en) * 2021-06-04 2023-08-08 广州大学 Fusion protein, base editing tool and application thereof
CN113403294A (en) * 2021-06-04 2021-09-17 广州大学 Fusion protein, base editing tool and application thereof
WO2023283585A2 (en) 2021-07-06 2023-01-12 Vor Biopharma Inc. Inhibitor oligonucleotides and methods of use thereof
WO2023015182A1 (en) 2021-08-02 2023-02-09 Vor Biopharma Inc. Compositions and methods for gene modification
WO2023049926A2 (en) 2021-09-27 2023-03-30 Vor Biopharma Inc. Fusion polypeptides for genetic editing and methods of use thereof
WO2023102393A1 (en) * 2021-11-30 2023-06-08 Pioneer Hi-Bred International, Inc. High efficiency large scale chromosomal genome manipulation
WO2024073751A1 (en) 2022-09-29 2024-04-04 Vor Biopharma Inc. Methods and compositions for gene modification and enrichment
CN116656528B (en) * 2023-03-15 2024-06-04 天津科技大学 Pseudomonas and method for preparing deodorizing nitrogen-fixing bacteria agent by utilizing same
CN116656528A (en) * 2023-03-15 2023-08-29 天津科技大学 Pseudomonas and method for preparing deodorizing nitrogen-fixing bacteria agent by utilizing same

Also Published As

Publication number Publication date
GB201618507D0 (en) 2016-12-14
WO2018083128A3 (en) 2019-02-14

Similar Documents

Publication Publication Date Title
WO2018083128A2 (en) Microbial genome editing
Mougiakos et al. Efficient genome editing of a facultative thermophile using mesophilic spCas9
AU2016432443B2 (en) Thermostable Cas9 nucleases
Lin et al. Consolidated bioprocessing of cellulose to isobutanol using Clostridium thermocellum
Sheng et al. Development and implementation of rapid metabolic engineering tools for chemical and fuel production in Geobacillus thermoglucosidasius NCIMB 11955
US20130052646A1 (en) Positive and negative selectable markers for use in thermophilic organisms
Taylor et al. Genetic tool development underpins recent advances in thermophilic whole‐cell biocatalysts
CN102421890A (en) Recombinant bacterium for producing ethanol and application thereof
JP2009511051A (en) Minimal bacterial genome
US11746361B2 (en) Metabolic engineering for simultaneous consumption of Xylose and glucose for production of chemicals from second generation sugars
KR20210136997A (en) Iterative genome editing in microorganisms
US20210332350A1 (en) Recombinase Genome Editing
KR20210137009A (en) Pooling Genome Editing in Microbes
US20210324378A1 (en) Multiplexed deterministic assembly of dna libraries
ES2828707T3 (en) Method to produce a recombinant microorganism
US8927254B2 (en) Pyrococcus furiosus strains and methods of using same
US20210115500A1 (en) Genotyping edited microbial strains
Joseph et al. Metabolic engineering and the synthetic biology toolbox for Clostridium
Smolinski et al. Gene editing technologies for biofuel production in thermophilic microbes
CN103620020A (en) Isolation of selected marker-free microorganisms with a known genetic element
Mougiakos Feel the burn: a collection of stories on hot’n’sharp DNA engineering
Skagestad Effect of hydrogen peroxide in the production of L-lysine-derived amino acids in Bacillus methanolicus
JP2020043824A (en) Obligately anaerobic acetic acid-producing microorganisms and recombinant microorganisms
WO2014096436A1 (en) Marker-free genetically-engineered double mutants of thermoanaerobacter
Grogan Genetic analysis in extremely thermophilic bacteria: an overview

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17811198

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17811198

Country of ref document: EP

Kind code of ref document: A2