WO2024096790A1 - Système crispr modulaire - Google Patents

Système crispr modulaire Download PDF

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WO2024096790A1
WO2024096790A1 PCT/SE2023/051082 SE2023051082W WO2024096790A1 WO 2024096790 A1 WO2024096790 A1 WO 2024096790A1 SE 2023051082 W SE2023051082 W SE 2023051082W WO 2024096790 A1 WO2024096790 A1 WO 2024096790A1
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crispr
binding peptide
cas
protein
cas9
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PCT/SE2023/051082
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Per Hofvander
Mariette Andersson
Niklas Olsson
Martin FRIBERG
Paul Vogel
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Soledits Ab
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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • 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 technology proposed herein generally relates to the field of genome editing using CRISPR-Cas systems.
  • DNA is a molecule that stores genetic information and functions as a blueprint for any living organism on earth. Manipulation of the genetic information can alter the organism. This has been indirectly done by humans to breed crop species with beneficial properties such as higher yield or better robustness. Plants that showed a preferred characteristic were selected for breeding. In this way plants with superior traits were created.
  • CRISPR-Cas systems introduce DSBs at specific positions, but the repair through non-homologous end-joining (NHEJ) is rather random and screening for the desired mutation can be laborious.
  • NHEJ non-homologous end-joining
  • Recent advancements enable more precise edits and changes.
  • Base editors BE
  • PE prime editors
  • First generation BEs use a catalytically dead Cas9 (dCas9), a variant in which both cleavage domains are inactivated by mutations but the RNA-programmable binding of DNA is retained.
  • dCas9 is then fused to a cytidine deaminase enzyme. Deamination of cytidine (C) results in uracil (U), which is recognized by DNA repair mechanisms and replaced with thymine (T).
  • U uracil
  • T thymine
  • this method converts a C:G base pair into A:T without introducing DSBs and is therefore called cytosine base editor (CBE).
  • nCas9 Cas9 nickase
  • ABE adenine base editors
  • PE adenosine deaminase
  • PE offers even higher versatility.
  • an nCas9 is fused to the murine leukemia virus (MMLV)-reverse transcriptase (RT).
  • MMLV murine leukemia virus
  • RT reverse transcriptase
  • the programmable RNA has a 3’ extension with a primer binding site (PBS) and is called prime editing guide RNA (pegRNA).
  • nCas9 dislocates the target strand through binding to the 5’ end of the pegRNA. After that, the non-target strand is cleaved by the RuvC nuclease domain of nCas9 introducing a nick in the DNA.
  • the PBS can now hybridize to the free 3’ end of the target DNA, acting as a primer for the RT, which reverse transcribe RNA to incorporate the desired sequence into the target DNA. Any sequence following the PBS of the pegRNA can thus be inserted into the DNA, which creates a powerful "search and replace" tool.
  • Engineering of the MMLV-RT, nicking the non-edited strand and including the PAM sequence into the edit yielded PE systems with significantly increased efficiency. Theoretically, any deletion up to 44 bp and insertion of up to 80 bp can be achieved. So far over 50 BEs and PEs have been developed, of which each has very specific advancements and a different scope of application.
  • a CRISPR-Cas system can be delivered to a cell in different forms: as DNA, as mRNA, and as a protein complex.
  • DNA can be transfected in a transient or stable manner.
  • the former refers to the introduction of the CRISPR-Cas system as DNA via a plasmid vector that does not integrate into the genomic DNA of the cell.
  • the plasmid DNA can be delivered for instance by the means of a gene gun or PEG transformation.
  • the plasmid is usually lost after cell divisions.
  • stable integration of transgenes results in continuous expression that is maintained through generations.
  • RNA encoding CRISPR-Cas enables faster expression and bypasses the risk of insertional mutagenesis. Because RNA is highly unstable and quickly degraded, delivery in the form of mRNA is however always transient.
  • CRISPR-Cas delivery in protein form allows for instantaneous CRISPR activity and avoids the risk of introducing recombinant DNA into the cell.
  • a CRISPR-Cas protein is loaded with guide RNA (gRNA) and then delivered to the cell as ribonucleoprotein (RNP).
  • gRNA guide RNA
  • RNP ribonucleoprotein
  • Means of delivery in plants include for instance biolistics, that allow for cell wall penetration, microinjection during sexual reproduction and protoplast transfection.
  • the main steps of the workflow for the latter one include: isolation of protoplasts from plant tissue, RNP assembly and delivery to the protoplasts, which are subsequently regenerated to whole plants. This transgene-free method is advantageous because unintended effects from recombinant DNA is preserved.
  • WO2021165508 discloses an expression cassette comprising a Type II or V CRISPR-Cas effector protein domain fused to a peptide tag and a reverse transcriptase domain fused to an affinity peptide.
  • US20200017869 discloses a recombinant vector comprising a first nucleic acid sequence comprising a plant promoter and that encodes a recombinant polypeptide comprising a nuclease-deficient CAS9 polypeptide (dCAS9) or fragment thereof and a multimerized epitope; a second nucleic acid sequence comprising a plant promoter and that encodes a recombinant polypeptide comprising a transcriptional activator and an affinity polypeptide that specifically binds to the epitope; and a third nucleic acid sequence comprising a promoter and that encodes a crRNA and a tracrRNA, or fusions thereof.
  • dCAS9 nuclease-deficient CAS9 polypeptide
  • BE base editing
  • PE prime editing
  • HDR homology directed repair
  • first binding peptide and the second binding peptide are configured to bind to each other and define a binding peptide pair.
  • the technology proposed herein is accordingly based on the recognition by the present inventors that the production of the fusion proteins of CRISPR-Cas and functional proteins is a limiting factor for using RNP delivery of a CRISPR-Cas system.
  • the modular CRISPR-Cas system is suitable for, or is arranged to be used in, ribonucleoprotein (RNP) delivery.
  • RNP ribonucleoprotein
  • the modular CRISPR-Cas system is an in vitro modular CRISPR-Cas system. Not only must the correct DNA sequences for the CRISPR-Cas protein and the functional protein be obtained and fused together, but the expression of the fused DNA sequence involves difficulties.
  • the CRISPR-Cas system addresses all these limitations.
  • the CRISPR-Cas protein can be manufactured, i.e. expressed and purified separately from the functional protein which greatly simplifies the production process and in particular the purification due to the smaller size of the proteins compared to the fusion protein.
  • the CRISPR-Cas protein separately from the functional protein it is easy to change or modify the function of the CRISPR-Cas protein by pairing it with different functional proteins. The pairing may be done by simply mixing the CRISPR-Cas protein with the functional protein. This thus allows for further expanding the capabilities of the CRISPR-Cas system as it allows for adding further functionality, including functionality not yet used with CRISPR-Cas systems based on fusion proteins. This provides novel and enhanced genome editing possibilities.
  • the technology proposed herein thus provides a modular CRISPR-Cas system where different CRISPR-Cas proteins and functional proteins can be combined to form ribonucleoproteins that are then provided to the cell to be modified.
  • the combination of the CRISPR-Cas protein and the functional protein is done outside the cell, e.g., in vitro and is thus easy to perform.
  • the modularity of the system is evident in that once various CRISPR-Cas proteins and functional proteins have been manufactured, they may be combined as desired to form different ribonucleoproteins without the need to edit or change the vectors and sequences used to express the CRISPR-Cas proteins and functional proteins.
  • the gRNA which guides the CRISPR-Cas system is also combined with the CRISPR-Cas protein in vitro, thus making it simple to choose and/or change the target gene to be modified.
  • the CRISPR-Cas protein and functional protein, and also the gRNA, in vitro it is possible to determine the sequential order of assembly of the ribonucleoprotein. This may be important if the functional protein itself has an RNA interaction or affinity in order to prevent premature complexation between the gRNA and the functional protein. Assembling the CRISPR-Cas-complex with gRNA in vitro, instead of in vivo, further may prevent the gRNA from interacting with other cell components such as protein, RNA, and DNA, that are present in a cell, i.e. , in vivo. Assembling the CRISPR-Cas-complex with gRNA in vitro further allows control over the molar ratio of the components.
  • the technology proposed herein therefore provides multiple advantages over e.g., WO2021165508 and US20200017869.
  • the modular CRISPR-Cas system is suitable for ribonucleoprotein (RNP) delivery or is arranged to be used in, ribonucleoprotein (RNP) delivery. Accordingly, the CRISPR-Cas protein and the functional protein are in a form suitable for forming a ribonucleoprotein (RNP) that in turn is in a form suitable to be delivered to a cell.
  • RNP ribonucleoprotein
  • RNP ribonucleoprotein
  • the equivalent terms “for ribonucleoprotein (RNP) delivery” and “suitable for ribonucleoprotein (RNP) delivery” and “arranged to be used in ribonucleoprotein (RNP) delivery” encompasses that ribonucleoprotein (RNP) delivery can be used with the modular CRISPR-Cas system.
  • the modular CRISPR-Cas system can be used with ribonucleoprotein (RNP) delivery.
  • CRISPR-Cas system and CRISPR system are used interchangeably.
  • the term modular when applied to the CRISPR-Cas system means that the CRISPR-Cas system encompasses different possible modules, where for example, the CRISPR-Cas protein with its first binding peptide defines a first module, and the functional protein with its second binding peptide defines a second module.
  • the system may comprise further modules such as a third module defined by a further functional protein also having the second binding peptide configured to bind to the first binding peptide.
  • the Modular CRISPR-Cas system thus allows for assembling a CRISPR-Cas-complex by choosing a desired CRISPR-Cas protein with its first binding peptide and assembling it with a desired functional protein with its second binding peptide, thereby assembling any desired CRISPR-Cas-complex tailored for e.g. base editing, prime editing, or other type of editing as desired.
  • the choosing and assembling of the CRISPR-Cas proteins and functional proteins is carried out on the proteins and not on genes encoding the proteins. This allows for ease of assembly, i.e. , by contacting such as by mixing, and does not require modification or the genes encoding the CRISPR-Cas proteins and functional proteins.
  • the CRISPR-Cas system may for example comprise a first plurality of first modules corresponding to different CRISPR-Cas proteins having different activity and a second plurality of second modules corresponding to functional proteins have different function, to allow the user of the system to obtain any desired combination of CRISPR-Cas protein and functional protein.
  • the CRISPR-Cas system may exist in assembled form as the CRISPR-Cas- complex, i.e. with the first binding peptide bound to the second binding peptide, or in nonassembled form, i.e. where the first binding peptide and the second binding peptide are not bound to each other and the CRISPR-Cas protein and the functional protein are thus not associated as a CRISPR-Cas-complex.
  • the CRISPR-Cas protein may for example be a catalytically dead Cas9 (dCas9) or a Cas9 nickase (nCas9).
  • the CRISPR-Cas protein may be any CRISPR-Cas- related protein, or part thereof, that is an RNA-guided DNA endonuclease enzyme such as CRISPR-Cas9, CRISPR-Cas12a or variants thereof.
  • the CRISPR-Cas protein may have both, one, or none, of the nuclease domains functional.
  • the CRISPR-Cas protein is fused, preferably by being expressed sequentially with, the first binding peptide.
  • the first binding peptide and the second binding peptide typically each have a length of 10-170 amino acids.
  • the first binding peptide and the second binding peptide are configured to bind to each other.
  • the binding may be the result of attractive forces between the side chains of the amino acids making up the respective binding peptide.
  • peptide covers peptides, polypeptides, and proteins.
  • the functional protein is a protein that is capable of altering or affecting the outcome of applying the CRISPR-Cas protein to DNA.
  • the functional protein may thus be configured to affect the functioning of the CRISPR-Cas protein.
  • the functional protein is configured to affect how the organism, whose genome or DNA is affected by the CRISPR-Cas protein, handles the effect, such as a break, caused by the action of the CRISPR-Cas protein.
  • the functional protein may thus for example affect how a break of the strands of DNA is repaired. Improving directed repair such as homology directed repair or homologous recombination are thus additional features that could be provided by a functional protein.
  • the first and second binding peptides together define a binding peptide pair.
  • Different binding peptide pairs are known in other technical fields. Binding peptide pairs may further be constructed synthetically, by designing sequences of the first and second binding peptides so as to bind to each other by the side chains.
  • the first and second binding peptides may be constructed so as to assume secondary structure, i.e. as alpha-helixes or beta sheets, suitable for binding.
  • the binding peptide pair preferably comprises a Cohesin/Dockerin system.
  • the Cohesin/Dockerin system i.e. in which one of the first and second binding peptide comprises or consists of Cohesin, and the other comprises of or consists of Dockerin, works well in the CRISPR-Cas system.
  • the binding peptide pair may comprise a first binding peptide, having at least 90% sequence identity, such as at least 95% or at least 99% sequence identity, to one of Cohesin and Dockerin, and a second binding peptide having at least 90% sequence identity, such as at least 95% or at least 99% sequence identity, to the other of Cohesin and Dockerin.
  • the binding peptide pair may comprise a first binding peptide, having at least 90% sequence identity, such as at least 95% or at least 99%, or 100% sequence identity to one of SEQ ID NO: 19 and SEQ ID NO 21 (see below) and a second binding peptide having at least 90% sequence identity, such as at least 95% or at least 99% or 100 % sequence identity to the other of SEQ ID NO: 19 and SEQ ID NO: 21.
  • Cohesin domain Cip7 is given by SEQ ID NO: 19: MAVRIKVDTVNAKPGDTVRIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIEPGELIVDPNPT KSFDTAVYPDRKMIVFLFAEDSGTGAYAITEDGVFATIVAKVKSGAPNGLSVIKFVEVGGF ANNDLVEQKTQFFDGGVNVG
  • the functional protein is selected among the group consisting of: a) deaminases, preferably modified deaminases, b) reverse transcriptases, c) methylases, and d) homologous recombination and homology directed repair enhancing proteins.
  • the functional protein preferably is a deaminase, a reverse transcriptase, a methylase, or a homologous recombination protein or a homology directed repair enhancing protein.
  • Deaminases are for example used in base editing (BE). Examples include cytidine deaminases which deaminates cytidine (C) resulting in uracil (II). This functional protein may be used together with a dead Cas9 (dCas9). Another example is adenosine deaminase, which may be used together with a nickase (nCas9).
  • the functional protein may be a reverse transcriptase, which is used together with a nickase (nCas9).
  • Methylases add methyl groups to adenine or cytosine bases within a recognition sequence and thus protect these bases.
  • Homologous recombination and homology directed repair enhancing proteins act by suppressing NHEJ repair mechanisms, improve the accessibility of DNA for homologous recombination or guide a repair template to the CRISPR-Cas interacting site.
  • the functional protein may alternatively be a RAD protein, such as a RAD51 protein.
  • RAD proteins such as RAD51 protein, may bind to the DNA at the site of a break and encase the DNA in a protein sheath.
  • RAD protein as functional protein, the effect of the CRISPR-Cas protein can thus be further modified.
  • a bacterial similar protein to RAD is the RecA protein.
  • the modular CRISPR-Cas system according to the first aspect of the technology proposed herein may further comprise:
  • the guide RNA sequence guides the CRISPR-Cas protein to the target
  • the guide RNA may be provided bound to the CRISPR-Cas protein, or may alternatively be provided separate from the CRISPR-Cas protein so that it can be assembled with the CRISPR-Cas protein by contacting the guide RNA and the CRISPR-Cas protein.
  • the guide RNA may further be functionalized by also providing a template for genome editing by prime editing, pegRNA.
  • the modular CRISPR-Cas system according to the first aspect of the technology proposed herein comprises:
  • kits may thus be provided which allows the user to perform both base editing and prime editing, by selecting the corresponding CRISPR- Cas protein and functional protein, from the same kit.
  • the system may thus be implemented as a kit comprising at least one CRISPR-Cas protein with its corresponding first binding peptide, more preferably at least two such as one dCas9 and one nCas9, and at least one functional protein with its corresponding second binding peptide, preferably at least two such as one deaminase and one reverse transcriptase.
  • the first and second container may be any type of container, but preferably a test tube such as a plastic test tube, for example an Eppendorf tube, or a glass test tube, that is capable of holding the CRISPR-Cas protein or the functional protein.
  • the CRISPR-Cas protein and the functional protein may thus be provided in a buffer or solution. It is further contemplated that the CRISPR-Cas protein and the functional protein may be provided in solid form, such as dried form or crystal form.
  • the modular CRISPR-Cas system according to the first aspect of the technology proposed herein comprises:
  • the modular CRISPR-Cas system preferably further comprises:
  • the third container may be of the same type as the second container described above.
  • the third binding peptide is configured to bind to the first binding peptide.
  • the third binding peptide may have an amino acid sequence that has a sequence identity of at least 90%, such as at least 95%, more preferably at least 99%, to the amino acid sequence of the second binding peptide. Even more preferably the third binding peptide has the same amino acid sequence as the second binding peptide.
  • the system comprises two or more sets of binding peptide pairs.
  • each set of binding peptide pairs comprises a first binding peptide fused to the CRISPR-Cas protein and a second binding protein fused to a functional protein as described above.
  • This allows the use of multiple functional proteins with the CRISPR-Cas protein.
  • the sequences of the first and second binding peptides of each set of binding peptide pairs are configured so that the first binding peptide of a first set of binding peptide pairs only binds to the second binding peptide of the same set of binding peptide pairs, and accordingly does not bind to the second binding peptide of another set of binding peptide pairs.
  • the further functional protein fused to the third binding peptide is provided in its own buffer or provided in solid form, such as dried form or crystal form.
  • the second aspect of the technology proposed herein concerns a method of assembling a CRISPR-Cas-complex comprising the steps of: i. providing the system according to the first aspect of the technology proposed herein, and ii. contacting the CRISPR Cas protein fused to the first binding peptide, and the functional protein fused to the second binding peptide, with each other, wherein the method preferably further comprises the step of: iii. contacting the CRISPR Cas protein with a guide RNA sequence.
  • Step iii may be performed prior to step ii.
  • the CRISPR-Cas complex is suitable for, or is arranged to be used in, ribonucleoprotein (RNP) delivery.
  • RNP ribonucleoprotein
  • the CRISPR-Cas complex is an in vitro modular CRISPR-Cas system.
  • the contacting of the CRISPR-Cas protein with its first binding peptide and the functional protein with its second binding peptide is preferably performed by mixing.
  • the CRISPR-Cas protein with its first binding peptide and the functional protein with its second binding peptide are thus preferably provided in separate buffer solutions, such that the buffer solutions can be mixed to assemble the CRISPR-Cas-complex.
  • the CRISPR-Cas protein with its first binding peptide and the functional protein with its second binding peptide may be provided in crystal form as solids, and mixed together in a buffer to assemble the CRISPR-Cas-complex.
  • the guide RNA sequence may be provided in a buffer for mixing with the CRISPR-Cas protein.
  • the guide RNA may be modified, e.g. as pegRNA.
  • Step ii of contacting the CRISPR-Cas protein with its first binding peptide and the functional protein with its second binding peptide is performed in vitro.
  • the contacting is performed outside the cell to which the CRISPR-Cas-complex is to be delivered as a ribonucleoprotein.
  • Step iii of contacting the CRISPR Cas protein with the guide RNA sequence may be performed prior to, or after step ii of contacting the CRISPR Cas protein fused to the first binding peptide, and the functional protein fused to the second binding peptide, with each other.
  • Step iii is performed in vitro. In other words, the contacting is performed outside the cell to which the CRISPR-Cas-complex is to be delivered as a ribonucleoprotein.
  • step iii before step ii may be advantageous when the functional protein itself has an RNA interaction or affinity in order to prevent complexation between the gRNA and the functional protein.
  • the third aspect of the technology proposed herein concerns a CRISPR-Cas complex obtained by the method according to the second aspect of the technology proposed herein, i.e. a CRISPR-Cas complex for ribonucleoprotein (RNP) delivery comprising:
  • the CRISPR-Cas complex preferably further comprises:
  • the CRISPR-Cas complex is suitable for, or is arranged to be used in, ribonucleoprotein (RNP) delivery.
  • RNP ribonucleoprotein
  • the CRISPR-Cas complex is an in vitro modular CRISPR-Cas system.
  • the CRISPR-Cas complex corresponds to the system according to the first aspect of the technology proposed herein when the first and the second binding peptides are bound to each other.
  • the CRISPR-Cas complex can be used to modify DNA in vivo or in vitro, and the DNA can be free (isolated DNA) or in a cell or an organism (genomic DNA).
  • the fourth aspect of the technology proposed herein concerns the use of the modular CRISPR-Cas system according to the first aspect of the technology proposed herein, or the CRISPR-Cas complex according to the third aspect of the technology proposed herein, for modifying the genome of an organism, wherein the organism preferably is a plant selected from the group consisting of a monocot and dicot plant, or the organism is a plant selected from the group consisting of maize, rice, sorghum, rye, barley, wheat, millet, oats, sugarcane, turfgrass, or switchgrass, soybean, canola, alfalfa, sunflower, cotton, tobacco, peanut, potato, sugar beet, grape, Arabidopsis and safflower cell, and wherein preferably the organism is a plant from the family Solanaceae, preferably from the genus Solanum, more preferably from the species Solanum tuberosum.
  • the use may comprise using the modular CRISPR-Cas system according to the first aspect of the technology proposed herein, or the CRISPR-Cas complex according to the third aspect of the technology proposed herein, for modifying the genome of a cell of the organism, such as a plant cell.
  • a plant having had its genome modified has been obtained, then the plant cell may be cultivated as known in the art to obtain a plant.
  • the finished plant can then in turn be propagated and/or cultivated to obtain further plants for planting and farming.
  • the organism is preferably not a human embryo.
  • the use according to the fourth aspect of the technology proposed herein preferably does not comprise modifying the germ-line genetic identity of humans. Further, the use according to the fourth aspect of the technology proposed herein preferably does not comprise a surgical or therapeutic method for the treatment of the human or animal body.
  • a fifth aspect of the technology proposed herein thus relates to a plant obtained by the use according to the fourth aspect of the technology proposed herein.
  • a sixth aspect of the technology proposed herein concerns a plant comprising or consisting of one or more plant cells having had its genome modified by the use according to the fourth aspect of the technology proposed herein.
  • a seventh aspect of the technology proposed herein concerns progeny of a plant according to the fifth or sixth aspect of the technology proposed herein.
  • An eight aspect of the technology proposed herein concerns a seed or spore obtained from a plant according to the fifth or sixth aspect of the technology proposed herein.
  • a ninth aspect of the technology proposed herein concerns a vegetatively propagation- capable object, such as a cutting or graft or tuber or bulb or callus, of a plant according to the fifth or sixth aspect of the technology proposed herein.
  • Fig. 1A shows a Cas9 protein assembled with a green fluorescent protein (GFP) by first and second binding peptides shown as a Dockerin peptide (D) and a cohesion peptide (C).
  • GFP green fluorescent protein
  • Fig. 1 B shows a Cas9 complex comprising a Cas9 protein assembled with a reverse transcriptase (RT) by first and second binding peptides shown as a Dockerin peptide (D) and a cohesion peptide (C).
  • RT reverse transcriptase
  • Fig. 2A shows a schematic overview of the three cloned constructs.
  • Cip7-RT Cip7 with 0.4 kbp corresponding to 15 kDa, RT (MMLV-RT) with 2.1 kbp corresponding to 77 kDa).
  • the T7 promoter controls the expression, allowing for inducible protein expression in Rosetta (DE3) E. coli strain. Kanamycin resistance (KanR) is used as a selection marker in all constructs.
  • KanR Kanamycin resistance
  • a nuclear localization sequence (NLS) is followed by the SpCas9 gene, which is fused to the first adaptor component Doc1 .
  • the second adaptor component Cip7 is fused to either GFP or Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT).
  • Fig. 2B shows a restriction analysis to verify the structure of the plasmid and successful transformation. Plasmid DNA was purified from transformed and untransformed (untransf.) Rosetta (DE3) cells, digested with Hindlll and Bglll and restriction patterns compared to the respective plasmid used for transformation. The band likely representing pRARE is indicated with an arrow.
  • Fig. 3 shows a Cas9 in-vitro cleavage assay to confirm nuclease activity
  • a Patatin 1 PCR fragment was incubated either with commercial TrueCut Cas9 or purified Cas9-Doc1 and gRNA. The reactions were run on an agarose gel.
  • Fig. 4 shows a PAGE showing interaction of Cip7-GFP and Cas9-Doc1.
  • Cip7-GFP and Cas9-Doc1 were loaded to a native gel, with increasing amounts of the latter one.
  • TrueCut Cas9 was used as a negative control. Depicted values are in pmol. After electrophoresis the gel was imaged for GFP signal.
  • Fig. 5 shows a Cas9 electrophoretic mobility shift assay (EMSA) with imaging for GFP.
  • the Cas9 in-vitro cleavage assay (Fig. 3) was adapted as an EMSA by removing the final heating step, which allows Cas9 to stay associated with the target DNA causing a mobility shift.
  • the gel was imaged for GFP signal and subsequently DNA was stained using a GelRed post-staining protocol.
  • the GFP band of the apparent interaction between Cip7-GFP + Cas9-Doc1 is indicated with an arrow.
  • Fig. 6 shows Cas9 electrophoretic mobility shift assay (EMSA) with Cip7-GFP and Cip7-RT.
  • the experimental setup is similar to Fig. 5 but less DNA and protein was used, and the running time was extended to achieve better separation.
  • the amount of Cas9-Doc1 was kept the same while increasing Cip7-GFP or Cip7-RT.
  • TrueCut Cas9 was used as a control.
  • Cas9-Doc1 or TrueCut Cas9 associated with the target DNA is indicated by the lower arrows in the respective panel, and the complexes with Cip7-RT or Cip7-GFP are indicated by the upper arrows in the respective panel.
  • Fig. 7A shows RNP transfected potato protoplasts in different conditions in bright field (left) and UV light (right), a, negative control (NC) displays no GFP signal, b, in Cip7-GFP transfected protoplasts GFP signal is clearly visible, c, protoplasts transfected with Cip7-GFP and Cas9-Doc1 showed more frequent green fluorescence in comparison to just Cip7-GFP. Scale bar 150 pm.
  • Fig. 7B shows potato protoplasts transfected with both Cip7-GFP and Cas9-Doc1 in bright field (left) and in UV light (right), a, intact protoplasts with strong autofluorescence did not give any apparent sign of GFP signal, b, protoplasts with less autofluorescence appeared more yellow to green in color which indicated the presence of GFP. c, broken protoplasts and cell debris showed increased green fluorescence. Scale bar 10 pm.
  • Oligonucleotides were diluted to 100 pM and 20 pL of both strands mixed with 20 pL 5M NaCI solution and 20 pL water (Sequences are listed in Table 1 below). The mix was heated to 95 °C and cooled down to room temperature. The annealed DNA fragments were diluted to suitable concentrations and used for cloning.
  • Table 1 The fragments and plasmids that were used for cloning pMJ806 (Addgene #39312), a T7 expression vector carrying the SpCas9 gene, was digested using ThermoFisher FastDigest restriction enzymes (RE) Xhol and Spel. The fragments were separated by 1.2% agarose gel electrophoresis and purified using the ThermoFisher GeneJET Gel Extraction Kit according to the manufacturer’s instructions. DNA concentration was determined by spectrophotometry using a DeNovix DS-11 device.
  • RE ThermoFisher FastDigest restriction enzymes
  • the 83 bp DNA fragment RBS-NLS (Table 1) created by oligonucleotide annealing was digested using Xhol and Spel and purified with the ThermoFisher GeneJet PCR Purification kit. Ligation was performed using the ThermoFisher T4 DNA ligase in a 20 pL reaction with a plasmid:insert ratio of 1:50. The ligation reaction was diluted 5 times and 2 pL were used to transform ThermoFisher OneShot Top10 chemically competent E. coli cells according to the manufacturer’s instructions by 42 °C heat-shock.
  • the IDT gBIock Doc1-NLS DNA fragment (Table 1) was inserted using Sacl and Xhol by digestion and ligation as mentioned previously. For ligation a plasmid:insert ratio of 1 :3 was used, creating the pMC:RBS-NLS-Cas9-Doc1 construct.
  • pMC:Cip7-GFP was created by removing undesired tags in pET30-R5-GFP (Addgene #17838) and replacing them with the 46 bp DNA fragment RBS (oligonucleotide annealing) using Xbal and Bglll with a plasmid:insert ratio of 1:50 as described earlier.
  • Cip7 from a synthesized DNA fragment (IDT gBIock) was introduced using Bglll and Kpnl as described earlier with a plasmid:insert ratio of 1 :3.
  • the MMLV-RT from pET-19b_MMLV-RT (Addgene #153312) was cut out using Ndel and Xhol and ligated into pMC:Cip7-GFP with plasmid:insert ratio of 1 :3, replacing the GFP gene and creating pMC:Cip7-RT. Plasmid sequences were confirmed by Sanger sequencing with primers listed in Table 2.
  • Freezer stocks of MilliporeSigma Novagen Rosetta (DE3) E. coli cells were streaked on LB agar plates and incubated overnight at 37 °C. Colonies were used to inoculate 5 mL LB cultures, that were incubated at 37 °C and 225 rpm overnight. The next day 50 mL LB medium was inoculated with 0.5 mL overnight culture and incubated for 2 h at 37 °C and 225 rpm.
  • Colonies from LB agar plates were picked and used for inoculation of 5 mL TB medium (Terrific Broth: 1.2% tryptone, 2.4% yeast extract, 0.5% glycerol, 89 mM), supplemented with 50 pg/mL kanamycin and incubated overnight at 37 °C and 225 rpm.
  • Protein expression was induced with 0.1 mM IPTG, the temperature reduced to 18 °C and cultures were grown for 20 h at 225 rpm.
  • Protein concentration was determined using the Thermo Scientific Coomassie (Bradford) protein assay kit according to the manufacturer’s instructions.
  • the standard microplate protocol with a working range of 100 - 1500 pg/mL was used and the absorbance measured at 595 nm in a Thermo Scientific Multiskan GO microplate spectrophotometer.
  • the purification process was evaluated utilizing sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE).
  • Cas9 ThermoFisher TrueCut Cas9 v2 as control or purified Cas9-Doc1
  • 30nM gRNA were mixed and incubated at 25 °C for 10 min in Cas9 nuclease reaction buffer (20mM HEPES, 100mM NaCI, 5mM MgCI2, 100nM EDTA).
  • substrate DNA was added in a final concentration of 3nM giving a ratio of 10:10:1 (Cas9:gRNA:substrate DNA) and the reaction incubated for 1 h at 37 °C.
  • 1 pL proteinase K was added and incubated at 65 °C for 10 min and finally the reaction was analyzed on a 2% agarose gel.
  • Protein samples were diluted to 0.5 - 2 pmol/pL concentrations, mixed with sample buffer (final concentration: 100mM Tris HCI, 10% glycerol, 0.000 25% bromophenol blue, pH 8.6) and incubated for 5 min at RT. Samples were loaded onto 10% Invitrogen Novex Trisglycine gels and run in native running buffer (25mM Tris base, 192mM glycine, pH 8.3) at 200 V on ice until the dye front reached the bottom of the gel. Imaging for GFP was performed using a Bio-Rad ChemiDoc Touch Imaging System in the Pro-Q Emerald 488 settings of the device.
  • the Cas9 in-vitro cleavage assay was adapted to investigate interaction of Cas9-Doc1 with Cip7-GFP and Cip7-RT.
  • To visualize for GFP 2 pmol of both Cas9-Doc1 and Cip7- GFP per 20 pL reaction was used in order to achieve a prominent GFP signal.
  • 1 pmol of Cas9-Doc1 and increasing concentrations of Cip7-GFP or Cip7-RT (1 , 2, 4, 6, 8 pmol) were used.
  • ThermoFisher TrueCut v2 Cas9 was used as a control.
  • Cas9-Doc1 , Cip7-GFP or Cip7-RT and gRNA were mixed and pre-incubated for 10 min at 25 °C in Cas9 reaction buffer.
  • Target DNA was added in a molar ratio 10:10:1 (Cas9:gRNA:DNA) after pre-incubation and the reaction incubated at 37 °C for exactly 30 min.
  • the tubes were immediately chilled on ice before loading on an 1% agarose gel, which was run at 4 V/cm.
  • the GFP signal was imaged using a Bio-Rad ChemiDoc Touch Imaging System in the Pro- Q Emerald 488 settings of the device. Post-staining was performed in 3X Biotium GelRed solution for 20 min after which the gel was imaged using the Biotium GelRed settings of the device.
  • Protoplasts were isolated, transfected and regenerated according to the protocol by Andersson et al., 2018. Briefly, leaves from 4 to 6 weeks old in-vitro S. tuberosum cv. Desiree plants were used for protoplast isolation. 1 - 2 g of leaves were incubated for 24 h at 4 °C in a petri dish with 20 mL conditioning medium (2.7 g/L MS salts, 0.001% Nitsch & Nitsch vits, 100 mg/L casein hydrolysate, 2 mg/L NAA, 0.5 mg/L BAP, pH 5.8). Leaves were cut into 1mm wide slices and incubated for 30 min in 20 mL plasmolysis solution (0.5M D-sorbitol).
  • the solution was replaced with 25mL enzyme solution (1x Macro, 1x Fe/EDTA, 1x Micro, 0.5x Vits 1 , 0.5x Vits 2, 0.5x Vits 3, 5x Nitsch & Nitsch vits, 1x Organics, 500 mg/L Casein hydrolysate, 0.205M D-glucose-monohydrate, 0.205M Mannitol, 20 g/L PVP-10, 5.37 pM 1 -Naphthaleneacetic acid, 1.78 pM 6- Benzylaminopurine, 10 g/L Cellulase R10, 2 g/L Maceroen-zyme R10, 6mM CaCI2 ⁇ 2H2O, pH 5.6) and incubated for 14 h at 25 °C.
  • enzyme solution 1x Macro, 1x Fe/EDTA, 1x Micro, 0.5x Vits 1 , 0.5x Vits 2, 0.5x Vits 3, 5x Nitsch & Nitsch vits, 1x Organics, 500
  • Protoplast solution was filtered through 100 pm and 70 pm filters before centrifuging for 5 min at 50xg.
  • the protoplast pellet was resuspended in 2 mL of wash solution (1x Macro, 6mM CaCI2 ⁇ 2H2O, 1x Micro, 240mM NaCI, 10.74 pM 1 -Naphthaleneacetic acid, 2.22 pM 6-Benzylaminopurine, 1x Fe/EDTA, pH 5.6) and carefully applied to tubes containing 6 mL sucrose solution (0.43M Sucrose). Centrifugation was done for 15 min at 50xg.
  • Transformation buffer 1 190mM Mannitol, 100mM Cacl2 ⁇ 2H2O, 0.5% MES, pH 5.6
  • transformation buffer 2 0.5M Mannitol, 15mM MgCI2 ⁇ 6H2O, 0.1% MES, pH 5.6
  • the protoplast were incubated in the dark at 25 °C for 5 days. In the following 14 days the light was stepwise increased to reach 10 pmol/m2/s.
  • the emerging calli were imaged and counted using the Analyze Particles tool in Fiji Imaged. 1.10 Statistical analysis
  • a T7 expression vector carrying the gene for SpCas9 undesired tags in the N-terminus were replaced with a short sequence including a Ribosomal Binding Site (RBS) and a Nuclear Localization Sequence (NLS).
  • RBS Ribosomal Binding Site
  • NLS Nuclear Localization Sequence
  • an RBS was added to the N-terminus along with the cohesin domain Cip7 which gave the second construct Cip7-GFP (Fig. 2A, middle).
  • the GFPuv was replaced with the Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT) creating Cip7-RT (Fig. 2A, bottom).
  • MMLV-RT Moloney Murine Leukemia Virus Reverse Transcriptase
  • T7 expression system these constructs were used to produce recombinant proteins in E. coli and subsequent RNP delivery in potato protoplasts. All three constructs are under the control of the T7 promoter that is recognized by the T7 RNA polymerase and allows for tightly controlled gene expression in E. coli strains carrying the gene for the T7 RNA polymerase. Furthermore, all three constructs have kanamycin resistance (KanR) for selection of successfully transformed colonies.
  • the first construct begins with a NLS to signal import of the recombinant protein into the cell nucleus.
  • the SpCas9 gene is adjacent to the NLS and codes for the SpCas9 protein, which is the core of the CRISPR system.
  • the dockerin domain Doc1 represents a first binding peptide, also called adapter herein, and will enable to couple other functional proteins to Cas9.
  • the 6x His-tag allows for protein purification.
  • the recombinant protein produced by this construct will hereafter be referred to as Cas9-Doc1.
  • the second construct starts with the 6xHis-tag for purification, which is followed by the second binding peptide or second adapter component, the cohesin domain Cip7 that can bind to the Doc1 domain.
  • GFPuv is a variant of the wild-type green fluorescent protein (GFP) and optimized for excitation by UV light. Here, it acts as a reporter in a proof-of-principle phase.
  • Cip7-GFP The respective recombinant protein will be named Cip7-GFP.
  • the last construct is similar to the second, but GFP is replaced with the MMLV-reverse transcriptase, that is used in prime editing systems. Hereafter, it will be called Cip7-RT.
  • Rosetta (DE3) E. coli cells were transformed with the constructed expression vectors and successful transformation was verified by restriction analysis (Fig. 2B). Plasmid DNA was isolated from transformed and untransformed Rosetta (DE3) cells. Isolated plasmid DNA was digested using Hindi II and Bglll together with the respective plasmid used for transformation as positive control. The restriction patterns of isolated DNA from Rosetta (DE3) cells were for all three constructs almost identical to the control. However, an additional band at about 10 000 bp could be observed in all samples isolated from Rosetta (DE3) cells including the untransformed control (Fig. 2B, indicated with an arrow). This band can most likely be attributed to the pRARE plasmid that the Rosetta (DE3) strain carries for expression of rare tRNAs. The results from the restriction analysis indicated successful transformation and allowed to start protein expression.
  • Recombinant protein was purified using immobilized metal affinity chromatography (IMAC) and the process monitored by SDS-PAGE.
  • the size of the purified proteins was predicted using the online tool ExPASy (Gasteiger, 2003, Table 4).
  • the molecular weight of the bands observed in SDS-PAGE were estimated using ThermoScientific PageRuler and the standard curve method.
  • For Cas9-Doc1 the estimation by standard curve was slightly lower than the ExPASy prediction, while for Cip7-GFP and Cip7-RT it was the other way around (Table 4).
  • the protein concentration in the elution fraction was determined by a Bradford assay.
  • Cip7-RT When Cip7-RT was grown without IPTG induction, the culture reached higher GD600 values and higher band intensity compared to the induced sample (data not shown). Impaired cell growth is expected upon induction with IPTG and was also observed for Cas9-Doc1 and Cip7-GFP, although not as severe. Basal expression of protein in the absence of IPTG could be seen for all three constructs (data not shown). However, for Cas9-Doc1 and Cip7-GFP the IPTG-induced samples the recombinant protein yield was much higher than the non-induced control, while Cip7-RT showed comparable and possibly even higher yield for non-induced samples against induced. Yields for the Cip7- RT non-induced were almost as high as Cas9-Doc1 induced, but the induced cultures yielded only about half of the protein amount.
  • Fig. 3A An in-vitro cleavage assay was performed (Fig. 3A).
  • a Patatin 1 PCR fragment was incubated either with commercial TrueCut Cas9 or purified Cas9-Doc1 and gRNA. The reactions were run on an agarose gel.
  • target DNA the Patatin 1 gene was amplified using PCR.
  • a previously validated gRNA was used to cleave the PCR fragment (Fig. 3B).
  • a commercial Cas9 protein was used as control. Correct cleavage of the target DNA would produce two cleaved fragments with 948 bp and 213 bp in size, as observed.
  • the assay clearly showed that the purified Cas9-Doc1 is active and cleaves efficiently the target DNA. There is no visible difference in the amount of cleaved DNA compared to the commercial Cas9.
  • the assay was repeated after storing the purified Cas9-Doc1 for 14 days at -20 °C in reaction buffer with 50% glycerol and no decrease in activity could be observed.
  • Cip7-RT and Cas9-Doc1 were repeated with longer running time and overall reduced amounts of DNA and protein to achieve higher band resolution (Fig. 6).
  • Cip7-GFP and Cas9-Doc1 were included.
  • the GFP imaging step was omitted, because the lower amounts of Cip7-GFP did not allow to observe the Cas9-Doc1 + Cip7-GFP band as for Fig. 5 and for Cip7-RT and Cas9-Doc1 a GFP signal is not expected.
  • the experimental procedure was kept the same but Cip7-RT or Cip7-GFP, respectively, were stepwise-increased this time.
  • Potato (Solanum tuberosum) protoplasts were transfected with the purified fusion proteins to investigate if the interaction of Cas9-Doc1 and Cip7-GFP that was already observed in- vitro can also be seen in-vivo. Furthermore, there was a need to assess if transfected protoplasts retained cell division ability and thus the grade of purity for the fusion proteins is sufficient. For this, protoplasts were isolated from potato leaves and transfected with 40% PEG. Protoplasts transfected with Cip7-GFP, Cip7-GFP + Cas9-Doc1 and the negative control without transfected protein were imaged using fluorescence microscopy with UV as light source (Fig. 7A and 7B).
  • Cas9-Doc1 has a NLS upstream of the SpCas9 gene which signals import into the nucleus. If Cip7-GFP couples to Cas9-Doc1 the complex might be imported and a fluorescence signal in the nucleus observed.
  • Chloroplasts are the light harnessing organelles of plant cells and carry a pigment called chlorophyll, which has an emission maximum at about 730 nm when excited with UV light. Emitted light at that wavelength is perceived by the human eye as red light.
  • the GFP variant GFPuv has an emission maximum at about 506 nm, which is perceived as green light. Note that bandpass filters for autofluorescence and GFP signal were not available during the experiment, which is why both can only be distinguished by the recorded color in the UV image.
  • the negative control seemed to display slightly more intact protoplasts than the samples that were transfected with the recombinant proteins (Fig. 7A, left panel, images representative for multiple observations).
  • a strong autofluorescence signal from chloroplasts could be seen in the spectrum of red light, and as expected, green light that would indicate GFP signal could not be observed (Fig. 7A, a).
  • Cip7-GFP together with the autofluorescence showed several green spots that indicates GFP signal (Fig. 7A, b).
  • Cip7-GFP + Cas9-Doc1 seemed to have a greater amount of GFP signal compared to just Cip7-GFP (Fig. 7A, c).
  • Protoplasts transfected with RNPs and functional proteins were embedded in alginate and allowed to divide over two weeks. The number of calli was counted as a measure of cell division ability, to evaluate if the purity of the purified recombinant proteins is sufficient for protoplast transfection and regeneration. High statistical significance in the difference for the number of calli was present in NC compared to the other samples with transfected proteins. Among the samples transfected with recombinant proteins no statistical significance could be observed. Interestingly, protoplasts transfected with only Cas9-Doc1 seemed to show a slightly higher cell division ability with 4 calli per 50 000 pps compared to the sample with Cas9-Doc1 and gRNA that had about 2 calli per 50 000 pps.
  • Protoplasts transfected with Cip7-GFP in addition to Cas9-Doc1 and gRNA did not show further impairment of regeneration having about 2 calli per 50 000 pps.
  • the Cas9 in-vitro cleavage assay showed that the purified Cas9-Doc1 is active and no difference in activity compared to the commercial Cas9 could be seen. Thus, the dockerin domain Doc1 does not impair the nuclease activity of Cas9.
  • Cip7-RT the non-induced protein yield reached almost the amounts of induced Cas9-Doc1 and the induced yield was only half of that.
  • the poor yield for induced Cip7- RT cultures might indicate toxicity of the protein in high amounts and marks the necessity for further optimization of the culture conditions.
  • Culture medium free of expressioninducing reagents as well as different temperatures and growth times might provide better yield for Cip7-RT.
  • the coexpression of T7 lysozyme has earlier been shown to give tighter control over protein expression (Spehr et al., 2000). T7 lysozyme can inhibit the T7 polymerase when the latter one is basally expressed, which allows for tighter control of and at the same time enables easier cell lysis using freeze-thaw cycles (Wanarska et al., 2007).
  • Cip7-RT seemed to cause a stronger mobility shift than the addition of Cip7-GFP demonstrated by the band width. This could be due to the smaller size of Cip7- GFP that is only about half of Cip7-RT (Table 4).
  • Potato protoplasts transfected with Cip7-GFP displayed distinct spots of green fluorescence, indicating that a sufficient amount of recombinant protein was used for transfection (Fig. 7A).
  • Protoplasts transfected with recombinant proteins clearly showed a reduced survivability compared to protoplasts treated with only 40% PEG. This likely demonstrates the stress that the procedure poses to the cells. Even though not significant, there seemed to be a tendency of the commercial TrueCut Cas9 achieving a higher cell division ability than the purified Cas9-Doc1. This could indicate higher purity of the commercial Cas9-Doc1 leading to better cell division ability, pointing to an advantage of further purification steps.
  • One plausible reason for this observation could be traces of bacterial compounds in the purified protein samples that cause an increased immune response by the protoplasts where pathogens triggered the immune response of protoplasts and induced cell death.
  • the modularly interactive CRISPR system can expand the molecular toolbox for RNP delivery methods.
  • the expression and purification of the first components i.e. the Cas9 protein with its dockerin binding peptide (Cas9-Doc1) and the functional protein (reverse transferase with its cohesion binding peptide (Cip7-RT) was successful and will pave the way for further advancements of the system.
  • the components can be produced in large scale in bacterial cells and thereby provide a better cost efficiency and thus increase the accessibility to CRISPR-Cas gene editing.

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Abstract

L'invention concerne un système CRISPR-Cas modulaire pour l'administration de ribonucléoprotéine (RNP), le système CRISPR-Cas comprenant une protéine CRISPR-Cas fusionnée à un premier peptide de liaison, et une protéine fonctionnelle fusionnée à un deuxième peptide de liaison, le premier peptide de liaison et le deuxième peptide de liaison étant conçus pour se lier l'un à l'autre et définir une paire de peptides de liaison. Un procédé d'assemblage d'un complexe CRISPR-Cas pour l'administration de ribonucléoprotéine (RNP) et d'un complexe CRISPR-Cas, ainsi que l'utilisation du système CRISPR-Cas modulaire ou du complexe CRISPR-Cas pour modifier le génome d'un organisme, sont également présentés.
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