Patent Application
20250115933
The present invention refers to a method for editing the genomic sequence of at least one cell at at least one specific target site with an endonuclease with ability of collateral cleavage of RNA and/or DNA, a kit comprising an endonuclease to perform the method according to the present invention and the use of an endonuclease with ability of collateral cleavage of RNA and/or DNA for editing the genomic sequence of at least one cell at a specific sequence site.
Genome editing using classical CRISPR/Cas nucleases such as Cas9 or Cpf1 is a well-established and straightforward process. Nevertheless, there are a number of problems and disadvantages related to this technology:
Shortcomings and disadvantages of state of the art technologies for genome editing are therefore the occurrence of undesired off-target edits and/or the occurrence of unedited and wrongly edited cells and/or the missing or limited possibility to directly select for correctly edited cells based on their phenotype. In order to fully circumvent these drawbacks and obtain cells that are correctly edited and do/do not contain off-target edits, extensive clone characterization and screening are required. This can result in significantly increased workloads, costs and timelines.
The described issues can have negative impacts on or even prohibit the application of edited cells in experimental or commercial set ups. Furthermore, they can clearly increase workloads, project costs and timelines, because extensive clone characterization and screening are required.
The technical problem underlying the present invention is to overcome the outlined disadvantages of the state of the art. The technical problem is specially to provide a method which allows for genome editing without off-target effects as well as for the automatic removal of unedited and wrongly edited cells, resulting in pure populations with target edited cells.
The present invention solves the underlying technical problem by the subject matter of the independent claims.
The present invention solves the underlying technical problem preferably by a method for editing the genomic sequence of at least one cell at at least one specific target site comprising the steps:
The inventors have found that advantageously endonucleases with ability of collateral cleavage of RNA and/or DNA can be used in methods for editing the genomic sequence of at least one cell at at least one specific target site. For example, certain CRISPR/Cas systems comprise nucleases with so called “collateral activity” (CA nucleases), which means the nonspecific degradation of RNA or DNA upon target recognition by the nuclease. For example, the most prominent example of CRISPR CA nucleases acting on RNA level is Cas13a, whereas so far, the only known CA nuclease acting on DNA level is BEC, a chimeric enzyme.
The mode of action of CA nucleases can be described in the following:
This mode of action can be used advantageously in genome editing according to the method of the present invention, that differs from approaches based on classical CRISPR Cas nucleases. In contrast to current technologies, CA nuclease-based gene editing is free from off-target effects and allows the automatic deletion of unedited or wrongly edited cells, resulting in pure cell populations consisting of target edited cells. The present invention allows therefore for the easy and efficient generation and selection of correctly edited, off-target free cells without the need to increase workloads, costs or timelines.
In a preferred embodiment of the invention, a culture of one cell or of at least 100 cells, more preferably of at least 10000 cells, even more preferably 1000000 cells is used. The method can be used accordingly for single cell cloning or for cloning of a population of cells.
In a preferred embodiment of the invention the at least one cell or the cells of the cell culture are mammalian cells. In an alternative embodiment of the invention the at least one cell or the cells of the cell culture are plant cells, yeast cells or bacterial cells.
In a preferred embodiment of the invention the at least one cell or the cells of the cell culture are cells of a cell line, preferably cells of a mammalian cell line. In a preferred embodiment of the invention the at least one cell or the cells of the cell culture are CHO cells.
In a preferred embodiment of the invention the CHO cell or cells are selected from the group consisting of CHO-K1 cells, CHO-S cells, CHO DG44 cells, CHO DUKX cells and CHO GS knock out cells. In a preferred embodiment of the invention the CHO cell or cells are CHO-K1 cells. In a preferred embodiment of the invention the CHO cell or cells are CHO-S cells. In a particularly preferred embodiment of the invention the CHO cell or cells are CHO DG44 cells. CHO DG44 has the advantage that both copies of the dihydrofolate reductase (dhfr) are deleted, which allows for selection of recombinant clones. In a preferred embodiment of the invention the CHO cell or cells are CHO DUKX cells. In a preferred embodiment of the invention the CHO cell or cells are CHO GS knock out cells.
In a preferred embodiment of the invention the method comprises the further step c) isolating the living cells.
In a preferred embodiment of the invention the endonuclease or a nucleotide with a sequence encoding the endonuclease, the at least one donor DNA comprising the edited sequence and the at least one guide RNA are introduced into the at least one cell via transfection. The skilled person knows suitable transfection systems and methods in the state of the art.
There are a number of options, in which form the individual components can be delivered to the cells: The donor DNA can be provided as plasmid, as linear dsDNA or as linear ssDNA. The gRNA can be provided as plasmid or as synthetic RNA, The CA nuclease can be provided as plasmid, as mRNA or as protein. The different form of delivering the donor DNA, gRNA and CA nuclease to the cells can be combined.
Also, there are a number of options for the delivery modes of the donor DNA, the gRNA and the CA nuclease. The delivery, preferably transfection, can be made in a single step, in two steps or in three steps.
In a single step delivery, preferably transfection, all three, the donor DNA, the gRNA and the CA nuclease are delivered together. In a twostep delivery, preferably transfection, preferably in a first step the donor DNA is delivered separately and in a second step the gRNA and the CA nuclease are delivered together. In a three-step delivery, preferably transfection, preferably in a first step the donor DNA is delivered separately in a second step the gRNA is delivered separately and in a third step the CA nuclease is delivered separately. Of course, also other combinations are possible.
In a preferred embodiment of the invention the endonuclease or a DNA or RNA with a sequence encoding the endonuclease, the at least one donor DNA comprising the edited sequence and the at least one guide RNA or a DNA coding for such a gRNA are introduced into the at least one cell together in one transfection step or in sequential separate steps in any order and combination.
In a preferred embodiment of the invention, in step a1) the at least one donor DNA is introduced into the at least one cell as linear ssDNA, wherein preferably in following step a2) the at least one guide RNA is introduced into the at least one cell as synthetic RNA together with the endonuclease protein.
In a preferred embodiment, the donor DNA is introduced as circular or linearized plasmid. In an alternative embodiment, the donor DNA is introduced as linear dsDNA or linear ssDNA.
A so-called guide RNA (gRNA) that is characterized by a sequence complementary to a sequence stretch of the targeted RNA/genomic DNA is designed. In a preferred embodiment of the invention the guide RNA is in antisense orientation, which is advantageous for collateral cleavage of DNA and/or RNA. In an alternative embodiment of the invention the guide RNA is in sense orientation. In a further embodiment of the invention a part of the guide RNA is in antisense orientation and a part of the guide RNA is in sense orientation.
In a preferred embodiment of the invention the endonuclease is a CRISPR-associated endonuclease.
In a preferred embodiment of the invention the endonuclease comprises collateral RNA cleavage activity, preferably is a type III or VI Cas nuclease, more preferably a type VI Cas nuclease, more preferably a Cas13 nuclease, most preferably a Cas13a nuclease.
Preferably a Cas13a nuclease is used as an endonuclease comprising collateral RNA cleavage activity.
In a preferred embodiment of the invention the endonuclease comprises at least collateral DNA cleavage activity, preferably is a BEC nuclease. In a preferred embodiment of the invention the endonuclease comprises collateral DNA cleavage activity, preferably is a BEC nuclease.
In a preferred embodiment of the invention the endonuclease is a BEC nuclease. The BEC nuclease is described in PCT/EP2021/000081 (cf. WO2022017633 A2).
The BEC nuclease can be a BEC85, a BEC67 or a BEC10 nuclease. The BEC nuclease is preferably a BEC85 nuclease. The BEC nuclease is preferably a BEC67 nuclease. The BEC nuclease is preferably a BEC10 nuclease. The BEC nuclease can be a BEC85 or a BEC10 nuclease. The BEC nuclease can be a BEC67 or a BEC10 nuclease. The BEC nuclease can be a BEC85 or a BEC67 nuclease.
The genes encoding the endonuclease, preferably BEC endonuclease, more preferably BEC85, BEC67 and BEC10, can be codon-optimized for expression in the target cell, preferably for expression in CHO cells.
In a preferred embodiment of the invention the endonuclease comprises collateral DNA cleavage activity, preferably is a BEC nuclease, wherein the at least one cell or the cells of the cell culture are CHO cells, preferably selected from the group consisting of CHO-K1 cells, CHO-S cells, CHO DG44 cells, CHO DUKX cells and CHO GS knock out cells, preferably CHO DG44.
In a preferred embodiment of the invention the endonuclease comprises at least collateral DNA cleavage activity. In a preferred embodiment of the invention the endonuclease comprises collateral DNA and collateral RNA cleavage activity. In an alternative embodiment of the invention the endonuclease comprises collateral DNA but no collateral RNA cleavage activity.
In a preferred embodiment of the invention the endonuclease comprises at least collateral DNA cleavage activity, preferably is a BEC nuclease, wherein the at least one cell or the cells of the cell culture are CHO cells, preferably selected from the group consisting of CHO-K1 cells, CHO-S cells, CHO DG44 cells, CHO DUKX cells and CHO GS knock out cells, preferably CHO DG44.
In a preferred embodiment of the invention the endonuclease comprises at least collateral DNA cleavage activity, preferably is a BEC nuclease, wherein the at least one cell or the cells of the cell culture are CHO cells, preferably selected from the group consisting of CHO-K1 cells, CHO-S cells, CHO DG44 cells, CHO DUKX cells and CHO GS knock out cells, preferably CHO DG44.
In a preferred embodiment of the invention the endonuclease comprises at least collateral DNA cleavage activity, preferably is a BEC nuclease, wherein the at least one cell or the cells of the cell culture are CHO cells, preferably selected from the group consisting of CHO-K1 cells, CHO-S cells, CHO DG44 cells, CHO DUKX cells and CHO GS knock out cells, preferably CHO DG44, and wherein the guide RNA is in antisense orientation.
In a preferred embodiment of the invention the endonuclease is a BEC nuclease, wherein the at least one cell or the cells of the cell culture are CHO cells, preferably selected from the group consisting of CHO-K1 cells, CHO-S cells, CHO DG44 cells, CHO DUKX cells and CHO GS knock out cells, preferably CHO DG44.
In a preferred embodiment of the invention the endonuclease is a BEC nuclease, wherein the at least one cell or the cells of the cell culture are CHO cells, preferably selected from the group consisting of CHO-K1 cells, CHO-S cells, CHO DG44 cells, CHO DUKX cells and CHO GS knock out cells, preferably CHO DG44, and wherein the guide RNA is in antisense orientation.
In a preferred embodiment of the invention the DNA or RNA with a sequence encoding the endonuclease has a sequence that encodes an amino acid sequence of which is at least 85% identical, more preferably at least 90% identical, more preferably at least 93% identical, more preferably at least 95% identical, even more preferably at least 96% identical and most preferably at least 97% identical to the amino acid sequence of a BEC endonuclease, preferably a BEC85, a BEC67 or a BEC10 nuclease, especially to the according amino acid sequences SEQ ID NO: 1, 3 or 29 disclosed in PCT/EP2021/000081 (cf. WO2022017633 A2) and the according sequence listing. It is here referred to the respective sections of PCT/EP2021/000081 (cf. WO2022017633 A2: pages 3 to 34, as well as claims 1 to 15, sequence listing), which are herein incorporated by reference.
In a preferred embodiment of the invention the DNA or RNA with a sequence encoding the endonuclease has a sequence that encodes an amino acid sequence of which is at least 85% identical, more preferably at least 90% identical, more preferably at least 93% identical, more preferably at least 95% identical, even more preferably at least 96% identical and most preferably at least 97% identical to the amino acid sequence of a BEC endonuclease, preferably a BEC85, a BEC67 or a BEC10 nuclease, especially to the according amino acid sequences SEQ ID NO: 1, 3 or 29 disclosed in PCT/EP2021/000081 (cf. WO2022017633 A2) and the according sequence listing. It is here referred to the respective sections of PCT/EP2021/000081 (cf. WO2022017633 A2: pages 3 to 34, as well as claims 1 to 15, sequence listing), which are herein incorporated by reference), wherein the at least one cell or the cells of the cell culture are CHO cells, preferably selected from the group consisting of CHO-K1 cells, CHO-S cells, CHO DG44 cells, CHO DUKX cells and CHO GS knock out cells, preferably CHO DG44.
In a preferred embodiment of the invention the DNA or RNA with a sequence encoding the endonuclease has a sequence that encodes an amino acid sequence of which is at least 85% identical, more preferably at least 90% identical, more preferably at least 93% identical, more preferably at least 95% identical, even more preferably at least 96% identical and most preferably at least 97% identical to the amino acid sequence of a BEC endonuclease, preferably a BEC85, a BEC67 or a BEC10 nuclease, especially to the according amino acid sequences SEQ ID NO: 1, 3 or 29 disclosed in PCT/EP2021/000081 (cf. WO2022017633 A2) and the according sequence listing. It is here referred to the respective sections of PCT/EP2021/000081 (cf. WO2022017633 A2: pages 3 to 34, as well as claims 1 to 15, sequence listing), which are herein incorporated by reference), wherein the at least one cell or the cells of the cell culture are CHO cells, preferably selected from the group consisting of CHO-K1 cells, CHO-S cells, CHO DG44 cells, CHO DUKX cells and CHO GS knock out cells, preferably CHO DG44, and wherein the guide RNA is in antisense orientation.
In a preferred embodiment, upon genomic integration of the edited sequence at the at least one target site via homologous recombination, the sequence of the at least one target site is changed in such a way that the at least one guide RNA sequence cannot bind to the modified at least one target site any more. In a preferred embodiment, upon genomic integration of the edited sequence at the at least one target site via homologous recombination, the sequence of the at least one target site is changed in such a way that that the collateral cleavage of the endonuclease gets activated.
In a preferred embodiment the sequence of the first homology arm is at least 81% identical to the genomic sequence upstream of the at least one target site and the sequence of the second homology arm is at least 81% identical to the genomic sequence downstream of the at least one target site. In a preferred embodiment the sequence of the first homology arm is at least 85% identical to the genomic sequence upstream of the at least one target site and the sequence of the second homology arm is at least 85% identical to the genomic sequence downstream of the at least one target site. In a preferred embodiment the sequence of the first homology arm is at least 90% identical to the genomic sequence upstream of the at least one target site and the sequence of the second homology arm is at least 90% identical to the genomic sequence downstream of the at least one target site. In a preferred embodiment the sequence of the first homology arm is at least 91% identical to the genomic sequence upstream of the at least one target site and the sequence of the second homology arm is at least 91% identical to the genomic sequence downstream of the at least one target site. In a preferred embodiment the sequence of the first homology arm is at least 95% identical to the genomic sequence upstream of the at least one target site and the sequence of the second homology arm is at least 95% identical to the genomic sequence downstream of the at least one target site. In a preferred embodiment the sequence of the first homology arm is at least 97% identical to the genomic sequence upstream of the at least one target site and the sequence of the second homology arm is at least 97% identical to the genomic sequence downstream of the at least one target site. In a preferred embodiment the sequence of the first homology arm is at least 98% identical to the genomic sequence upstream of the at least one target site and the sequence of the second homology arm is at least 98% identical to the genomic sequence downstream of the at least one target site. In a preferred embodiment the sequence of the first homology arm is at least 99% identical to the genomic sequence upstream of the at least one target site and the sequence of the second homology arm is at least 99% identical to the genomic sequence downstream of the at least one target site. In a preferred embodiment the sequence of the first homology arm is 100% identical to the genomic sequence upstream of the at least one target site and the sequence of the second homology arm is 100% identical to the genomic sequence downstream of the at least one target site.
In a preferred embodiment the sequence of the first homology arm is at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the genomic sequence upstream of the at least one target site.
In a preferred embodiment the sequence of the second homology arm is at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the genomic sequence downstream of the at least one target site.
There can be at least one target site, i.e. there can be more than one target sites. In a preferred embodiment there is one target site.
In a preferred embodiment the sequence of the first homology arm is at least 80% identical to the genomic sequence upstream of the target site and the sequence of the second homology arm is at least 80% identical to the genomic sequence downstream of the target site.
In a preferred embodiment the sequence of the first homology arm is at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the genomic sequence upstream of the target site.
In a preferred embodiment the sequence of the second homology arm is at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the genomic sequence downstream of the target site.
In an alternative embodiment there can be also 2 target sites, 3 target sites, 4 target sites, 5 target sites or more than 5 target sites.
The method according to the present invention can be used for example also in genome editing approaches that require or benefit from the absence of off-target effects and/or are suffering from low editing efficiencies, e.g. in case of knock-in or complex knock-out edits and/or do not allow the direct selection of edited cells based on phenotypes and/or require or benefit from the automatic removal of unedited and wrongly edited cells, leading to pure populations consisting of target edited cells.
The present invention refers also to a kit comprising an endonuclease with ability of collateral cleavage of RNA and/or DNA and specific gRNA and/or a donor DNA. Preferably, the endonuclease is an endonuclease as described herein. Preferably, the endonuclease is an endonuclease as described herein, preferably an endonuclease with ability of collateral cleavage of DNA or at least DNA, more preferably a BEC endonuclease. Preferably the at least one cell is a CHO cell.
Preferably the kit according to the present invention is for use in a method according to the present invention. Preferably the kit according to the present invention is for use in a method according to the present invention, wherein the cells are CHO cells.
Preferably, the kit comprises a manual for use of the kit in a method according to the present invention.
The present invention refers also to a use of an endonuclease with ability of collateral cleavage of RNA and/or DNA for editing the genomic sequence of at least one cell at a specific sequence site. Preferably, the endonuclease is an endonuclease as described herein, preferably an endonuclease with ability of collateral cleavage of DNA or at least DNA, more preferably a BEC endonuclease. Preferably the at least one cell is a CHO cell. Preferred is a use of an endonuclease with ability of collateral cleavage of DNA or at least DNA for the selection of editing events in the genomic sequence of at least one cell at a specific sequence site, wherein the at least one cell is a CHO cell.
Preferred embodiments of the invention are also disclosed in the dependent claims.
The invention will be explained in more detail in the examples and the figures.
The described approach allows for genome editing without off-target effects as well as for concomitant removal of unedited and wrongly edited cells, leading to enriched or essentially pure populations consisting of target edited cells.
The goal of the present experiment is the generation and selection of engineered CHO cells with inactivated fucosyltransferase, by the insertion of stop codons in Exon 3 of the FUT8 (fucosyltransferase 8) gene. The donor DNA is synthesized as ssDNA fragment (SEQ ID No. 2) in which the stop codon sequence (SEQ ID No. 1) is flanked by ca. 750 bp FUT8 sequences surrounding the desired integration site as the 5′ and 3′ homology arms. 2E6 CHO DG44 cells are transfected with 150 pmol BEC10 nuclease, 600 pmol synthetic gRNA (SEQ ID No. 3) and 4 μg donor DNA by electroporation using a Neon system from Thermo Fisher. For comparison reasons, two additional transfections are executed as follows:
Following transfection, the cells are transferred into culture medium and cultivated. After 3 days, genomic DNA is isolated and amplified by PCR using primers flanking the target site followed by Sanger sequencing. The generated sequencing data are analyzed using the ICE analysis tool provided by Synthego (https://ice.synthego.com/#/) in order to quantify the portion of correctly edited cells and compare the three different transfection approaches with each other and to measure editing efficiencies and confirm successful editing.
As expected, the ratio of edited cells was significantly higher with BEC10 as compared to MAD7. Moreover, the overall number of cells was drastically reduced by BEC10, since only correctly edited cells survive, which carry an inactivated FUT-8. With donor DNA (negative control) no editing and no reduction of cell number occurred.
The goal of the present experiment is the generation and selection of engineered CHO cells with inactivated fucosyltransferase, by the insertion of stop codons in Exon 3 of the FUT8 gene. The donor DNA is synthesized as ssDNA fragment (SEQ ID NO 2) in which the stop codon sequence (SEQ ID No. 1) is flanked by ca. 750 bp FUT8 sequences surrounding the desired integration site as the 5′ and 3′ homology arms. 2E6 CHO DG44 cells are transfected with Cas13 nuclease, synthetic gRNA (SEQ ID NO: 11) and 4 μg donor DNA by electroporation using a Neon system from ThermoFisher. For comparison reasons, two additional transfections are executed as follows:
Following transfection, the cells are transferred into culture medium and cultivated. After 3 days, genomic DNA is isolated and amplified by PCR using primers flanking the target site followed by Sanger sequencing. The generated sequencing data are analyzed using the ICE analysis tool provided by Synthego (https://ice.synthego.com/#/) in order to quantify the portion of correctly edited cells and compare the three different transfection approaches with each other and to measure editing efficiencies and confirm successful editing.
As expected, the ratio of edited cells was significantly higher with Cas13 as compared to MAD7. Moreover, the overall number of cells was drastically reduced by Cas13, since only correctly edited cells survive, which carry an inactivated FUT-8. With donor DNA (negative control) no editing and no reduction of cell number occurred.
The goal of the present experiment is the generation and selection of engineered CHO cells with inactivated LPL (lipoprotein lipase), by the insertion of stop codons in Exon 3 of the LPL gene. The donor DNA is synthesized as ssDNA fragment (SEQ ID No. 5) in which the stop codon sequence (SEQ ID No. 1) is flanked by ca. 60 bp LPL sequences surrounding the desired integration site as the 5′ and 3′ homology arms. 2E6 CHO DG44 cells are transfected with 150 pmol BEC85 nuclease, 600 pmol synthetic gRNA (SEQ ID No. 6) and 4 ag donor DNA by electroporation using a Neon system from Thermo Fisher. For comparison reasons, two additional transfections are executed as follows:
Following transfection, the cells are transferred into culture medium and cultivated. After 3 days, genomic DNA is isolated and amplified by PCR using primers flanking the target site followed by Sanger sequencing. The generated sequencing data are analyzed using the ICE analysis tool provided by Synthego (https://ice.synthego.com/#/) in order to quantify the portion of correctly edited cells and compare the three different transfection approaches with each other and measure editing efficiencies and confirm successful editing.
As expected, the ratio of edited cells was significantly higher with BEC85 as compared to MAD7. Moreover, the overall number of cells was drastically reduced by BEC85, since only correctly edited cells survive, which carry an inactivated LPL. With donor DNA (negative control) no editing and no reduction of cell number occurred.
The goal of the present experiment is the generation and selection of engineered CHO cells with inactivated BGN (biglycan), by the insertion of stop codons in Exon 2 of the BGN gene. The donor DNA is synthesized as ssDNA fragment (SEQ ID No. 8) in which the stop codon sequence (SEQ ID No. 1) is flanked by ca. 500 bp BGN sequences surrounding the desired integration site as the 5′ and 3′ homology arms. 2E6 CHO DG44 cells are transfected with 150 pmol BEC67 nuclease, 600 pmol synthetic gRNA (SEQ ID No. 9) and 4 μg donor DNA by electroporation using a Neon system from Thermo Fisher. For comparison reasons, two additional transfections are executed as follows:
Following transfection, the cells are transferred into culture medium and cultivated. After 3 days, genomic DNA is isolated and amplified by PCR using primers flanking the target site followed by Sanger sequencing. The generated sequencing data are analyzed using the ICE analysis tool provided by Synthego (https://ice.synthego.com/#/) in order to quantify the portion of correctly edited cells and compare the three different transfection approaches with each other and measure editing efficiencies and confirm successful editing.
As expected, the ratio of edited cells was significantly higher with BEC67 as compared to MAD7. Moreover, the overall number of cells was drastically reduced by BEC67, since only correctly edited cells survive, which carry an inactivated BGN. With donor DNA (negative control) no editing and no reduction of cell number occurred.
| Number | Date | Country | Kind |
|---|---|---|---|
| 22152438.2 | Jan 2022 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2023/051394 | 1/20/2023 | WO |
