Cytoplasmic delivery of genome editing tools

Abstract

The first aspect of the present invention relates to the use of saponin in the in vitro delivery of nucleic acid into cells. Typically, the nucleic acid is a plasmid DNA having a relatively large size, for example at least 5.5 kbp. The second aspect of the present invention relates to a method for delivering a nucleic acid encoding a CRISPR / Cas construct into a cell in vitro. Typically, the nucleic acid is a plasmid DNA having a relatively large size, for example at least 5.5 kbp. The third aspect of the present invention relates to a kit of parts for delivering a nucleic acid encoding a CRISPR / Cas construct into a cell in vitro. In addition, the present invention also relates to nanoparticles suitable for the in vitro delivery of nucleic acid into cells, the nanoparticles comprising or consisting of a nucleic acid encoding a CRISPR / Cas construct, a polylysine peptide, and optionally a saponin.

Description

[Technical field]

[0001] The first aspect of the present invention relates to the use of saponin in the in vitro delivery of relatively large nucleic acids into cells, the nucleic acids comprising more than 5.5 kilobase pairs (kbp). The second aspect of the present invention relates to a method for delivering such nucleic acids encoding CRISPR / Cas constructs into cells in vitro in the presence of saponin. The third aspect of the present invention relates to a kit of parts for delivering nucleic acids encoding CRISPR / Cas constructs into cells in vitro in the presence of saponin. In addition, the present invention also relates to nanoparticles suitable for the in vitro delivery of nucleic acids into cells, the nanoparticles comprising or consisting of a nucleic acid encoding a CRISPR / Cas construct, a polylysine peptide, and optionally saponin. [Background technology]

[0002] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 technology - often called "genetic scissors" - is a tool that has revolutionized the field of genetic technology in the last few years. Its simple, cheap and fast manipulation allows precise excision or insertion of DNA sequences or even genes from genomes (genome editing). These properties make CRISPR-Cas9 a useful and promising technology for gene therapy that outperforms other already existing gene editing tools. The real purpose of CRISPR-Cas9 is the antiviral defense mechanism of the bacterium Streptococcus pyogenes against bacteriophages.

[0003] CRISPR-Cas9 consists of three components: Cas9 is a bacterial enzyme that induces strand breaks (a nuclease for double-strand breaks and a nickase for single-strand breaks). To help the nuclease locate its enzymatic activity, the trans-activating CRISPR-RNA (tracrRNA) and the CRISPR-RNA (crRNA) form a guide RNA (gRNA), which provides the Cas9 enzyme with the information it needs for precise cleavage.

[0004] During bacteriophage infection, the crRNA possesses or acquires the sequence of the phage sequence and thereby the information to induce double-strand breaks and prevent infection. The purpose of the tracrRNA is to stabilize the crRNA and together form the CRISPR-Cas9 complex, and after annealing of the crRNA sequence to its target, the Cas9 nuclease can cut the phage sequence. The cell's repair mechanism then leads to mutation, resulting in gene inactivation (knockout).

[0005] This discovered defense mechanism set in motion a revolution in genome editing when the idea arose to target selected genes by designing artificial crRNAs, allowing CRISPR-Cas9-based knock-out or insertion ("knock-in") of genes, distinguishing itself from other genome editing techniques by its simple, rapid, and cost-effective applicability.

[0006] The introduction of Cas9 nuclease together with gRNA into the cytosol is possible in the form of DNA or RNA encoding the enzyme. However, the transfection efficiency is poor, and the use of Cas9 nuclease in vitro by introducing (plasmid) DNA encoding Cas9 is hindered due to the low transfection efficiency of relatively large DNA constructs, such as large plasmid DNA. Therefore, Cas9 nuclease / protein itself is usually delivered with the help of commercial reagents (e.g. Lipofectamine™ CRISPRMAX™ Cas9 transfection reagent). Because this method shows more efficient transfection and less off-target effects, and the poor DNA transfection efficiency is prevented by the in vitro delivery of protein into cells.

[0007] To the best of our knowledge, there is no in vitro non-viral transfection enhancer that can deliver relatively large DNA constructs, such as Cas9 DNA plasmids, i.e., plasmid DNAs that code for Cas9 and have a size of at least 5.5 kilobase pairs (kbp), in a simple and efficient manner.Moreover, no universal transfection enhancer that can deliver DNA, such as plasmid DNA, is available to practitioners in the field of in vitro Cas9 DNA delivery into cells. Summary of the Invention

[0008] The present inventors have surprisingly found that efficient in vitro delivery of relatively large plasmids, such as CRISPR / Cas9 nucleic acid constructs, can be established by applying plant defense molecules, i.e. saponins, during cell transfection. A certain group of triterpenoid saponins (bisdesmosidic triterpenoid 12,13-dehydrooleanane saponins), such as SO1861 and GE1741, can significantly increase the transfection efficiency of oligolysine-based nanoplexes (Sama et al., 2017; Sama et al., 2018). By this invention, the inventors have now established: (i) the feasibility of saponin-based transfection (coined by the inventors as sapofection) of nucleic acids and polylysine-containing peptide-based nanoparticles; (ii) the universal complexation of nucleic acids such as DNA, mRNA, and minicircle DNA; and (iii) the use of non-toxic concentrations of saponin, i.e., 1 μg / mL to 5 μg / mL, makes saponin a suitable component of DNA-containing compositions for application in the field of transfection of large plasmid DNA.

[0009] A first aspect of the present invention relates to the use of triterpenoid 12,13-dehydrooleanane-type saponins of the bisdesmosidic type, in particular saponins according to formula (I), in the in vitro delivery of nucleic acids into cells:

[0010] [ka]

[0011] During the ceremony, R 1 is a xylose, arabinose, or glucose residue, which is attached by its C1 atom to the corresponding xylose residue of formula (I), R 2 is different from other R on the same molecule. 2 independently of the residue, H, an acetyl residue, or a xylose residue bound by its C1 atom to the corresponding quinovose residue of formula (I), provided that at least two acetyl residues or at least one acetyl residue and a xylose residue bound to the quinovose residue are present in the saponin; The nucleic acid comprises at least 5.5 kilobase pairs (kbp) or more, e.g., a first portion of nucleic acid encoding a CRISPR-associated endonuclease (Cas), and optionally a second portion of nucleic acid encoding or being or comprising a guide RNA (gRNA).

[0012] The present inventors have previously demonstrated that plant secondary metabolites from the carnation family enhance the intracellular delivery of relatively small sized plasmid DNA (<5 kbp), minicircle DNA (~3 kbp), and mRNA (<1 kbp). The DNA transfection technique using triterpene saponins has been coined sapofection, suggesting that saponins are used as a tool to improve the transfection process. A disadvantage of commonly used transfection reagents other than saponins is their low efficiency when relatively large plasmids such as CRISPR / Cas9 constructs have to be transfected into target cells. The present inventors now show that plant secondary metabolites from Dypsophila elegans M. beeve and Saponaria officinalis L. improve the intracellular delivery of large nucleic acid constructs (e.g., plasmid DNA with a size >8,000 bp), such as CRISPR / Cas9 constructs. The inventors have surprisingly established that these saponin compounds, e.g. GE1741 and SO1861, which are bisdesmosidic triterpenoid 12,13-dehydrooleanane-type saponins, contribute to the efficient delivery of complexed CRISPR / Cas9 constructs into eukaryotic cells.

[0013] Triterpene saponins are plant-derived secondary metabolites synthesized by a significant number of plants. We have previously demonstrated that characterized triterpene saponins, such as SO1861 from Saponaria officinalis L., GE1741 from Gypsophila elegans M. Beebe, and AG1856 from Agrostemma gitago L., significantly increase the transfection efficiency of oligolysine-based nanoplexes containing (small-sized) plasmid DNA, minicircle DNA, and mRNA (Clochard et al., 2020; Sama et al., 2018a; Sama et al., 2017). The simple feasibility of these transfections makes triterpene saponins a novel and promising player in the field of transfection.

[0014] CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 technology allows precise deletion of genes on chromosomal DNA and insertion of DNA sequences into genomes. CRISPR / Cas9 consists of three components: Cas9 is an endonuclease that induces strand breaks; tracerRNA (trRNA) and CRISPR-RNA (crRNA) guide Cas9-nuclease to the desired cleavage site. The number of non-viral transfection reagents suitable for delivery of relatively large Cas9DNA plasmid constructs (plasmid DNA size 7,000bp or larger) is limited or even non-existent.

[0015] The present inventors have now demonstrated that GE1741 and SO1861 are powerful facilitators and tools for the delivery of large nucleic acids, including CRISPR / Cas9 DNA plasmid constructs, into eukaryotic cells.In addition, the present inventors have also demonstrated that efficient knockout experiments were successfully established by GFP donor sequences transfected as part of large plasmid DNA (greater than 7,000 bp in size) when transfection was in the presence of saponins such as GE1741.

[0016] A second aspect of the invention relates to a method of delivering a nucleic acid comprising more than 5.5 kilobase pairs (kbp), e.g., encoding a CRISPR / Cas construct, into a cell in vitro, comprising the steps of: (i) providing a nucleic acid and providing a saponin according to formula (I);

[0017] [ka]

[0018] During the ceremony, R 1 is a xylose, arabinose, or glucose residue, which is attached by its C1 atom to the corresponding xylose residue of formula (I), R 2 is different from other R on the same molecule. 2 independently of the residue, H, an acetyl residue, or a xylose residue bound to the corresponding quinovose residue of formula (I) by its C1 atom, provided that at least two acetyl residues or at least one acetyl residue and a xylose residue bound to the quinovose residue are present in the saponin; and (ii) incubating the nucleic acid and the cells in the presence of saponin; Here, for example, the nucleic acid encoding the CRISPR / Cas construct includes at least a first portion of nucleic acid that encodes a Cas, and the nucleic acid optionally includes a second portion of nucleic acid that encodes or comprises or is a gRNA.

[0019] For complexation of the large plasmid DNA CMV-CAS9-2A-GFP (8.2 kbp) (Sigma-Aldrich, Taufkirchen, Germany), polylysine (K 16 Nanoplexes based on P(peptide)D(Cas9-GFP plasmid) were formulated (Figure 1). The average size and PDI (polydispersity index) of P(peptide)D(Cas9-GFP plasmid) nanoplexes formulated at different mass ratios were measured using a Malvern Nano Zetasizer (Malvern Instruments Ltd., UK). A trend towards lower size could be observed at higher mass ratios. The PDI showed a certain monodisperse distribution, n > 3. We also determined the complexation efficiency of the nanoplexes by applying agarose-based gel electrophoresis and Quant-IT-PicoGreen assays known in the art (Sama et al., 2017, Figure 3). By applying gel retention assay and quantitative complexation efficiency, the DNA retention potential of the PD nanoplex formulations served as a qualitative measure of DNA complexation efficiency. The nanoplex formulations retained DNA and did not allow it to migrate on the gel. The quantitative complexation assay showed efficient complexation for all formulations containing plasmid DNA and polylysine peptide.

[0020] After the biophysical characterization established that large nanoplexes of plasmid DNA and polylysine peptide were formed, cell experiments, transfections were performed. Due to the peptide / DNA (N / P) ratio, the tests performed by the inventors revealed toxic effects of nanoplexes on cells at peptide to plasmid DNA mass ratios of 16:1 or higher, in addition, further tests performed by the inventors revealed inefficient nucleic acid delivery at peptide to plasmid DNA mass ratios of 2:1 or lower. Without wishing to be bound by any theory, the latter effect is most likely due to insufficient complexation of DNA. Thus, the inventors demonstrate the benefits of the present invention by examples performed with PD nanoplexes formulated at 4:1 and 8:1 ratios. JIMT-1 cells (DSMZ, no.ACC589) and Neuro-2A cells (DSMZ, no.ACC148) were transfected with nanoplexes containing relatively large Cas9-GFP plasmids, with and without GE1741 or SO1861 co-application, at concentrations that are non-toxic when the cells to be transfected are considered. The transfection efficiency of all transfection conditions was measured after 48 hours of incubation time using flow cytometry, in terms of fluorescence intensity, compared to negative controls (e.g., cells contacted with nanoparticles in the absence of saponin). The efficiency of GE1741-based and SO1861-based transfection was also compared to the efficiency of nanoparticle Lipofectamine3000™-based transfection. Transfection was performed according to protocols commonly known and applied in the art (Clochard et al., 2020; Sama et al., 2018a; Sama et al., 2017; Sama et al., 2018b). Since transfection efficiency did not increase from a peptide / DNA ratio of 4.1 to a ratio of 8:1 in Neuro-2A cells, a ratio of 4:1 was used for further testing and examples with JIMT-1 cells.The transfection efficiency of GE1741- and SO1861-mediated PD transfection in Neuro-2A and JIMT-1 cells was evaluated. Cells were transfected with PD nanoplexes containing Cas9-GFP DNA with and without GE1741 or SO1861 (final concentration of saponin was 2 μg / ml). In Neuro-2A and JIMT-1 cells, the transfection efficiency varied between 30% and 40%. Lipofectamin3000 served as a reference, n≧3. Significant differences were evident compared to transfection without GE1741 or transfection without SO1861. U test, p≦0.05.

[0021] Compared with SO1861, GE1741 showed slightly better transfection regulation properties. In further knockout experiments, GE1741 and Neuro-2A cells were tested. Integration of a linear donor sequence (LDS) containing the gene for GFP onto genomic DNA was evaluated using three kits from Origene (Herford, Germany): Slc19a3-KN2.0 (CAT#KN515911), Slc25a24-KN2.0 (CAT#KN515970), and Slc26a4-KN2.0 (CAT#KN516006). All three of these kits were non-homology-mediated CRISPR mouse gene knockout kits. The kits contained an all-in-one plasmid encoding Cas9 and target-specific sgRNA. The Slc26a4 kit targeted solute transporter family 26 member 4 on chromosome 12. Indel efficiency by targeting the Slc26a4 locus could already be shown in Neuro-2A cells (Ryu et al., 2018). And for the linear donor kit (Slc19a3, Slc25a24), two different sgRNA sequences were tested. In the Examples section, Figure 9 demonstrates the transfection efficiency in the presence of GE1741. GFP-mediated fluorescence indicates efficient delivery and integration of the linear donor sequence onto chromosomal DNA under the influence of the presence of saponin. Therefore, to compare the efficiency of different targets (genes to be knocked out) and different integration techniques for knocking out different solute transporter genes (Slc) in Neuro-2A cells by CRISPR / Cas9, Neuro-2A cells were transfected with different kits (see Figures 8 and 10). A significant increase in transfection efficiency was observed for cells treated with GE1741 compared to transfection without GE1741. U test, p ≤ 0.05 (n ≥ 3).

[0022] These examples therefore surprisingly established that bisdesmosidic triterpenoid 12,13-dehydrooleanane type saponins, in particular saponin GE1741 from Gypsophila elegans M. Beebe and SO1861 from Saponaria officinalis L., are suitable for efficient delivery of relatively large DNA, such as CRISPR / Cas9 constructs (e.g. plasmid DNA), having a size of 6.5 kbp or more, up to 8.2 kbp or more, into eukaryotic cells in a simple and efficient manner.

[0023] A third aspect of the invention relates to a kit of parts for delivering a nucleic acid containing more than 5.5 kbp, preferably more than 8 kbp, e.g. encoding a CRISPR / Cas construct, into a cell in vitro, comprising: A first combination comprising or consisting of: (a) a first container comprising a first plasmid DNA, e.g., encoding Cas9; (b) optionally, a second container containing a second plasmid DNA, e.g., encoding a gRNA; (c) Polylysine K 16 a third container containing a peptide; (d) a fourth container containing at least one bisdesmosidic triterpenoid 12,13-dehydrooleanane-type saponin, preferably one of GE1741 and SO1861; (e) Instructions for use; Here, the use includes at least polylysine K 16 Preparation of nanoplexes of a peptide and a first plasmid DNA, and optionally a saponin; The first plasmid has a size of at least 5.5 kbp, or A second combination comprising or consisting of: (a) a first container comprising a third plasmid DNA, e.g., encoding Cas9 and encoding a gRNA; (b) Polylysine K 16 a second container containing a peptide; (c) a third container containing at least one bisdesmosidic triterpenoid 12,13-dehydrooleanane-type saponin, preferably one of GE1741 and SO1861; (d) Instructions for use; Here, the use includes at least polylysine K 16 preparation of nanoplexes of a peptide and a third plasmid DNA, and optionally a saponin; The third plasmid has a size of at least 5.5 kbp, or A third combination comprising or consisting of: (a) a first container comprising a first plasmid DNA, e.g., encoding Cas9; (b) optionally a second container containing oligonucleotides, such as RNA oligonucleotides, e.g., RNA oligonucleotides of a gRNA; (c) Polylysine K 16 a third container containing a peptide; (d) a fourth container containing at least one bisdesmosidic triterpenoid 12,13-dehydrooleanane-type saponin, preferably one of GE1741 and SO1861; (e) Instructions for use; Here, the use includes at least polylysine K 16 Preparation of nanoplexes of a peptide and a first plasmid DNA, and optionally a saponin; The first plasmid has a size of at least 5.5 kbp.

[0024] The present invention also relates to a composition comprising nanoparticles suitable for in vitro delivery of a nucleic acid, e.g., encoding a CRISPR / Cas construct, into a cell, wherein the nanoparticle comprises or consists of: (i) a nucleic acid comprising more than 5.5 kilobase pairs (kbp), e.g., encoding a CRISPR / Cas construct; (ii) a polylysine peptide, and (iii) saponins that are triterpenoid 12,13-dehydrooleanane-type saponins of the bisdesmosidic type; In some cases, wherein the nucleic acid encoding the CRISPR / Cas construct comprises at least a first portion of nucleic acid encoding a CRISPR-associated endonuclease (Cas), and the nucleic acid optionally comprises a second portion of nucleic acid encoding or being or comprising a guide RNA (gRNA).

[0025] A preferred composition is a composition comprising nanoparticles suitable for in vitro delivery of a nucleic acid encoding a CRISPR / Cas construct into a cell of the invention, further comprising a saponin according to formula (I):

[0026] [ka]

[0027] During the ceremony, R 1 is a xylose, arabinose, or glucose residue, which is attached by its C1 atom to the corresponding xylose residue of formula (I), R 2 is different from other R on the same molecule. 2 Independently of the residue, it is H, an acetyl residue, or a xylose residue bound to the corresponding quinovose residue of formula (I) by its C1 atom, provided that at least two acetyl residues or at least one acetyl residue and a xylose residue bound to the quinovose residue are present in the saponin.

[0028] The nucleic acid encoding the CRISPR / Cas construct comprises at least a first portion of nucleic acid encoding a CRISPR-associated endonuclease (Cas), and the nucleic acid optionally comprises a second portion of nucleic acid encoding or being or comprising a guide RNA (gRNA). Preferably, the saponin is GE1741 or SO1861, more preferably GE1741. Preferably, the polylysine peptide consists of 16 lysine residues. Preferably, the nucleic acid is provided as part of a plasmid DNA. Preferably, the encoded Cas is Cas9 (SEQ ID NO: 1), or a Cas having at least 90% sequence identity to Cas9.

[0029] While the present invention will be described with respect to specific embodiments, the invention is not limited thereto but only by the claims. The embodiments of the invention described herein can be combined and worked together unless otherwise specified.

[0030] definition The terms first, second, third, and the like are used in the specification and claims not necessarily to describe a sequential or chronological order, but rather, for example, to distinguish between similar elements, compositions, components of compositions, or separate method steps. The terms are interchangeable under appropriate circumstances, and embodiments of the invention may function in sequences other than those described or exemplified herein, unless otherwise specified.

[0031] The embodiments of the invention described herein can be combined and worked together unless otherwise specified.

[0032] Furthermore, references to various embodiments as "preferably" or "eg" or "for example" or "particularly" and the like should not be construed as limiting the scope of the invention, but rather as exemplary ways in which the invention may be implemented.

[0033] The term "comprises" used in the claims should not be construed as being limited to the subsequently listed elements or composition, method, use, method step or component. It does not exclude other elements or method steps or components in a certain composition. It needs to be construed as specifying the presence of the claimed feature, object, (method) step, or component referred to, but does not preclude the presence or addition of one or more other features, objects, steps, or components, or groups thereof. Thus, the scope of the expression "a method comprising steps A and B" should not be limited to a method consisting of only steps A and B, but rather, for the present invention, the only recited steps of the method are A and B, and the claims should be construed to encompass equivalents of those method steps. Thus, the scope of the expression "a composition comprising components A and B" should not be limited to a composition consisting of only components A and B, but rather, for the present invention, the only recited components of the composition are A and B, and the claims should be construed to encompass equivalents of those components.

[0034] In addition, the reference to an element or component by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element or component is present, unless the context clearly requires that there is only one of the element or component. The indefinite article "a" or "an" therefore normally means "at least one". [Brief description of the drawings]

[0035] [Figure 1] Table presenting the average size and PDI of PD nanoplexes formulated at different mass ratios. Peptide / DNA (PD) nanoplexes were formulated at different mass ratios to evaluate optimal formulations with low particle size and preferably monodisperse size distribution. A trend towards lower size was observed at higher mass ratios. PDI showed a constant monodisperse distribution. n>3. [Figure 2A]Size distribution of DNA-loaded (D) oligolysine-based (P) nanoplexes. PD nanoplexes loaded with Cas9DNA (A), GFPDNA (B), or GFP-Cas9DNA (C) (4:1 mass ratio for peptide mass:DNA mass) showed a major size distribution around 120 nm. The percentage of aggregated particles was low (small peak in the size range of 1,000–10,000 nm). [Figure 2B] Size distribution of DNA-loaded (D) oligolysine-based (P) nanoplexes. PD nanoplexes loaded with Cas9DNA (A), GFPDNA (B), or GFP-Cas9DNA (C) (4:1 mass ratio for peptide mass:DNA mass) showed a major size distribution around 120 nm. The percentage of aggregated particles was low (small peak in the size range of 1,000–10,000 nm). [Figure 2C] Size distribution of DNA-loaded (D) oligolysine-based (P) nanoplexes. PD nanoplexes loaded with Cas9DNA (A), GFPDNA (B), or GFP-Cas9DNA (C) (4:1 mass ratio for peptide mass:DNA mass) showed a major size distribution around 120 nm. The percentage of aggregated particles was low (small peak in the size range of 1,000–10,000 nm). [Figure 3A]Gel retention assay for a series of nanoplex formulations encompassing quantitative complexation efficiency. The DNA retention potential of the PD nanoplex formulations served as a qualitative measure of DNA complexation efficiency. Formulated nanoplexes were applied to agarose gel pockets, after which voltage was applied. Well-complexed nanoplexes showed low ethidium bromide staining and no migration on the gel. Compared to free migrating DNA (left lane in Figure 3A and Figure 3B, 0:1), none of the nanoplex formulations exited their pockets in any of lanes 2-8 from left to right. The signal of PD (GFP-DNA; last three lanes in Figure 3A and Figure 3B, lanes 6-8 from left) appeared weaker, indicating better complexation of the smaller DNA. Quantitative complexation assays showed efficient complexation for all formulations. GFP-loaded nanoplexes were best complexed, the larger sized plasmid (Cas9-GFP-DNA; Figure 3B, lanes 2-5 from left) was slightly less complexed, and the complexation efficiency for the PD(Cas9DNA) plasmid (Figure 3A, lanes 2-5 from left) was approximately between those of the other two. [Figure 3B]Gel retention assay for a series of nanoplex formulations encompassing quantitative complexation efficiency. The DNA retention potential of the PD nanoplex formulations served as a qualitative measure of DNA complexation efficiency. Formulated nanoplexes were applied to agarose gel pockets, after which voltage was applied. Well-complexed nanoplexes showed low ethidium bromide staining and no migration on the gel. Compared to free migrating DNA (left lane in Figure 3A and Figure 3B, 0:1), none of the nanoplex formulations exited their pockets in any of lanes 2-8 from left to right. The signal of PD (GFP-DNA; last three lanes in Figure 3A and Figure 3B, lanes 6-8 from left) appeared weaker, indicating better complexation of the smaller DNA. Quantitative complexation assays showed efficient complexation for all formulations. GFP-loaded nanoplexes were best complexed, the larger sized plasmid (Cas9-GFP-DNA; Figure 3B, lanes 2-5 from left) was slightly less complexed, and the complexation efficiency for the PD(Cas9DNA) plasmid (Figure 3A, lanes 2-5 from left) was approximately between those of the other two. [Figure 4A]Transfection efficiency of saponin-mediated PD (Cas9-GFP) transfection in Neuro-2A cells (Figure 4A) and JIMT-1 cells (Figure 4B). Neuro-2A cells (A) and JIMT-1 cells (B) were transfected by PD nanoplexes carrying GFP-DNA (i.e., a plasmid containing GFP DNA) or by PD nanoplexes carrying Cas9-GFP-DNA, with and without the most non-toxic saponin co-administration (GE1741 co-administration at 2 μg / ml, or SO1861 co-administration at 2 μg / ml). GFP-DNA transfection reached a high efficiency of around 70%, and approximately 40% of all cells expressed GFP after Cas9-GFP transfection. Lipofectamin3000 served as a positive control and showed only effective GFP-DNA delivery. GFP-Cas9-DNA was delivered only at low doses, N≧3. *: significant difference compared to transfection without saponin (nuclease only), U test. [Figure 4B] Transfection efficiency of saponin-mediated PD (Cas9-GFP) transfection in Neuro-2A cells (Figure 4A) and JIMT-1 cells (Figure 4B). Neuro-2A cells (A) and JIMT-1 cells (B) were transfected by PD nanoplexes carrying GFP-DNA (i.e., a plasmid containing GFP DNA) or by PD nanoplexes carrying Cas9-GFP-DNA, with and without the most non-toxic saponin co-administration (GE1741 co-administration at 2 μg / ml, or SO1861 co-administration at 2 μg / ml). GFP-DNA transfection reached a high efficiency of around 70%, and approximately 40% of all cells expressed GFP after Cas9-GFP transfection. Lipofectamin3000 served as a positive control and showed only effective GFP-DNA delivery. GFP-Cas9-DNA was delivered only at low doses, N≧3. *: significant difference compared to transfection without saponin (nuclease only), U test. [Figure 5A]Confluence measurements of Neuro-2A cells during PD(Cas9-GFP) transfection ± saponin administration. Visual monitoring of confluence was performed during Cas9-GFP transfection (Figure 5A,E,F) and GFP-DNA transfection (Figure 5A,C,D) of Neuro-2A cells. Presentation of confluence during incubation time was provided by Cytosmart algorithm. No clear toxic effects could be observed in comparison to the untreated negative control (Figure 5A,B). Saponin GE1741 was absent (Figure 5A,C,E) or present (Figure 5A,D,F) during confluency evaluation over time. Figure 5B-F shows Neuro-2A cell confluency after 95 h incubation time. [Figure 5B] Confluence measurements of Neuro-2A cells during PD(Cas9-GFP) transfection ± saponin administration. Visual monitoring of confluence was performed during Cas9-GFP transfection (Figure 5A,E,F) and GFP-DNA transfection (Figure 5A,C,D) of Neuro-2A cells. Presentation of confluence during incubation time was provided by Cytosmart algorithm. No clear toxic effects could be observed in comparison to the untreated negative control (Figure 5A,B). Saponin GE1741 was absent (Figure 5A,C,E) or present (Figure 5A,D,F) during confluency evaluation over time. Figure 5B-F shows Neuro-2A cell confluency after 95 h incubation time. [Figure 5C]Confluence measurements of Neuro-2A cells during PD(Cas9-GFP) transfection ± saponin administration. Visual monitoring of confluence was performed during Cas9-GFP transfection (Figure 5A,E,F) and GFP-DNA transfection (Figure 5A,C,D) of Neuro-2A cells. Presentation of confluence during incubation time was provided by Cytosmart algorithm. No clear toxic effects could be observed in comparison to the untreated negative control (Figure 5A,B). Saponin GE1741 was absent (Figure 5A,C,E) or present (Figure 5A,D,F) during confluency evaluation over time. Figure 5B-F shows Neuro-2A cell confluency after 95 h incubation time. [Figure 5D] Confluence measurements of Neuro-2A cells during PD(Cas9-GFP) transfection ± saponin administration. Visual monitoring of confluence was performed during Cas9-GFP transfection (Figure 5A,E,F) and GFP-DNA transfection (Figure 5A,C,D) of Neuro-2A cells. Presentation of confluence during incubation time was provided by Cytosmart algorithm. No clear toxic effects could be observed in comparison to the untreated negative control (Figure 5A,B). Saponin GE1741 was absent (Figure 5A,C,E) or present (Figure 5A,D,F) during confluency evaluation over time. Figure 5B-F shows Neuro-2A cell confluency after 95 h incubation time. [Figure 5E]Confluence measurements of Neuro-2A cells during PD(Cas9-GFP) transfection ± saponin administration. Visual monitoring of confluence was performed during Cas9-GFP transfection (Figure 5A,E,F) and GFP-DNA transfection (Figure 5A,C,D) of Neuro-2A cells. Presentation of confluence during incubation time was provided by Cytosmart algorithm. No clear toxic effects could be observed in comparison to the untreated negative control (Figure 5A,B). Saponin GE1741 was absent (Figure 5A,C,E) or present (Figure 5A,D,F) during confluency evaluation over time. Figure 5B-F shows Neuro-2A cell confluency after 95 h incubation time. [Figure 5F] Confluence measurements of Neuro-2A cells during PD(Cas9-GFP) transfection ± saponin administration. Visual monitoring of confluence was performed during Cas9-GFP transfection (Figure 5A,E,F) and GFP-DNA transfection (Figure 5A,C,D) of Neuro-2A cells. Presentation of confluence during incubation time was provided by Cytosmart algorithm. No clear toxic effects could be observed in comparison to the untreated negative control (Figure 5A,B). Saponin GE1741 was absent (Figure 5A,C,E) or present (Figure 5A,D,F) during confluency evaluation over time. Figure 5B-F shows Neuro-2A cell confluency after 95 h incubation time. [Figure 6A]GFP knockout in GFP-expressing Neuro-2A-GFP cells by PDD (Cas9-DNA, gRNA(GFP)-DNA) nanoplexes ± saponin administration (co-transfection). GFP-expressing Neuro-2A cells were transfected by co-transfection of Cas9-DNA and gRNA(GFP)-DNA plasmids with 500 ng of each DNA / well and 4,000 ng of polylysine K16 peptide per well in a ratio of 4:0.5:0:5. A decrease in FITC-H median was observed with transfection with and without saponin GE1741. Saponin co-administration reduced FITC-H by 10% (Figure 6D) and increased the amount of cells not considered "fluorescent" by 7.5% compared to the negative control (Figure 6E), n >= 3. Figure 6A: FACS results from negative control. Figure 6B: FACS results with PDD (Cas9-DNA, gRNA(GFP)-DNA), 4:1. Figure 6C: FACS results with PDD (Cas9-DNA, gRNA(GFP)-DNA), 4:1+GE1741. Figure 6D: Median FITC-H values ​​for test samples and control Lipofectamine3000DD (Cas9-DNA) in Figures 6A-C. Figure 6E: Amount of non-fluorescent cells in test samples and control Lipofectamine3000DD (Cas9-DNA) in Figures 6A-C. [Figure 6B]GFP knockout in GFP-expressing Neuro-2A-GFP cells by PDD (Cas9-DNA, gRNA(GFP)-DNA) nanoplexes ± saponin administration (co-transfection). GFP-expressing Neuro-2A cells were transfected by co-transfection of Cas9-DNA and gRNA(GFP)-DNA plasmids with 500 ng of each DNA / well and 4,000 ng of polylysine K16 peptide per well in a ratio of 4:0.5:0:5. A decrease in FITC-H median was observed with transfection with and without saponin GE1741. Saponin co-administration reduced FITC-H by 10% (Figure 6D) and increased the amount of cells not considered "fluorescent" by 7.5% compared to the negative control (Figure 6E), n >= 3. Figure 6A: FACS results from negative control. Figure 6B: FACS results with PDD (Cas9-DNA, gRNA(GFP)-DNA), 4:1. Figure 6C: FACS results with PDD (Cas9-DNA, gRNA(GFP)-DNA), 4:1+GE1741. Figure 6D: Median FITC-H values ​​for test samples and control Lipofectamine3000DD (Cas9-DNA) in Figures 6A-C. Figure 6E: Amount of non-fluorescent cells in test samples and control Lipofectamine3000DD (Cas9-DNA) in Figures 6A-C. [Figure 6C]GFP knockout in GFP-expressing Neuro-2A-GFP cells by PDD (Cas9-DNA, gRNA(GFP)-DNA) nanoplexes ± saponin administration (co-transfection). GFP-expressing Neuro-2A cells were transfected by co-transfection of Cas9-DNA and gRNA(GFP)-DNA plasmids with 500 ng of each DNA / well and 4,000 ng of polylysine K16 peptide per well in a ratio of 4:0.5:0:5. A decrease in FITC-H median was observed with transfection with and without saponin GE1741. Saponin co-administration reduced FITC-H by 10% (Figure 6D) and increased the amount of cells not considered "fluorescent" by 7.5% compared to the negative control (Figure 6E), n >= 3. Figure 6A: FACS results from negative control. Figure 6B: FACS results with PDD (Cas9-DNA, gRNA(GFP)-DNA), 4:1. Figure 6C: FACS results with PDD (Cas9-DNA, gRNA(GFP)-DNA), 4:1+GE1741. Figure 6D: Median FITC-H values ​​for test samples and control Lipofectamine3000DD (Cas9-DNA) in Figures 6A-C. Figure 6E: Amount of non-fluorescent cells in test samples and control Lipofectamine3000DD (Cas9-DNA) in Figures 6A-C. [Figure 6D]GFP knockout in GFP-expressing Neuro-2A-GFP cells by PDD (Cas9-DNA, gRNA(GFP)-DNA) nanoplexes ± saponin administration (co-transfection). GFP-expressing Neuro-2A cells were transfected by co-transfection of Cas9-DNA and gRNA(GFP)-DNA plasmids with 500 ng of each DNA / well and 4,000 ng of polylysine K16 peptide per well in a ratio of 4:0.5:0:5. A decrease in FITC-H median was observed with transfection with and without saponin GE1741. Saponin co-administration reduced FITC-H by 10% (Figure 6D) and increased the amount of cells not considered "fluorescent" by 7.5% compared to the negative control (Figure 6E), n >= 3. Figure 6A: FACS results from negative control. Figure 6B: FACS results with PDD (Cas9-DNA, gRNA(GFP)-DNA), 4:1. Figure 6C: FACS results with PDD (Cas9-DNA, gRNA(GFP)-DNA), 4:1+GE1741. Figure 6D: Median FITC-H values ​​for test samples and control Lipofectamine3000DD (Cas9-DNA) in Figures 6A-C. Figure 6E: Amount of non-fluorescent cells in test samples and control Lipofectamine3000DD (Cas9-DNA) in Figures 6A-C. [Figure 6E]GFP knockout in GFP-expressing Neuro-2A-GFP cells by PDD (Cas9-DNA, gRNA(GFP)-DNA) nanoplexes ± saponin administration (co-transfection). GFP-expressing Neuro-2A cells were transfected by co-transfection of Cas9-DNA and gRNA(GFP)-DNA plasmids with 500 ng of each DNA / well and 4,000 ng of polylysine K16 peptide per well in a ratio of 4:0.5:0:5. A decrease in FITC-H median was observed with transfection with and without saponin GE1741. Saponin co-administration reduced FITC-H by 10% (Figure 6D) and increased the amount of cells not considered "fluorescent" by 7.5% compared to the negative control (Figure 6E), n >= 3. Figure 6A: FACS results from negative control. Figure 6B: FACS results with PDD (Cas9-DNA, gRNA(GFP)-DNA), 4:1. Figure 6C: FACS results with PDD (Cas9-DNA, gRNA(GFP)-DNA), 4:1+GE1741. Figure 6D: Median FITC-H values ​​for test samples and control Lipofectamine3000DD (Cas9-DNA) in Figures 6A-C. Figure 6E: Amount of non-fluorescent cells in test samples and control Lipofectamine3000DD (Cas9-DNA) in Figures 6A-C. [Figure 7A]GFP knockout of GFP-expressing Neuro-2A cells by PD(Cas9-gRNA(GFP)-DNA) nanoplex ± saponin administration (all-in-one transfection). GFP-expressing Neuro-2A cells were transfected by "all-in-one" transfection with Cas9-gRNA(GFP)-DNA plasmids at a ratio of 4:1. 500 ng of each DNA / well. No FITC-H could be observed (Figure 7D). However, the amount of cells that appeared fluorescent increased by 11% at a PD mass ratio of 8:1 (Figure 7E). n≧3. Figure 7A: FACS results with negative controls. Figure 7B: FACS results with PD (Cas9-gRNA (GFP)-DNA, 4:1. Figure 7C: FACS results with PD (Cas9-gRNA (GFP)-DNA, 4:1 + GE1741. Figure 7D: Median FITC-H values ​​of test samples in Figures 7A-C and PD (Cas9-gRNA (GFP)-DNA, 8:1 and PD (Cas9-gRNA (GFP)-DNA, 8:1 + GE1741. Figure 7E: Amount of non-fluorescent cells in test samples in Figures 7A-C and PD (Cas9-gRNA (GFP)-DNA, 8:1 and PD (Cas9-gRNA (GFP)-DNA, 8:1 + GE1741. [Figure 7B]GFP knockout of GFP-expressing Neuro-2A cells by PD(Cas9-gRNA(GFP)-DNA) nanoplex ± saponin administration (all-in-one transfection). GFP-expressing Neuro-2A cells were transfected by "all-in-one" transfection with Cas9-gRNA(GFP)-DNA plasmids at a ratio of 4:1. 500 ng of each DNA / well. No FITC-H could be observed (Figure 7D). However, the amount of cells that appeared fluorescent increased by 11% at a PD mass ratio of 8:1 (Figure 7E). n≧3. Figure 7A: FACS results with negative controls. Figure 7B: FACS results with PD (Cas9-gRNA (GFP)-DNA, 4:1. Figure 7C: FACS results with PD (Cas9-gRNA (GFP)-DNA, 4:1 + GE1741. Figure 7D: Median FITC-H values ​​of test samples in Figures 7A-C and PD (Cas9-gRNA (GFP)-DNA, 8:1 and PD (Cas9-gRNA (GFP)-DNA, 8:1 + GE1741. Figure 7E: Amount of non-fluorescent cells in test samples in Figures 7A-C and PD (Cas9-gRNA (GFP)-DNA, 8:1 and PD (Cas9-gRNA (GFP)-DNA, 8:1 + GE1741. [Figure 7C]GFP knockout of GFP-expressing Neuro-2A cells by PD(Cas9-gRNA(GFP)-DNA) nanoplex ± saponin administration (all-in-one transfection). GFP-expressing Neuro-2A cells were transfected by "all-in-one" transfection with Cas9-gRNA(GFP)-DNA plasmids at a ratio of 4:1. 500 ng of each DNA / well. No FITC-H could be observed (Figure 7D). However, the amount of cells that appeared fluorescent increased by 11% at a PD mass ratio of 8:1 (Figure 7E). n≧3. Figure 7A: FACS results with negative controls. Figure 7B: FACS results with PD (Cas9-gRNA (GFP)-DNA, 4:1. Figure 7C: FACS results with PD (Cas9-gRNA (GFP)-DNA, 4:1 + GE1741. Figure 7D: Median FITC-H values ​​of test samples in Figures 7A-C and PD (Cas9-gRNA (GFP)-DNA, 8:1 and PD (Cas9-gRNA (GFP)-DNA, 8:1 + GE1741. Figure 7E: Amount of non-fluorescent cells in test samples in Figures 7A-C and PD (Cas9-gRNA (GFP)-DNA, 8:1 and PD (Cas9-gRNA (GFP)-DNA, 8:1 + GE1741. [Figure 7D]GFP knockout of GFP-expressing Neuro-2A cells by PD(Cas9-gRNA(GFP)-DNA) nanoplex ± saponin administration (all-in-one transfection). GFP-expressing Neuro-2A cells were transfected by "all-in-one" transfection with Cas9-gRNA(GFP)-DNA plasmids at a ratio of 4:1. 500 ng of each DNA / well. No FITC-H could be observed (Figure 7D). However, the amount of cells that appeared fluorescent increased by 11% at a PD mass ratio of 8:1 (Figure 7E). n≧3. Figure 7A: FACS results with negative controls. Figure 7B: FACS results with PD (Cas9-gRNA (GFP)-DNA, 4:1. Figure 7C: FACS results with PD (Cas9-gRNA (GFP)-DNA, 4:1 + GE1741. Figure 7D: Median FITC-H values ​​of test samples in Figures 7A-C and PD (Cas9-gRNA (GFP)-DNA, 8:1 and PD (Cas9-gRNA (GFP)-DNA, 8:1 + GE1741. Figure 7E: Amount of non-fluorescent cells in test samples in Figures 7A-C and PD (Cas9-gRNA (GFP)-DNA, 8:1 and PD (Cas9-gRNA (GFP)-DNA, 8:1 + GE1741. [Figure 7E]GFP knockout of GFP-expressing Neuro-2A cells by PD(Cas9-gRNA(GFP)-DNA) nanoplex ± saponin administration (all-in-one transfection). GFP-expressing Neuro-2A cells were transfected by "all-in-one" transfection with Cas9-gRNA(GFP)-DNA plasmids at a ratio of 4:1. 500 ng of each DNA / well. No FITC-H could be observed (Figure 7D). However, the amount of cells that appeared fluorescent increased by 11% at a PD mass ratio of 8:1 (Figure 7E). n≧3. Figure 7A: FACS results with negative controls. Figure 7B: FACS results with PD (Cas9-gRNA (GFP)-DNA, 4:1. Figure 7C: FACS results with PD (Cas9-gRNA (GFP)-DNA, 4:1 + GE1741. Figure 7D: Median FITC-H values ​​of test samples in Figures 7A-C and PD (Cas9-gRNA (GFP)-DNA, 8:1 and PD (Cas9-gRNA (GFP)-DNA, 8:1 + GE1741. Figure 7E: Amount of non-fluorescent cells in test samples in Figures 7A-C and PD (Cas9-gRNA (GFP)-DNA, 8:1 and PD (Cas9-gRNA (GFP)-DNA, 8:1 + GE1741. [Figure 8]CRISPR-Cas9-based gene knockout and reporter gene knock-in (OriGene). Gene knockout and knock-in experiments are performed by co-transfection of plasmid DNA (top left) of an all-in-one DNA plasmid carrying Cas9 and gRNA sequences ("linear donor" containing EF1a (i.e., EF1 alpha promoter), GFP sequence, P2A sequence (i.e., 2A self-cleaving peptide), and puromycin ("Puro") sequence) together with a linear donor sequence (LDS) encoding GFP and puromycin. The gene of solute carrier (Slc) was selected as gRNA target ("target sequence"). After Cas9-induced strand break (Slc knockout), the LDS is integrated in forward or reverse orientation by non-homologous recombination (GFP-puromycin knock-in). Selection of modified cell clones is achieved by the inserted puromycin resistance gene. [Figure 9A] Transfection efficiency of saponin-mediated PDD (Cas9-gRNA(Slc26a4)-DNA, LDSDNA) transfection. A: Neuro-2A cells were transfected with PDD nanoplexes (Cas9-gRNA(Slc26a4)-DNA, GFP-puromycin LDS, 4:1 and 8:1 mass ratios when peptide and DNA masses are considered) with and without GE1741 co-administration. Application of GE1741 led to transfection efficiencies of 20% and 30% for 4:1 and 8:1 mass ratios, respectively. Lipofectamin3000 served as a positive control. B-D: Clear fluorescence intensity increase of Neuro-2A cells after saponin-mediated GFP gene knock-in. n≧3. *Significant difference compared to transfection without saponin (nuclease only). U test was performed. Fig. 9B: Negative control. Figure 9C: PDD (Cas9-gRNA(Slc26a4)-DNA, Puro-GFP-LDS), 4:1). Figure 9D: PDD(Cas9-gRNA(Slc26a4)-DNA, Puro-GFP-LDS), 4:1)+GE1741. [Figure 9B] Transfection efficiency of saponin-mediated PDD (Cas9-gRNA(Slc26a4)-DNA, LDSDNA) transfection. A: Neuro-2A cells were transfected with PDD nanoplexes (Cas9-gRNA(Slc26a4)-DNA, GFP-puromycin LDS, 4:1 and 8:1 mass ratios when peptide and DNA masses are considered) with and without GE1741 co-administration. Application of GE1741 led to transfection efficiencies of 20% and 30% for 4:1 and 8:1 mass ratios, respectively. Lipofectamin3000 served as a positive control. B-D: Clear fluorescence intensity increase of Neuro-2A cells after saponin-mediated GFP gene knock-in. n≧3. *Significant difference compared to transfection without saponin (nuclease only). U test was performed. Fig. 9B: Negative control. Figure 9C: PDD (Cas9-gRNA(Slc26a4)-DNA, Puro-GFP-LDS), 4:1). Figure 9D: PDD(Cas9-gRNA(Slc26a4)-DNA, Puro-GFP-LDS), 4:1)+GE1741. [Figure 9C]Transfection efficiency of saponin-mediated PDD (Cas9-gRNA(Slc26a4)-DNA, LDSDNA) transfection. A: Neuro-2A cells were transfected with PDD nanoplexes (Cas9-gRNA(Slc26a4)-DNA, GFP-puromycin LDS, 4:1 and 8:1 mass ratios when peptide and DNA masses are considered) with and without GE1741 co-administration. Application of GE1741 led to transfection efficiencies of 20% and 30% for 4:1 and 8:1 mass ratios, respectively. Lipofectamin3000 served as a positive control. B-D: Clear fluorescence intensity increase of Neuro-2A cells after saponin-mediated GFP gene knock-in. n≧3. *Significant difference compared to transfection without saponin (nuclease only). U test was performed. Fig. 9B: Negative control. Figure 9C: PDD (Cas9-gRNA(Slc26a4)-DNA, Puro-GFP-LDS), 4:1). Figure 9D: PDD(Cas9-gRNA(Slc26a4)-DNA, Puro-GFP-LDS), 4:1)+GE1741. [Figure 9D]Transfection efficiency of saponin-mediated PDD (Cas9-gRNA(Slc26a4)-DNA, LDSDNA) transfection. A: Neuro-2A cells were transfected with PDD nanoplexes (Cas9-gRNA(Slc26a4)-DNA, GFP-puromycin LDS, 4:1 and 8:1 mass ratios when peptide and DNA masses are considered) with and without GE1741 co-administration. Application of GE1741 led to transfection efficiencies of 20% and 30% for 4:1 and 8:1 mass ratios, respectively. Lipofectamin3000 served as a positive control. B-D: Clear fluorescence intensity increase of Neuro-2A cells after saponin-mediated GFP gene knock-in. n≧3. *Significant difference compared to transfection without saponin (nuclease only). U test was performed. Fig. 9B: Negative control. Figure 9C: PDD (Cas9-gRNA(Slc26a4)-DNA, Puro-GFP-LDS), 4:1). Figure 9D: PDD(Cas9-gRNA(Slc26a4)-DNA, Puro-GFP-LDS), 4:1)+GE1741. [Figure 10]Figure 10 (1-3). Reporter gene knock-in technology-homologous recombination vs. non-homologous recombination. Figure 10 presents a knock-in technology that applies homologous recombination, using a circular donor template DNA as part of a DNA vector, here pUC. For comparison, Figure 8 presents a knock-in technology that applies non-homologous recombination, using the LDS of a linear donor template DNA to knock-in the target nucleic acid. The homologous recombination kit (1+2) consists of a circular DNA donor sequence (2: circular donor template DNA containing homologous arms and a functional cassette) and a plasmid DNA encoding Cas9 and gRNA (1: target sequence cloned into the pCas-Guide vector), as presented in Figure 8. Here, the donor sequence is integrated by homologous repair (3:1 co-transfection of pUC vector serving as plasmid DNA carrying pCas-Guide plasmid DNA + 2 circular donor template DNA results in genomic integration of the donor sequence (GFP-Puro (puromycin)) to provide an edited chromosome with the knocked-in gene. The target gene is knocked out, GFP is under the native gene promoter, and the puromycin gene (Puro) is under the PGK promoter in this example. Although the circular donor template DNA (2) is less degradable than the linear donor sequence (see, for example, Figure 8 upper right), the integration process appears to be a more complex process, as is evident when gene integration efficiency is assessed by measuring the degree of GFP fluorescence. [Figure 11A]Knockout of different solute transporters (Slc) in Neuro-2A cells - Homologous versus non-homologous recombination. To compare the efficiency for different targets and different integration techniques in nanoplexes containing K16 peptides in the absence or presence of saponin GE1741, Neuro-2A cells were transfected with NHR kit and with HR kit. All NHR knock-in kits (Slc26a4, Slc19a3, and Slc25a24) showed a significant increase in transfection efficiency, but GFP expression could not be observed with HR knock-in kits. n >= 3. *Significant difference compared to transfection without saponin (nuclease only). U test. (A) Co-transfection of plasmid DNA containing nucleic acids encoding Cas9 and gRNA (Slc26a4) and LDS DNA encoding puromycin and green fluorescent protein (Puro-GFP-LDS). (B) Co-transfection of plasmid DNA containing nucleic acids encoding Cas9 and gRNA (Slc) and donor template DNA encoding puromycin and green fluorescent protein (Puro-GFP-DS) provided as a DNA vector. [Figure 11B]Knockout of different solute transporters (Slc) in Neuro-2A cells - Homologous versus non-homologous recombination. To compare the efficiency for different targets and different integration techniques in nanoplexes containing K16 peptides in the absence or presence of saponin GE1741, Neuro-2A cells were transfected with NHR kit and with HR kit. All NHR knock-in kits (Slc26a4, Slc19a3, and Slc25a24) showed a significant increase in transfection efficiency, but GFP expression could not be observed with HR knock-in kits. n >= 3. *Significant difference compared to transfection without saponin (nuclease only). U test. (A) Co-transfection of plasmid DNA containing nucleic acids encoding Cas9 and gRNA (Slc26a4) and LDS DNA encoding puromycin and green fluorescent protein (Puro-GFP-LDS). (B) Co-transfection of plasmid DNA containing nucleic acids encoding Cas9 and gRNA (Slc) and donor template DNA encoding puromycin and green fluorescent protein (Puro-GFP-DS) provided as a DNA vector. [Figure 12A] Puromycin selection after knock-in of GFP-puromycin LDS. After knockout of solute transporter Slc26a4 and integration of GFP-puromycin donor sequence, cells were cultured for 7 passages before puromycin selection was initiated. Different concentrations of puromycin were applied and cell viability was observed (A, B). 1 μg / mL and 2 μg / mL showed a decrease in confluence with several viable cell populations due to puromycin resistance. (B) Green fluorescence is evident by whitening in the wells of the cell culture plate shown. Plate wells in rows A-C are numbered 1-4 from left to right. Wells C3-4 were empty. [Figure 12B]Puromycin selection after knock-in of GFP-puromycin LDS. After knockout of solute transporter Slc26a4 and integration of GFP-puromycin donor sequence, cells were cultured for 7 passages before puromycin selection was initiated. Different concentrations of puromycin were applied and cell viability was observed (A, B). 1 μg / mL and 2 μg / mL showed a decrease in confluence with several viable cell populations due to puromycin resistance. (B) Green fluorescence is evident by whitening in the wells of the cell culture plate shown. Plate wells in rows A-C are numbered 1-4 from left to right. Wells C3-4 were empty. [Figure 13] FITC-H analysis after isolation of Cas9-mediated Slc26a4 knockout-GFP knockin cells. Grown Neuro-2A cell clones were trypsinized and applied to flow cytometry. Cells were analyzed in terms of fluorescence (FITC-H). Untreated Neuro-2A cells and Neuro-2A-GFP cells were used as negative and positive controls, respectively. Two types of cell clones were observed. Some clones showed no fluorescence and did not demonstrate stable GFP expression, while other clones showed two cell populations. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] While the present invention will be described with respect to specific embodiments, the invention is not limited thereto but only by the claims. The embodiments of the invention described herein can be combined and worked together unless otherwise specified.

[0037] It is a primary goal of the present invention to provide improved methods for the in vitro delivery of relatively large nucleic acids, such as plasmid DNA having a size of, for example, 5.5 kbp or larger, into cells.

[0038] It is an object of the present invention to provide a method for providing target cells in vitro with a relatively large nucleic acid (i.e., a nucleic acid having a size of at least 5.5 kbp), preferably encoding at least Cas, which is accompanied by less onerous manufacturing requirements and improved nucleic acid transfection efficacy, for example when the method steps for preparing a composition comprising the nucleic acid are concerned and / or when the relative transfection efficiency is concerned, when the transfection according to the present invention is compared with the transfection methods commonly applied in the art. It is also an object of the present invention to provide a tool, such as a kit, applicable for combining the components of the kit with a selected nucleic acid for the purpose of in vitro delivery (transfection) of the nucleic acid into the target cell. Such nucleic acids are typically larger than 5,000 bp, or even as large as 8,000-9,000 bp, or larger.

[0039] At least one of the above objects is achieved by providing an in vitro method for providing a target cell with a relatively large nucleic acid (i.e., a nucleic acid having a size of at least 5.5 kbp) preferably encoding at least one of the Cas of the present invention. At least a further object is achieved by providing a kit of the present invention comprising a transfection efficacy enhancing reagent, such as saponin GE1741 and / or saponin SO1861.

[0040] Although the present invention will be described with respect to specific embodiments, the present invention is not limited thereto but only by the claims. Although the present invention has been described in terms of several embodiments, it is contemplated that alternatives, modifications, permutations, and equivalents thereof will become apparent to those skilled in the art upon reading the specification. The present invention is in no way limited to the illustrated embodiments. Changes may be made without departing from the scope defined by the appended claims.

[0041] A first aspect of the present invention relates to the use of bisdesmosidic triterpenoid 12,13-dehydrooleanane-type saponins in the in vitro delivery of relatively large nucleic acids, i.e. having a larger size such as at least 5.5 kbp or at least 8 kbp, into cells.

[0042] The term "saponin" as used herein has its formal meaning established in the art and refers herein to a group of amphiphilic glycosides that contain one or more hydrophilic sugar chains covalently attached to a lipophilic backbone structure referred to as an aglycone core or sapogenin. Saponins can be naturally occurring or synthetic (i.e., not naturally occurring). The term "saponin" as used herein should be interpreted to include naturally occurring saponins as well as saponins synthesized de novo by chemical and / or biotechnological synthetic routes. Bisdesmosidic triterpenoid 12,13-dehydrooleanane type saponins have a triterpene, i.e., a pentacyclic C30 terpene aglycone core, which is of the 12,13-dehydrooleanane type, and has two sugar chains attached thereto (i.e., of the bisdesmosidic type).

[0043] A particularly advantageous embodiment of the present invention relates to the use of saponins in the in vitro delivery of relatively large, i.e. larger, size such as at least 5.5 kbp or at least 8 kbp, nucleic acids into cells, wherein the saponins are according to formula (I):

[0044] [ka]

[0045] During the ceremony, R 1 is a xylose, arabinose, or glucose residue, which is attached by its C1 atom to the corresponding xylose residue of formula (I), R 2 is different from other R on the same molecule. 2Independently of the residue, it is H, an acetyl residue, or a xylose residue bound to the corresponding quinovose residue of formula (I) by its C1 atom, provided that at least two acetyl residues or at least one acetyl residue and a xylose residue bound to the quinovose residue are present in the saponin.

[0046] Typically, the use of the present invention is characterized in that the nucleic acid comprises at least 6 kbp, preferably at least 7 kbp, more preferably at least 7.5 kbp, most preferably at least 8 kbp, or even at least 9 kbp or more, or 10 kbp or more, and is preferably a plasmid DNA, such as a plasmid DNA encoding a CRISPR-associated endonuclease (Cas).

[0047] Advantageously, the use of the invention is characterized in that the nucleic acid comprises a first portion of nucleic acid encoding a CRISPR-associated endonuclease (Cas), the nucleic acid optionally comprises a second portion of nucleic acid encoding a guide RNA (gRNA) or being or comprising a gRNA, preferably the nucleic acid is a plasmid DNA encoding a Cas, more preferably the nucleic acid is a plasmid DNA for expressing at least a Cas, even more preferably the nucleic acid is a plasmid DNA for expressing a Cas and for expressing a gRNA.

[0048] Thus, a particular embodiment of the present invention relates to the use of a saponin according to formula (I) in the in vitro delivery of nucleic acids into cells:

[0049] [ka]

[0050] During the ceremony, R 1 is a xylose, arabinose, or glucose residue, which is attached by its C1 atom to the corresponding xylose residue of formula (I), R 2 is different from other R on the same molecule.2 independently of the residue, H, an acetyl residue, or a xylose residue bound by its C1 atom to the corresponding quinovose residue of formula (I), provided that at least two acetyl residues or at least one acetyl residue and a xylose residue bound to the quinovose residue are present in the saponin; Here, the nucleic acid comprises at least a first portion of nucleic acid that encodes a CRISPR-associated endonuclease (Cas), and the nucleic acid optionally comprises a second portion of nucleic acid that encodes a guide RNA (gRNA) or is or comprises a gRNA.

[0051] Typically, the use according to the invention is characterized in that the cell is a eukaryotic cell.

[0052] The use according to the invention is preferably carried out in the presence of a saponin according to formula (I) 1 are xylose residues and / or the saponin has exactly two acetyl groups.

[0053] Preferred is a use according to the invention characterized in that: the acetyl groups of the saponin according to formula (I) are bonded to the oxygen atoms in the C3 and C4 positions of the corresponding quinovose residue of the saponin.

[0054] Also preferred is a use according to the invention characterized in that R of a saponin according to formula (I) 2 One of the residues is a xylose residue, R 2 One of the residues is an acetyl group, R 2 One of the residues is H.

[0055] According to some embodiments, the use according to the invention is characterized in that the xylose residue of the saponin according to formula (I) is bound to an oxygen atom in the C3 position of the corresponding quinovose residue, wherein an acetyl group is bound to an oxygen atom in the C4 position of the corresponding quinovose residue of the saponin.

[0056] Uses according to the invention are preferred which are characterized by the following: R1 is a xylose residue, where the two R 2 The groups are acetyl groups, which are bonded to the oxygen atoms at the C3 and C4 positions of the corresponding quinovose residues, where the third R 2 The group is H.

[0057] Particularly preferred is the use according to the invention which is characterized in that: The saponin is GE1741 according to formula (II):

[0058] [ka]

[0059] In the formula, R 1 is xylose, Or, the saponin is SO1861 according to formula (III):

[0060] [ka]

[0061] The use of the invention may advantageously be characterized by: the nucleic acid forms part of a nanoparticle, which nanoparticle further comprises a nanoparticle-forming compound, preferably the nanoparticle-forming compound comprises or consists of a polylysine peptide, preferably the polylysine peptide consists of 5 to 25 lysine residues, more preferably the polylysine peptide consists of 16 lysine residues.In a specific embodiment, the use may be characterized by: the nanoparticle comprises the nanoparticle-forming compound and the nucleic acid in a mass ratio ranging from 3:1 to 15:1, preferably ranging from 3:1 to 9:1.

[0062] By combining saponin SO1861 or GE1741, preferably GE1741, with plasmid DNA that has been nanoplexed with a polylysine peptide (saponin-based "sapofection" technology, referring to nanoplexed structures, i.e. nanoplexes). 16 The inventors have surprisingly found that, by using a nucleic acid, preferably in the form of a peptide, preferably in the form of a plasmid DNA, and a saponin, preferably SO1861 or GE1741, more preferably GE1741, the present invention provides a method for the production of a peptide-based (polylysine K) polypeptide, which encodes, for example, the Cas9 protein, in e.g. mammalian cells (e.g. JIMT-1 cancer cells) and in murine cancer cells (Neuro-2A). 16 The saponins, such as SO1861 or GE1741, were mixed with preformed nanoplexes consisting of plasmid DNA and polylysine peptides, thereby effectively delivering relatively large nucleic acids (>6,500 bp, e.g., plasmid DNA having a size of 7.0 kbp to 8.3 kbp) in the form of DNA plasmids incorporated into the nanoplexes into cells in vitro. 16A composition is provided that includes nanoplexes made of peptides and includes a saponin such as GE1741. Such a mixture is contacted with a target cell that is to be transfected with a nucleic acid. Alternatively, both a first composition containing a saponin and a second composition containing a nanoplex are contacted with the cell without prior mixing of the first composition and the second composition. Here, for example, the first composition and the second composition are added to a cell culture medium in which cells selected for transfection with a nucleic acid are cultured. For example, co-transfection of a double-stranded DNA donor sequence, such as a donor sequence of green fluorescent protein (GFP), either combined or not combined with a donor DNA sequence of puromycin inserted into a Cas9 cleavage site, has shown efficient donor sequence-derived gene expression. The transfection efficiency achieved by the transfection method of the present invention, by the use of relatively large nanoplexes of DNA plasmids and polylysine in the presence of saponins such as GE1741 and SO1861, either added as part of a composition comprising the nanoplex structure or separately to cells selected for transfection with the DNA plasmid according to the present invention, was higher than can be obtained by the current gold standard Lipofectamine3000™. For example, reference is made to the Examples section, Figures 4-7, 9, 11, and 12. As mentioned above, according to the present invention, either the saponin is premixed with the nanoparticles composed of polylysine and nucleic acid before the mixture is brought into contact with the cells, or the cells are transfected by co-adding the nanoparticles and saponin separately to the cells, for example by adding the nanoparticles and saponin to the cell culture medium.

[0063] By applying the method of the invention and by using according to the invention, the inventors have achieved an in vitro transfection efficiency of at least 15% when considering the transfection of plasmid DNA having a size of about 7 kbp or even 8.2 kbp. Preferably, the transfection efficiency is at least 20%, more preferably at least 25%, most preferably at least 30%. Indeed, by applying the method of the invention and by using according to the invention, a transfection efficiency of at least 30% was obtained (see also FIG. 11). Typically, such a high efficiency, for example at least 3 times higher than the transfection efficiency that can be obtained by transfecting plasmid DNA with the aid of Lipofectamin3000, can be achieved by transfecting a plasmid DNA having a size of at least 7 kbp, such as a plasmid DNA encoding a Cas enzyme, such as at least Cas9 having an amino acid sequence according to SEQ ID NO: 1, Cas12a having an amino acid sequence according to SEQ ID NO: 2, or Cas13a having an amino acid sequence according to SEQ ID NO: 3, preferably Cas9, under the influence of the presence of saponin during transfection. Typically, the plasmid DNA also codes for gRNA. Typically, such plasmid DNA has a size of at least 8 kbp and, due to the presence of saponin during transfection, still can be efficiently transfected in vivo by applying the method of the present invention and by the use according to the present invention. Alternatively and equally, the following use according to the present invention is preferred: the nucleic acid codes for Cas, such as Cas9, where gRNA is provided as a second nucleic acid encoding gRNA (second plasmid DNA encoding gRNA) or as the RNA sequence of gRNA.

[0064] Typically, the use of the invention encompasses a nucleic acid encoding, preferably said Cas9, having an amino acid sequence according to SEQ ID NO: 1, a Cas12a having an amino acid sequence according to SEQ ID NO: 2, or a Cas13a having an amino acid sequence according to SEQ ID NO: 3. In addition, the use according to the invention is also preferred, characterized in that the nucleic acid encoding a Cas encodes Cas9 (SEQ ID NO: 1), Cas12a (SEQ ID NO: 2), Cas13a (SEQ ID NO: 3), or a Cas having at least 90% amino acid residue identity to any one of Cas9 of Streptococcus pyogenes serovar M1, Cas12a of Francisella tularensis subsp. novicida (strain U112), and Cas13a of Leptotrichia buccalis (strain ATCC14201 / DSM1135 / JCM12969 / NCTC10249 / C-1013-b).

[0065] An embodiment is a use of the present invention, wherein the nucleic acid, preferably a plasmid DNA, comprises at least a first portion of a nucleic acid encoding a CRISPR-associated endonuclease (Cas), preferably Cas9 (SEQ ID NO: 1), or a Cas having at least 90% sequence identity to said Cas9, and the nucleic acid optionally comprises any one or more of the following: a second portion of the nucleic acid that is a non-coding guide RNA comprising a CRISPR-RNA (crRNA) and a trans-activating CRISPR-RNA (tracrRNA), the encoded guide RNA being a single guide RNA or a two-piece RNA, and the tracrRNA comprising a binding site for Cas encoded by the first portion of the nucleic acid. Alternatively, an embodiment is a use of the present invention, wherein the Cas, such as Cas9, is encoded by a first nucleic acid, such as a first plasmid DNA, and the gRNA is encoded by a second nucleic acid, such as a second plasmid DNA. A use according to the present invention, wherein the nucleic acid encoding the Cas and the nucleic acid encoding the gRNA are both encompassed by a single nucleic acid, such as a single plasmid DNA, is preferred.

[0066] For the purpose of providing cells with a knocked-in gene (stably transfected) encoding a protein of interest, the following uses of the present invention are preferred: the in vitro delivery of nucleic acid involves the delivery of a single plasmid DNA containing a nucleic acid encoding a Cas, such as Cas9, and encoding a gRNA, together with an additional nucleic acid that is a DNA donor template encoding the gene selected for knocking into the genome of the cell, or the delivery of two separate plasmid DNAs, the first encoding a Cas and the second encoding a gRNA. The donor template DNA is either a linear donor sequence or a circular donor template DNA, e.g. a DNA vector. The inventors have established that stable transfection and the formation of knocked-in cells are more efficient when a linear DNA donor sequence is applied for the use of the method, compared to the application of a circular donor template DNA. Thus, the following uses are preferred: the in vitro delivery of nucleic acid into cells involves the in vitro delivery of a CRISPR / Cas construct into cells, including the delivery of a linear donor sequence.

[0067] Preferred is a use according to the invention characterized in that: the Cas encoded by the in vitro delivered nucleic acid is Cas9 (SEQ ID NO: 1), Cas12a (SEQ ID NO: 2), Cas13a (SEQ ID NO: 3), or said nucleic acid encodes a Cas having at least 90% amino acid residue identity to any one of these Cas9, Cas12a, and Cas13a, or an endonuclease having a Cas-like endonuclease activity similar to the endonuclease activity of any one of these Cas9, Cas12a, and Cas13a and capable of binding to tracrRNA when Cas9 is considered, and having at least 90% amino acid residue identity to any one of these Cas9, Cas12a, and Cas13a.

[0068] The inventors have established that polylysine (10-20 lysine residues, typically 16 lysine residues) efficiently forms nanoparticles (nanoplexes) with nucleic acids such as plasmid DNA having a size of at least 5.5 kbp, for example at least 7 kbp, or even at least 8 kbp. The inventors have established that such nanoparticles are particularly efficiently transfected into cells in the presence of 1-5 micrograms / mL of a saponin such as SO1861 or GE1741 during transfection of cells when the mass ratio between the polylysine peptide and the nucleic acid is less than 16:1, for example 3:1 to 15:1. The inventors have also established that for efficient nucleic acid transfection, the mass ratio between the polylysine peptide and the nucleic acid in the nanoparticle should be higher than 2:1, for example 3:1 to 15:1. In the use of the invention, it is preferred that the selected nucleic acid for in vitro delivery into a cell is provided as part of a nanoplex (nanoparticle), where the nanoplex comprises a nucleic acid, preferably in the form of plasmid DNA, typically having a size of at least 5.5 kbp, such as at least 6.0 kbp, for example 6-9 kbp, and a polylysine peptide, e.g. K 12 Peptide~K 20 Peptides (polylysine peptides consisting of any one of 12 to 20 lysine residues), preferably K 16 During transfection of cells with such nucleic acids, a saponin is also added to the cells, or the saponin is first premixed with the nanoparticles and then added to the cells. The following use of the invention is optional: a selected nucleic acid for in vitro delivery into a cell is provided as part of a nanoplex (nanoparticle), wherein the nanoplex comprises a nucleic acid, preferably in the form of plasmid DNA, typically having a size of at least 5.5 kbp, such as at least 6.0 kbp, for example 6-9 kbp, a saponin, such as SO1861 or GE1741, preferably GE1741, and a polylysine peptide, such as K 12 Peptide~K 20Peptides (polylysine peptides consisting of any one of 12 to 20 lysine residues), preferably K 16 Typically, in the use of the present invention, the nanoparticles comprise a nanoparticle-forming compound, such as a polylysine peptide, and a nucleic acid in a mass ratio ranging from 3:1 to 15:1, preferably from 3:1 to 9:1, because at a mass ratio of 2:1 transfection is inefficient and at a mass ratio of 16:1 the nanoparticles (nanoplexes) are toxic to cells.

[0069] A second aspect of the present invention relates to a method for delivering a nucleic acid containing more than 5.5 kilobase pairs (kbp) into a cell in vitro, comprising the steps of: (i) providing a nucleic acid; and (ii) providing a saponin which is a triterpenoid 12,13-dehydrooleanane-type saponin of the bisdesmosidic type; and (iii) incubating the nucleic acid and the cells in the presence of saponin.

[0070] In a preferred embodiment of the second aspect of the present invention there is provided a method wherein the saponin conforms to formula (I):

[0071] [ka]

[0072] During the ceremony, R 1 is a xylose, arabinose, or glucose residue, which is attached by its C1 atom to the corresponding xylose residue of formula (I), R 2 is different from other R on the same molecule. 2 Independently of the residue, it is H, an acetyl residue, or a xylose residue bound to the corresponding quinovose residue of formula (I) by its C1 atom, provided that at least two acetyl residues or at least one acetyl residue and a xylose residue bound to the quinovose residue are present in the saponin.

[0073] In a particular embodiment, the second aspect of the invention may relate to a method for delivering a nucleic acid encoding a CRISPR / Cas construct into a cell in vitro, comprising the steps of: (i) providing a nucleic acid and providing a saponin according to formula (I);

[0074] [ka]

[0075] During the ceremony, R 1 is a xylose, arabinose, or glucose residue, which is attached by its C1 atom to the corresponding xylose residue of formula (I), R 2 is different from other R on the same molecule. 2 independently of the residue, H, an acetyl residue, or a xylose residue bound to the corresponding quinovose residue of formula (I) by its C1 atom, provided that at least two acetyl residues or at least one acetyl residue and a xylose residue bound to the quinovose residue are present in the saponin; and (ii) incubating the nucleic acid and the cells in the presence of saponin; wherein the nucleic acid encoding the CRISPR / Cas construct comprises at least a first portion of nucleic acid encoding a Cas, wherein the nucleic acid optionally comprises a second portion of nucleic acid encoding or comprising or being a gRNA.

[0076] Advantageously, a method is provided which is characterized in that the nucleic acid is a plasmid DNA.

[0077] A method according to the invention is preferred which is characterized in that: the nucleic acid forms part of a nanoparticle, which nanoparticle further comprises a nanoparticle-forming compound.

[0078] Particularly preferred is a method according to the invention characterized in that the nanoparticles comprise a nanoparticle-forming compound polylysine peptide, whereby preferably the polylysine peptide consists of 5 to 25 lysine residues, more preferably the polylysine peptide consists of 16 lysine residues. For example, a preferred polylysine peptide is K 16 Also referred to as peptides.

[0079] It is part of the invention that the method or use of the invention is characterized in that the nanoparticles comprise a nanoparticle-forming compound, such as a peptide, e.g. polylysine, and a nucleic acid in a mass ratio ranging from 3:1 to 15:1, preferably from 3:1 to 9:1. Typically, the nanoparticle-forming compound is a polylysine peptide, e.g. K 16 The following method is preferred: the nucleic acid is a plasmid DNA, encoding at least Cas, preferably Cas9 (SEQ ID NO: 1) and optionally also gRNA, and wherein the nanoparticles, in addition to the plasmid DNA, contain at least one polylysine peptide, preferably polylysine K. 16 Alternatively, the polylysine peptide is a peptide consisting of 8 to 25 lysine residues, preferably 10 to 20, and more preferably 12 to 18. Plasmid DNA, K 16High in vitro transfection efficiency is obtained by the method of the present invention when nanoparticles incorporating peptides are prepared and applied and contacted with cells in the presence of a saponin selected from GE1741 and SO1861, preferably the saponin is GE1741. Typically, the saponin concentration during transfection of plasmid DNA into selected target cells in vitro is 1 μg / mL to 5 μg / mL, preferably 2 to 4 μg / mL. The inventors have established that when saponin is applied to cells together with nanoplexes at a saponin concentration of 1 μg / mL to 5 μg / mL, the saponin does not show toxic activity against cells with which the nanoplexes are contacted in the presence of a saponin such as GE1741 or SO1861. The cells are typically contacted with a composition comprising the nanoplexes and a separate composition comprising a saponin, preferably GE1741. Thus, typically, the nanoparticles comprise a polylysine peptide and a nucleic acid, e.g., plasmid DNA, encoding at least a Cas, preferably Cas9 (SEQ ID NO:1), or a Cas having at least 90% sequence identity, preferably 90-99.5% sequence identity, e.g., 92%, 93%, 94%, 95%, or 95-99%, to this Cas9.

[0080] A method according to the invention is preferred, characterized in that the saponin concentration is between 1 μg / mL and 5 μg / mL in step (ii) of the method. Typically, the concentration of saponin during transfection of the cells is between 1.5 and 4.5 μg / mL, for example 2, 2.5, 3, 3.5 or 4 μg / mL, or any concentration therebetween. Such saponin concentrations do not impose toxicity on the cells selected for transfection of the nucleic acid according to the uses and methods of the invention. Such saponin concentrations, where the saponin is preferably GE1741 or SO1841, more preferably the saponin is GE1741, are suitable for efficient transfection of a nucleic acid, preferably a plasmid DNA encoding at least a Cas, preferably Cas9 (SEQ ID NO: 1). Alternatively, the encoded Cas is Cas12a (SEQ ID NO: 2) or Cas13a (SEQ ID NO: 3).

[0081] The present inventors have determined that the cell contains a plasmid DNA having a size of at least 5.5 kbp, for example, 6.5 kbp to 8.5 kbp, for example, 7 kbp to 8.3 kbp, and 16 It has been established that relatively high transfection efficiencies were obtained when nanoplexes comprising polylysine peptides, such as peptides, were contacted in vitro in the presence of saponins SO1861 or GE1741. GE1741 is preferred, since by application of GE1741 much improved transfection efficiencies were achievable compared to SO1861. In this case, the efficiency was compared to the results obtained by transfection applying the "gold standard" Lipofectamin3000 according to the manufacturer's recommendations. Thus, the application of GE1741 in the method of the invention and for use according to the invention is particularly preferred. Nanoplexes are capable of transfecting nucleic acids, such as plasmid DNA, having a size of 5.5 kbp or larger, for example 5.5-9.5 kbp, with K. 16After such nanoplexes of nucleic acid and polylysine are obtained, these nanoplexes are optionally first further mixed with a saponin such as SO1861 or GE1741 before the nanoplexes consisting of nucleic acid and polylysine peptide are applied to cells in the presence of saponin. Alternatively, the nanoparticles and saponin are added separately to the cell culture, so that the cells are transfected, for example, in the presence of saponin in the cell culture medium.

[0082] An embodiment is a method according to the invention characterized in that: The saponin is GE1741 according to formula (II):

[0083] [ka]

[0084] In the formula, R 1 is xylose, Or, the saponin is SO1861 according to formula (III):

[0085] [ka]

[0086] The method of the invention or the use according to the invention is preferred where the saponin is GE1741. Since efficient transfection is achieved by nanoplexes combining with GE1741 or SO1861 during transfection of target cells, saponins having similar molecular structures according to the embodiments described herein are also suitable for improving nucleic acid transfection efficiency according to the use of the invention or according to the method of the invention.

[0087] Typically, in the method or according to the use of the invention, saponin is applied at a concentration between 1 and 5 μg / ml of transfection of target cells, since at such a concentration, saponin is non-toxic to cells while transfection efficacy remains optimal, when considering the positive effect of the presence of saponin on the transfection efficiency of the nucleic acid contained in the nanoplex (nanoparticle) of the invention. Preferably, the nanoplex contains polylysine peptide K 16 Of course, as long as cytotoxicity is not induced under the influence of alternative lengths of polylysine peptides, and the transfection efficiency is at least 15%, preferably at least 20%, more preferably at least 25%, and most preferably at least 30%. 16 Shorter and longer polylysine peptides are equally suitable, unless prevented by the application of polylysine peptides having a length different from that of the . Typically, when transfection efficiency is considered for transfecting plasmid DNA of a size of at least 5.5 kbp, e.g., 5.5 to 10 kbp or 6.5 to 9.0 kbp, the K 16 The application of peptides is highly beneficial. In the nanoplexes applied in the method of the invention or for use according to the invention, therefore, K 16 The presence of peptides is preferred, but polylysine peptides of shorter or longer length, e.g., K 10 ~K 20 is also suitable.

[0088] Advantageously, in the method of the invention, the nucleic acid is a nucleic acid encoding a CRISPR / Cas construct comprising at least a first portion encoding a Cas, wherein the nucleic acid will optionally comprise a second portion encoding or comprising or being a gRNA. Particularly preferred is a use of the invention or a method according to the invention characterized in that the nucleic acid encoding the CRISPR / Cas construct is provided as part of a plasmid DNA, preferably wherein at least a portion of the nucleic acid encoding the Cas portion of the CRISPR / Cas construct is provided as part of a plasmid DNA, more preferably wherein the plasmid DNA is for expressing the Cas and gRNA of the CRISPR / Cas construct. Preferably, the plasmid DNA encodes Cas9 (SEQ ID NO: 9) or a Cas having at least 90% sequence identity to this Cas9 protein. Typically, the plasmid DNA has a size of at least 6.0 kbp, for example 6.5 to 8.8 kbp. Typically, when the plasmid DNA contains a nucleic acid encoding Cas, preferably Cas9, and also contains a nucleic acid encoding gRNA, the size of the plasmid DNA is greater than 7.0 kbp, for example, 7.5-9.5 kbp, or 8-8.5 kbp. It is beneficial to apply a plasmid DNA containing a nucleic acid encoding Cas, for example Cas9 (SEQ ID NO: 1), and a nucleic acid encoding gRNA. Because a single plasmid DNA contains a polylysine peptide, preferably K 16This is because it only has to involve nanoplex formation with the peptide and then forming a mixture with saponin (either before adding nanoparticles to the cells or by simultaneously adding a separate composition containing nanoparticles and a separate composition containing saponin to the cells), such as SO1861 or GE1741, preferably GE1741. Consistent batch-to-batch production is made more convenient and easier when only a single plasmid DNA has to be nanoplexed with the peptide (and optionally premixed with saponin before being added to the cells selected for transfection with the nanoplexed nucleic acid). However, the method and use of the present invention is also suitable and applicable for the application of a first plasmid DNA encoding Cas and a second plasmid DNA encoding gRNA. In an embodiment, the method and use of the present invention is applied with a plasmid DNA encoding Cas9 and green fluorescent protein (GFP).

[0089] Preferred is a use or method of the invention characterized in that the plasmid DNA comprises more than 6 kbp, preferably at least 7 kbp, more preferably at least 7.5 kbp, most preferably at least 8 kbp. For example, a plasmid DNA comprising a nucleic acid encoding Cas9 is typically 6.5-7.5 kbp, and a plasmid DNA comprising a nucleic acid encoding a Cas9-GFP construct is typically 7.5-8.5 kbp. 16Such plasmid DNA sizes can be efficiently transfected when nanoplexes are prepared with polylysine peptides such as peptides, optionally subsequently mixed with a saponin such as GE1741, and these nanoplexes are contacted with cells selected for transfection in the presence of the (premixed) saponin. The inventors have previously demonstrated that, in a general sense, toxic effects were evident when cells were contacted with nanoplexes at a mass ratio of 16:1 (16 parts peptide, 1 part nucleic acid based on the total weight of the nanoplex (nanoparticles). In addition, the inventors have previously demonstrated that, in a general sense, inefficient nucleic acid delivery was evident when cells were contacted with nanoplexes at a mass ratio of 2:1 (peptide:nucleic acid). Thus, in view of the absence of toxicity and in view of the apparent nucleic acid delivery efficiency, nanoplexes are formulated at a mass ratio of peptide mass to nucleic acid mass of 3:1 to 12:1 based on the total weight of the nanoplex (nanoparticles). Preferably, the mass ratio of nucleic acid, e.g., total peptide to total plasmid DNA, is 4:1 to 8:1, e.g., 4:1, 5:1, 6:1, 7:1, or 8:1, based on the total weight of the nanoplex. Under the influence of a saponin, e.g., SO1861 or GE1741, preferably GE1741, a polylysine peptide, e.g., K 14 ~K 18 Peptides, preferably K 16 In nanoplexes (nanoparticles) comprising a single first plasmid DNA comprising a peptide and comprising a nucleic acid encoding a Cas, such as Cas9 (SEQ ID NO:1), and comprising a nucleic acid encoding a gRNA, or comprising a first plasmid DNA encoding said Cas and a second plasmid DNA encoding said gRNA, the transfection efficiency is typically at least 20%.

[0090] Preferred is a use of the invention or a method according to the invention, characterized in that the nucleic acid encoding a Cas encodes Cas9 (SEQ ID NO: 1), Cas12a (SEQ ID NO: 2), Cas13a (SEQ ID NO: 3), or a Cas having at least 90% amino acid residue identity to any one of Cas9 of Streptococcus pyogenes serovar M1, Cas12a of Francisella tularensis subsp. novicida (strain U112), and Cas13a of Leptotrichia buccalis (strain ATCC14201 / DSM1135 / JCM12969 / NCTC10249 / C-1013-b). Preferably, the nucleic acid encoding a Cas is part of a plasmid DNA, for example a plasmid DNA having a size of 6-9 kbp, typically 6.5-8.5 kbp. For Cas9, the gRNA encodes a tracrRNA and a CRISPR-RNA (crRNA), but when the nucleic acid encodes Cas12a (SEQ ID NO:2) or Cas13a (SEQ ID NO:3), the nucleic acid encoding the gRNA encodes only the crRNA. When the nucleic acid applied in the method or involved in the use of the present invention encodes Cas9, or a Cas having at least 90% sequence identity to Cas9 according to SEQ ID NO:1, the encoded gRNA comprises a CRISPR-RNA (crRNA) and comprises a trans-activating CRISPR-RNA (tracrRNA), where the gRNA is a single guide RNA (sgRNA) or a two-piece RNA, where the tracrRNA comprises a binding site for Cas9.

[0091] A third aspect of the invention relates to a kit of parts for delivering a nucleic acid containing more than 5.5 kilobase pairs (kbp) into a cell in vitro, comprising: A first combination comprising or consisting of: (a) a first container containing a first plasmid DNA; (b) optionally, a second container containing a second plasmid DNA; (c) Polylysine K 16 a third container containing a peptide; (d) a fourth container containing a triterpenoid 12,13-dehydrooleanane-type saponin of the bisdesmosidic type, preferably one saponin, most preferably at least one saponin selected from GE1741 and SO1861; (e) Instructions for use, Here, the use includes at least polylysine K 16 Preparation of nanoplexes of a peptide and a first plasmid DNA, and optionally a saponin; wherein the first plasmid has a size of at least 5.5 kbp; or A second combination comprising or consisting of: (a) a first container containing a third plasmid DNA; (b) Polylysine K 16 a second container containing a peptide; (c) a third container containing a triterpenoid 12,13-dehydrooleanane-type saponin of the bisdesmosidic type, preferably one saponin, most preferably at least one saponin selected from GE1741 and SO1861; (d) Instructions for use; Here, the use includes at least polylysine K 16 preparation of nanoplexes of a peptide and a third plasmid DNA, and optionally a saponin; wherein the third plasmid has a size of at least 5.5 kbp; or A third combination comprising or consisting of: (a) a first container containing a first plasmid DNA; (b) optionally, a second container containing an RNA oligonucleotide; (c) Polylysine K 16 a third container containing a peptide; (d) a fourth container containing at least one saponin, a bisdesmosidic triterpenoid 12,13-dehydrooleanane type saponin, preferably a saponin, most preferably selected from GE1741 and SO1861; (e) Instructions for use; Here, the use includes at least polylysine K 16 Preparation of nanoplexes of a peptide and a first plasmid DNA, and optionally a saponin; wherein the first plasmid has a size of at least 5.5 kbp; Preferably, wherein the first plasmid DNA encodes Cas9, the second plasmid DNA encodes a gRNA, and / or the RNA oligonucleotide is an RNA oligonucleotide of a gRNA, or wherein the third plasmid DNA encodes Cas9 and encodes a gRNA.

[0092] Alternatively, a third aspect of the invention may relate to a kit of parts for delivering a nucleic acid encoding a CRISPR / Cas construct into a cell in vitro comprising: A first combination comprising or consisting of: (a) a first container comprising a first plasmid DNA encoding Cas9; (b) optionally, a second container containing a second plasmid DNA encoding a gRNA; (c) Polylysine K 16 a third container containing a peptide; (d) a fourth container containing at least one saponin, preferably one of GE1741 and SO1861 saponins; (e) Instructions for use; Here, the use includes at least polylysine K 16 Preparation of nanoplexes of a peptide and a first plasmid DNA, and optionally a saponin; wherein the first plasmid has a size of at least 5.5 kbp; or A second combination comprising or consisting of: (a) a first container comprising a third plasmid DNA encoding Cas9 and encoding a gRNA; (b) Polylysine K 16 a second container containing a peptide; (c) a third container containing at least one saponin, preferably one of GE1741 and SO1861 saponins; (d) Instructions for use; Here, the use includes at least polylysine K 16 preparation of nanoplexes of a peptide and a third plasmid DNA, and optionally a saponin; wherein the third plasmid has a size of at least 5.5 kbp; or A third combination comprising or consisting of: (a) a first container comprising a first plasmid DNA encoding Cas9; (b) optionally, a second container containing RNA oligonucleotides for a gRNA; (c) Polylysine K 16 a third container containing a peptide; (d) a fourth container containing at least one saponin, preferably one of GE1741 and SO1861 saponins; (e) Instructions for use; Here, the use includes at least polylysine K 16 Preparation of nanoplexes of a peptide and a first plasmid DNA, and optionally a saponin; Here, the first plasmid has a size of at least 5.5 kbp.

[0093] The kit optionally includes at least one further container containing: a diluent or solvent for diluting or dissolving the components contained in any one or more of the first, second, third and, if present, fourth containers of the first, second or third combination, and / or for diluting or dissolving the components of the nanoplexes comprising at least plasmid DNA and a polylysine peptide consisting of 16 lysine residues, and optionally at least one saponin, preferably either GE1741 or SO1861, more preferably GE1741. Preferably, the kit contains a container or vial containing GE1741 as the only saponin. Typically, the plasmid DNA has a size of 5.5 kbp to 10 kbp, e.g. 6.5 kbp to 7.5 kbp, typical of the first or third combination, or 7.5 kbp to 8.5 kbp, typical of the second combination. The plasmid DNA preferably includes a portion of DNA encoding Cas9 according to the amino acid sequence of SEQ ID NO:1. Alternatively, the kit comprises a plasmid DNA encoding a Cas having at least 90% identity to the amino acid sequence of Cas9 (SEQ ID NO:1; see, e.g., www.uniprot.org / uniprot / Q99ZW2).

[0094] The encoded non-coding guide RNA of the first or second combination of the kit or the gRNA included in the third combination of the kit comprises a CRISPR-RNA (crRNA) and comprises a trans-activating CRISPR-RNA (tracrRNA), where the guide RNA is a single guide RNA or a two-piece RNA, where the tracrRNA comprises a binding site for Cas9 encoded by the first or third plasmid DNA.

[0095] The use and / or method and / or kit of the present invention is particularly suitable for obtaining cells with in vitro knocked-in genes, preferably in vitro stably transfected knock-in cells. Preferably, the nucleic acid encoding the protein to be knocked-in to the genome of the target cell is a DNA of the type of linear donor sequence (LDS), but circular DNA donor templates are also applicable for knocking in the encoding nucleic acid into the genome of the target cell by applying the method of the present invention or according to the use of the present invention, by using the kit of the present invention. The LDS or circular DNA donor template, for example in the form of a vector or plasmid DNA, is co-transfected with a nucleic acid encoding Cas, preferably Cas9 (SEQ ID NO: 1), preferably as part of a plasmid DNA, which plasmid DNA also preferably comprises a nucleic acid encoding a gRNA, but in the co-transfection the gRNA can also be provided as RNA or as a further plasmid DNA encoding the gRNA. According to the present invention, the nucleic acid encoding Cas and gRNA, preferably one or two plasmid DNAs, more preferably a single plasmid DNA, is co-transfected with a plasmid DNA, a polylysine peptide, preferably K 16The present invention is provided as nanoparticles (nanoplexed components forming nanoparticles) that preferably comprise or consist of a peptide and optionally a saponin selected from SO1861 and GE1741, and preferably the saponin is GE1741. Nanoparticles that comprise nucleic acid and polylysine peptide are preferred, and these nanoparticles are mixed with saponin during transfection of target cells with nucleic acid. Cells selected for knock-in by (stable) transfection of linear or circular DNA donor template, preferably LDS, are contacted with saponin and nanoparticles and DNA donor template, so that the nucleic acid encoding Cas, preferably Cas9 (SEQ ID NO: 1), the nucleic acid encoding gRNA, and DNA donor template, preferably LDS, are transfected into cells in vitro, so that the donor DNA is subsequently knocked into the target location of the genome of the cell. Preferably, the use of the invention, the method of the invention, the application of the kit of the invention and / or the application of the nanoparticles of the invention in combination with saponin results in efficient transfection of the target cells, where "efficient" refers to a transfection rate of at least 20%, such as at least 25% or at least 30% or at least 35%, and preferably the knock-in of the selected nucleic acid into the genome of the cell is a stable transfection, i.e. the knocked-in nucleic acid remains in the genome of the cell after one or more passages of the cell.

[0096] A fourth aspect of the invention relates to a composition comprising nanoparticles suitable for in vitro delivery of nucleic acids into cells, wherein the nanoparticles comprise or consist of: (i) a nucleic acid containing more than 5.5 kilobase pairs (kbp); (ii) a polylysine peptide, and (iii) Saponins that are bisdesmosidic triterpenoid 12,13-dehydrooleanane-type saponins.

[0097] Certain specific embodiments of the fourth aspect of the invention may advantageously relate to a composition comprising nanoparticles suitable for the in vitro delivery of a nucleic acid encoding a CRISPR / Cas construct into a cell, wherein the nanoparticles comprise or consist of: (i) a nucleic acid encoding a CRISPR / Cas construct; (ii) a polylysine peptide, wherein the nucleic acid encoding the CRISPR / Cas construct comprises at least a first portion of nucleic acid encoding a CRISPR-associated endonuclease (Cas), and wherein the nucleic acid optionally comprises a second portion of nucleic acid encoding or being or comprising a guide RNA (gRNA).

[0098] Compositions according to the invention comprising a saponin according to formula (I) are preferred:

[0099] [ka]

[0100] During the ceremony, R 1 is a xylose, arabinose, or glucose residue, which is attached by its C1 atom to the corresponding xylose residue of formula (I), R 2 is different from other R on the same molecule. 2 Independently of the residue, it is H, an acetyl residue, or a xylose residue bound to the corresponding quinovose residue of formula (I) by its C1 atom, provided that at least two acetyl residues or at least one acetyl residue and a xylose residue bound to the quinovose residue are present in the saponin.

[0101] According to the present invention, the composition can be first mixed with saponin before it is applied to cells selected for transfection with a selected nucleic acid, such as a CRISPR / Cas construct, that contains more than 5.5 kbp. Alternatively, the composition contacts the cells and saponin is also added to the cells by applying a first composition containing saponin to the cells and applying a composition of the present invention to the cells.

[0102] One embodiment is a composition according to the invention comprising a saponin, wherein R of the saponin according to formula (I) 1 is a xylose residue and / or the saponin has exactly two acetyl groups.

[0103] One embodiment is a composition according to the invention comprising a saponin, wherein the acetyl groups of the saponin according to formula (I) are bonded to oxygen atoms at the C3 and C4 positions of the corresponding quinovose residue of the saponin.

[0104] One embodiment is a composition according to the invention comprising a saponin, wherein R of the saponin according to formula (I) 2 One of the residues is a xylose residue, R 2 One of the residues is an acetyl group, R 2 One of the residues is H.

[0105] One embodiment is a composition according to the invention comprising a saponin, wherein a xylose residue of the saponin according to formula (I) is bonded to an oxygen atom at the C3 position of the corresponding quinovose residue and an acetyl group is bonded to an oxygen atom at the C4 position of the corresponding quinovose residue of the saponin.

[0106] One embodiment is a composition according to the invention comprising a saponin, wherein R of the saponin according to formula (I) 1 is a xylose residue, and the two R 2 The R groups are acetyl groups, which are attached to the oxygen atoms at the C3 and C4 positions of the corresponding quinovose residues, and the third R 2 The group is H.

[0107] An embodiment is a composition according to the present invention comprising a saponin, wherein the saponin is GE1741 according to formula (II):

[0108] [ka]

[0109] In the formula, R 1 is xylose, Or, wherein the saponin is SO1861 according to formula (III):

[0110] [ka]

[0111] Preferred is a composition according to the invention, wherein the polylysine peptide consists of 5 to 25 lysine residues, more preferably wherein the polylysine peptide consists of 16 lysine residues.

[0112] Typically, in the composition according to the invention, the nanoparticles comprise polylysine peptide and nucleic acid in a mass ratio ranging from 3:1 to 15:1, preferably ranging from 3:1 to 9:1. The composition either comprises or does not comprise saponin. Typically, the composition of the invention does not comprise saponin, and when the composition is applied to transfect cells with the construct, the cells are here co-incubated with the composition of the invention and saponin in the embodiments outlined above.

[0113] Compositions according to the invention in which the nucleic acid is part of a plasmid DNA are preferred. As explained above, in certain embodiments, the following compositions are preferred: the nucleic acid comprises at least a first portion of a nucleic acid encoding a Cas, where the nucleic acid optionally comprises a second portion of a nucleic acid encoding or comprising or being a gRNA.

[0114] Particularly preferred are compositions of the invention wherein the plasmid DNA comprises more than 6 kbp, preferably at least 7 kbp, more preferably at least 7.5 kbp, and most preferably at least 8 kbp.

[0115] Preferred are compositions according to the invention wherein the Cas-encoding first portion of the nucleic acid encodes Cas9 (SEQ ID NO:1), Cas12a (SEQ ID NO:2), Cas13a (SEQ ID NO:3), or a Cas having at least 90% amino acid residue identity to any one of Cas9 of Streptococcus pyogenes serovar M1, Cas12a of Francisella tularensis subsp. novicida (strain U112), and Cas13a of Leptotrichia buccalis (strain ATCC14201 / DSM1135 / JCM12969 / NCTC10249 / C-1013-b).

[0116] A fifth aspect of the invention relates to nanoparticles suitable for in vitro delivery of nucleic acids into cells, the nanoparticles comprising or consisting of: (i) a nucleic acid encoding a CRISPR / Cas construct; (ii) a polylysine peptide, and (iii) a saponin according to formula (I),

[0117] [ka]

[0118] During the ceremony, R 1 is a xylose, arabinose, or glucose residue, which is attached by its C1 atom to the corresponding xylose residue of formula (I), R 2 is different from other R on the same molecule. 2 independently of the residue, H, an acetyl residue, or a xylose residue bound by its C1 atom to the corresponding quinovose residue of formula (I), provided that at least two acetyl residues or at least one acetyl residue and a xylose residue bound to the quinovose residue are present in the saponin; wherein the nucleic acid encoding the CRISPR / Cas construct comprises at least a first portion of nucleic acid encoding a CRISPR-associated endonuclease (Cas), and wherein the nucleic acid optionally comprises a second portion of nucleic acid encoding or being or comprising a guide RNA (gRNA).

[0119] An embodiment of the present invention is a nanoparticle according to the present invention, comprising R 1 is a xylose residue and / or the saponin has exactly two acetyl groups.

[0120] The following nanoparticles according to the invention are preferred: the acetyl groups of the saponin according to formula (I) are linked to oxygen atoms in the C3 and C4 positions of the corresponding quinovose residue of the saponin.

[0121] The following nanoparticles according to the invention are also suitable: R 2 One of the residues is a xylose residue, R 2 One of the residues is an acetyl group, R 2 One of the residues is H.

[0122] In certain embodiments, a nanoparticle according to the invention comprises a saponin of formula (I), wherein a xylose residue of the saponin according to formula (I) is bonded to an oxygen atom at the C3 position of the corresponding quinovose residue and an acetyl group is bonded to an oxygen atom at the C4 position of the corresponding quinovose residue of the saponin.

[0123] In certain embodiments, the nanoparticles according to the invention comprise a saponin according to formula (I), wherein R 1 is a xylose residue, where the two R 2 The R groups are acetyl groups, which are attached to the oxygen atoms at the C3 and C4 positions of the corresponding quinovose residues, and the third R 2 The group is H.

[0124] The following nanoparticles according to the invention are preferred: The saponin is GE1741 according to formula (II):

[0125] [ka]

[0126] In the formula, R 1 is xylose, Or the saponin is SO1861 according to formula (III):

[0127] [ka]

[0128] Preferably, the nanoparticles comprise SO1861 or GE1741, more preferably GE1741.

[0129] Preferably, in the nanoparticles according to the invention, the polylysine peptide consists of 5 to 25 lysine residues, more preferably the polylysine peptide consists of 16 lysine residues.

[0130] Typically, in the nanoparticles according to the invention, the nanoparticles comprise the nucleic acid and the polylysine peptide in a mass ratio ranging from 3:1 to 15:1, preferably ranging from 3:1 to 9:1. Preferably, the range is selected from 4:1 to 8:1.

[0131] Preferred are nanoparticles according to the invention, in which the nucleic acid is part of a plasmid DNA. The plasmid DNA preferably comprises a first portion of nucleic acid encoding a Cas, preferably Cas9 (SEQ ID NO: 1), and a second portion of nucleic acid encoding a gRNA.

[0132] Typically, in nanoparticles according to the present invention, plasmid DNA comprises more than 5.5 kbp, preferably at least 6 kbp, more preferably at least 7 kbp, most preferably at least 7.5 kbp.Plasmid DNA comprises nucleic acid encoding CRISPR / Cas construct.CRISPR / Cas construct encoding nucleic acid comprises at least a first part of nucleic acid encoding CRISPR-associated endonuclease (Cas), where nucleic acid optionally comprises a second part of nucleic acid encoding guide RNA (gRNA) or being gRNA or comprising gRNA.

[0133] In the nanoparticles according to the invention, it is preferred that the first portion of the nucleic acid encoding a Cas encodes Cas9 (SEQ ID NO:1), Cas12a (SEQ ID NO:2), Cas13a (SEQ ID NO:3), or a Cas having at least 90% amino acid residue identity to any one of Cas9 of Streptococcus pyogenes serovar M1, Cas12a of Francisella tularensis subsp. novicida (strain U112), and Cas13a of Leptotrichia buccalis (strain ATCC14201 / DSM1135 / JCM12969 / NCTC10249 / C-1013-b). More preferably, the Cas is Cas9.

[0134] The composition of the invention comprising nanoparticles suitable for in vitro delivery of nucleic acids encoding CRISPR / Cas constructs into cells or the nanoparticles of the invention are particularly suitable for application in a method according to the invention for transfecting cells with nucleic acids in the presence of saponin. The use of the invention typically involves such a composition of the invention or such a nanoparticle of the invention, where the saponin is co-administered to the cells to be transfected or the saponin is part of the composition or part of the nanoparticle.

[0135] The present invention is further illustrated by the following examples, which should not be construed as limiting the present invention in any way. Modifications and alternative implementations of some parts or compounds are possible and are encompassed within the scope of protection defined in the appended claims. EXAMPLES

[0136] Nanoplex characterization The inventors have achieved nontrivial conjugation of relatively large plasmid DNA (8,216 bp) to oligolysine-based nanoplexes (nanoparticles). Such conjugation was previously achieved only with plasmids in the 4-5 kbp range (Sama et al., 2017). 16 Nanoplexes consisting of peptide and DNA plasmid, so-called "PD-nanoplexes", were formulated at different mass ratios (Fig. 1, Fig. 2) and DNA complexation was determined qualitatively (gel retention assay) and quantitatively (fluorescence intensity measurement) (Fig. 3). The plasmid DNA encoded Cas9. The Cas9 amino acid sequence is provided here as SEQ ID NO: 1 in the list of sequences below. The Cas9 sequence is Cas9 from Streptococcus pyogenes serovar M1, the sequence was retrieved from www.uniprot.org / uniprot / Q99ZW2, where it is listed as >sp|Q99ZW2|CAS9_STRP1 CRISPR-associated endonuclease Cas9 / Csn1.

[0137] Size and size distribution PD nanoplexes loaded with Cas9 and Cas9-GFP DNA were complexed at different mass ratios. Size and PDI (polydispersity index) provide an initial assessment of the mass ratio for the optimal formulation in terms of stability and transfection efficiency (Figure 1). When the mass ratio between the mass of applied peptide and the mass of applied nucleic acid is considered, the size values ​​showed a trend towards smaller particles at higher mass ratios. PDI indicated the suspension of monodisperse particles for all formulations. This was confirmed by an exemplary size distribution evaluation shown for a 4:1 mass ratio (peptide:nucleic acid) formulation (Figure 2).

[0138] Gel Retention Without wishing to be bound by any theory, in gel electrophoresis, dyes such as ethidium bromide or SYBR Safe DNA gel stain have the purpose of making double-stranded nucleic acids visible by fluorescence after intercalation. If DNA is compactly incorporated within the nanoplex, the nucleic acid does not flow toward the anode after applying a voltage. The DNA within the nanoplex remains in the gel pocket because cancellation of the negative charges of the positively charged oligopeptides is achieved.

[0139] Therefore, DNA plasmids encoding Cas9, Cas9-GFP, and comparative GFP were complexed at different mass ratios and transferred into gel pockets. After completing electrophoresis, it was observed that DNA complexation was possible and achieved even for large plasmids (Cas9 about 7 kbp, Cas9-GFP about 8 kbp) (Figure 3).

[0140] To quantify the complexation or determine the complexation efficiency, a fluorescence-based quantitative assay was performed after nanoplex formulation. Using a fluorescent dye (Quant-IT™ PicoGreen™, Thermo Fisher) after binding to DNA, the amount of free DNA was calculated - the complexed DNA is not accessible by the dye - and thereby the complexation efficiency was calculated.

[0141] The experiment was carried out using a peptide (polylysine K 16 A slightly increased efficiency was observed with increasing amounts of glycerol (peptide), which was generally efficient (84-98%) for all mass ratios. In addition, less efficient complexation was observed with larger plasmid sizes (Figure 3).

[0142] The inventors previously demonstrated that, in a general sense, toxic effects were evident when cells were contacted with nanoplexes at a 16:1 mass ratio. In addition, the inventors previously demonstrated that, in a general sense, inefficient nucleic acid delivery was evident when cells were contacted with nanoplexes at a 2:1 mass ratio due to the N / P ratio. Thus, toxicity and nucleic acid delivery efficiency were evaluated for PD nanoplexes formulated at 4:1 and 8:1 ratios.

[0143] Cell transfection Cas9-GFP-DNA transfer JIMT-1 cells (human breast cancer) and Neuro-2A cells (murine neuroblastoma) were transfected with nanoplexes containing Cas9-GFP plasmid with and without saponin GE1741 co-application or saponin SO1861 co-application (i.e., saponin is added to the cell culture together with the addition of nanoparticles containing nucleic acids to be transfected in the presence of saponin) at a non-toxic concentration when saponin was considered. The mass ratio for peptide and nucleic acid (plasmid DNA) in the nanoparticles was selected taking into account dynamic light scattering (DLS) measurements and taking into account previously evaluated toxicity and previously performed efficiency studies as mentioned above. The transfection efficiency of all transfection conditions was measured in terms of fluorescence intensity by flow cytometry after 48 hours of incubation time, by comparison with a negative control (Figure 4). As a comparison, the efficiency of saponin-based transfection was compared with the commercially available transfection enhancer Lipofectamine3000™. The brand Lipofectamin™ is seen as the gold standard in the field of non-viral transfection. The determination of transfection efficiency was possible by the GFP (Green Fluorescent Protein) gene, which was co-transfected as the Cas9-GFP fusion protein gene in the presence or absence of saponin during contacting the cells with the nucleic acid. Successful transfection of the DNA plasmid containing Cas9-GFP, and thus indirect delivery of the Cas9 protein by intracellular transcription of Cas9-DNA in successfully transfected cells, would be indicated by green fluorescence caused by the co-transfected GFP DNA.

[0144] During the transfection process of Neuro-2A cells (seeding, transfection, incubation), the cells were extensively imaged by a live cell imaging device (Cytosmart-Omni) and analyzed for confluence. By means of a cloud-based algorithm that processed the visual images, Cytosmart provided a useful measure of viability in terms of confluence. In this concurrent experiment, no significant toxic effects were observed for transfection with and without saponin co-application (Figure 5).

[0145] The performed test provided the surprising finding that large DNA plasmids (having a size of at least 7 kbp) could be efficiently transfected into cell lines when saponin was present during cell transfection. And the results of the performed test acquire more impact when considering the smaller amount of nucleic acid molecules transfected, compared to the relatively small GFP plasmids, having a size of less than 5 kbp. Although the applied amount of nucleic acid remains at 500 ng / well, the size of the transfected DNA plasmid, and therefore the molecular mass of the plasmid, varies significantly, so the efficiency is even higher than presented.

[0146] Cas9-DNA transfer After establishing the successful delivery of Cas9-DNA by GFP-driven fluorescence, we next tested the functionality of Cas9-transfection. Thus, GFP-expressing Neuro-2A-GFP cells were obtained. The aim of the test was to demonstrate and visualize the knockout of GFP gene by CRISPR-Cas9 gene knockout. Thus, Cas9-DNA (i.e., plasmid DNA) was either co-transfected with gRNA DNA plasmid (PDD) or delivered by one plasmid DNA ("all-in-one plasmid") that codes for Cas9 and gRNA (PD) and expresses both Cas9 and guide RNA (Figure 8, Figure 9). The effect of GE1741 co-application during cell transfection was examined by the decrease in fluorescence intensity (FITC-H median and separation of dot plots on quadrants for FITC-H and FSC). Both experiments revealed a reduction in fluorescence (10% and 11% of all cells, respectively, were not considered fluorescent) (Figure 6, Figure 7). Considering that 30% of all cells were successfully transfected by Cas9-DNA, the results showed efficient and significant gene knockout to some extent. To improve gene knockout efficiency, several different gRNA sequences were tested to select the most efficient target sequence for the gRNA. The initial and further applied gRNA sequences were obtained from databases with efficiency information. Since many GFP sequences are available, the GFP gene sequence of the transfected cell line must be known for the selection of the optimal gRNA sequence. Furthermore, gene knockout requires a highly sensitive assay in which minimal changes can be detected. In highly fluorescent cells driven by promoters, single gene knockouts could not be identified in some cases.

[0147] Cas9-gRNA-DNA and LDS transfer The incorporation of linear donor sequence (LDS, also referred to as linear DNA donor template) into genomic DNA immediately after strand break is a method for knocking in with a gene of interest, thereby stably transfecting cells. In this manner, knockout and knockin can be established by CRISPR-Cas9 technology. The LDS coding for a reporter gene such as GFP and a resistance gene such as puromycin facilitates the detection of successful Cas9 modification and the isolation of each successfully transfected cell. The inventors provide a first kit, which includes: a first container containing an all-in-one plasmid (first plasmid DNA) coding for Cas9 and gRNA, and a second container containing an LDS (linear donor sequence DNA serving as DNA donor template) coding for GFP and puromycin. The all-in-one plasmid and the LDS are selected for co-transfection into cells (Figure 8), and the kit further includes instructions for transfecting the first plasmid and the LDS into target cells. Similarly, a second kit is provided by the present inventors, comprising: a container containing an all-in-one plasmid (first plasmid) encoding Cas9 and gRNA, and containing an LDS encoding GFP and puromycin. The all-in-one plasmid and the LDS are selected for co-transfection into cells (FIG. 8), and the kit further comprises instructions for transfecting the first plasmid and the LDS into target cells. The first kit and the second kit further comprise polylysine K for mixing with the first plasmid and optionally for mixing with the LDS, or for mixing with a combination of the first plasmid and the LDS, to provide nanoplexes of peptide and the first plasmid and (optionally) the LDS. 16 Alternatively, the first kit or the second kit may further contain polylysine K to provide nanoplexes of the peptide and the first plasmid and (optionally) LDS. 16 A first container containing a peptide or further comprising polylysine K 16The peptide-containing vessel is included. GFP-mediated fluorescence proves the delivery of the GFP-encoded LDS into the nucleus. Transfected cells selected by puromycin selection pressure will express GFP fluorescence over multiple passages as evidence of stable LDS integration into the genome after Cas9-induced strand breaks.

[0148] Co-transfection of Neuro-2A cells with plasmid DNA encoding Cas9 and gRNA and LDS encoding GFP and puromycin was performed using 4:1 and 8:1 polylysine K 16 The peptide-nucleic acid ratio was adjusted so that the plasmid DNA and LDS were equally complexed (500 ng / well). The cells were transfected with and without GE1741 in the cell culture medium in which the cells were cultured. The transfection efficiency under the influence of the absence or presence of saponin was compared to the transfection under the influence of the presence of Lipofectamin3000 during the transfection of the cells. The expression of fluorescence for more than 30% of all transfected cells indicates the successful delivery of LDS and consequent expression of GFP when saponin was present during the transfection. Lipofectamin3000 did not achieve comparable results (<10%) (Figure 9).

[0149] The integration of the donor sequence into the genome can be performed by non-homologous recombination ("NHR") and homologous recombination ("HR"). Linear donor sequences can be integrated in forward and reverse orientations (Figure 8), while homologous integration (homologous recombination "HR") is only possible through the homologous arms, resulting in exclusively forward integration (Figure 10). Both techniques can present advantages and disadvantages. Linear donor sequence (LDS) DNA can be integrated into the strand break more easily by NHR compared to integration applying HR. However, linearized nucleic acids are degraded relatively quickly by DNAse. The more complex homologous integration of the donor sequence faces the stability of the vector. Besides the tested knockout kit for solute transporter 26a4 (Figure 9), two other LDS donor kits (Slc19a3, Slc25a24) and two HR-based kits were tested (Slc4a4, Slc11a2). For each kit, two different gRNA sequences were examined. The results show that no fluorescence expression could be detected in transfections using homologous recombination (Figure 11). However, transfections with LDS showed a significant increase in efficiency when saponin was present during transfection. The highest efficiency level was reached after transfection with the Slc26a4 target.

[0150] Subsequent to co-transfection of Cas9-gRNA plasmid and LDS in the presence of GE1741, selection mediated by puromycin allows the isolation of transfected cells and facilitates the identification of donor sequence integration on the genome. For this purpose, different puromycin concentrations were applied to the transfected cells in the presence of GE1741. The optimal puromycin concentration should optimally kill untransfected / non-resistant cells while sparing cells that received resistance from the donor sequence. Puromycin at 0.5 μg / mL achieved no significant effect on Neuro-2A cells, 1 μg / mL only achieved incomplete selection, whereas 2-3 μg / mL depleted close to all cells and was therefore adopted for further steps (Figure 12).

[0151] Cells were grown by maintaining the puromycin concentration. Genomic DNA was extracted when confluence was reached. The presence of the integrated donor cassette by the designed primers (forward primer at the beginning of Slc26a4, reverse primer at the end of Slc26a4) would indicate targeted integration by CRISPR-Cas9. Fluorescence could still be seen 5 days after transfection.

[0152] Another knock-in transfection under the influence of the presence of GE1741 was performed, and the positive population was sorted and cultured by fluorescence-activated cell sorting (FACS). The positive population and the new Neuro-2A clones were further monitored for confluence and fluorescence. Fluorescence-activated cell sorting (FACS) for isolation of modified cells was performed as an alternative isolation method of genetically modified cells. Highly fluorescent Neuro-2A cells were selected by FACS, isolated on 96-well plates, and grown to confluence. Confluent wells were trypsinized and applied to flow cytometry. Untreated Neuro-2A cells and untreated Neuro-2A-GFP cells served as negative and positive controls. Some cell clone populations showed no fluorescence, while others revealed two distinct populations (Figure 13).

[0153] After detailed examination of the examples and tests, the following benefits were revealed to be achieved by applying the method of the present invention and by using nanoplexes in combination with saponin according to the method and use of the present invention, or by applying a composition comprising the nanoparticles of the present invention or the nanoplexes of the present invention, including co-transfection with saponin: Increased delivery of Cas9 DNA by simultaneous application of triterpenoid saponins, such as GE1741 or SO1861, preferably GE1741, is evident. When considering the mass ratio of polylysine to nucleic acid, the present inventors establish formulation ratios and concentrations that show just enough low cytotoxicity and show improved and efficient cell entry. A ratio selected from 2:1 to 15:1 is preferred, and a ratio selected from 4:1 to 8:1 is preferred. The timing of application of plasmid DNA (and saponin) to cells was precise enough to establish gene knockout and gene knockin. In the examples, gRNA sequences were used that provide efficient gene knockout. A highly sensitive assay can identify cells that are single knockouts within a cell population.

[0154] Therefore, the surprisingly increased delivery of large constructs such as Cas9 DNA (>8 kbp) under the influence of the presence of saponin during transfection of nucleic acids into cells in vitro represents a novel property of saponin that has not existed and been evident until now to the knowledge of the inventors. Cells capable of saponin-based in vitro transfection (e.g. Neuro-2A, JIMT-1) can now be transfected by any nucleic acid, regardless of their size, due to this contribution by the inventors. Importantly, saponin-based transfection of plasmid DNA with a plasmid size larger than 5 kbp, for example even larger than 7-8 kbp, reaches a higher efficiency when compared to one of the methods currently used in the field using the transfection agent Lipofectamin3000. The problems that plague the art when the desired transfection of relatively large plasmid DNA, e.g., 5.5 kbp, or higher, is considered, are exemplified, for example, by the following quote taken from the Thermo Fisher website in December 2020: "Despite similar uptake efficiencies in cationic lipid-mediated transfection, nuclear delivery of large plasmids is challenging compared to small plasmid molecules. This effect was observed using constructs of different sizes of comparable mass or molar concentration, suggesting that nuclear delivery of plasmids may be limited by the rate of transit within the cell and that small plasmids escape degradation not by saturation of cellular defenses but rather by rapid transit through the cytoplasm (Lukacs et al., 2000; McLenachan et al., 2007)." Taken from www.thermofisher.com / de / de / home / references / gibco-cell-culture-basics / transfection-basics / guidelines-for-plasmid-dna-transfection.html.

[0155] The technique of the present invention, in which saponin is applied as a transfection booster for large constructs such as plasmid DNA, offers a solution to the seemingly hindered transfection efficiency currently seen in the art.

[0156] Saponin offers various applications in the field of transfection. The tests carried out and the examples presented and the tests carried out previously have demonstrated that this applies in in vitro environments and experiments. The successful complexation of large DNA plasmids (plasmid DNA) to polylysine-based nanoplexes and the previously carried out nanoplex formulations with smaller sized plasmid DNA (having a plasmid DNA size of up to 4-5 kbp), mRNA and minicircle DNA, combined with the presence of saponin when the nanoparticles are brought into contact with the cells that should be transfected with the nucleic acid, have been demonstrated. 16 The transfection triad consisting of nanoparticles composed of peptides and nucleic acids has now been shown according to the present invention to be a universal tool for efficient and non-toxic in vitro gene delivery.

[0157] Polylysine K 16 Surprisingly, DNA plasmids as large as 8.2 kbp could be complexed and transfected into cells in vitro in the presence of GE1741 or SO1861 (1-5 micrograms / mL) by peptide-based nanoplexes. Based on the analysis performed, it was evident that polylysine peptides of larger size could also form stable nanoplexes (gel retention assay). This provides flexibility in the amount of positively charged peptide, which can be adjusted.

[0158] reference Clochard, J., Jerz, G., Schmieder, P., Mitdank, H., Troger, M., Sama, S., Weng, A. (2020) A new acetylated triterpene saponin from Agrostemma githago L. modulates gene delivery efficiently and shows a high cellular tolerance. International Journal of Pharmaceutics, Vol. 589, p. 119822. Ryu, N., Kim, MA, Park, D., Lee, B., Kim, YR, Kim, KH, Baek, JI, Kim, WJ, Lee, KY, Kim, UK, (2018) Effective PEI-mediated delivery of CRISPR-Cas9 complex for targeted gene therapy. Nanomedicine, Vol. 14, p. 2095-2102. Sama, S., Jerz, G., Schmieder, P., Joseph, JF, Melzig, MF, Weng, A. (2018a) Plant derived triterpenes from Gypsophila elegans M.Bieb. enable non-toxic delivery of gene loaded nanoplexes. Journal of Biotechnology, 284, 131-139. Sama, S., G. Jerz, P. Schmieder, E. Woith, MF Melzig, and A. Weng. 2017. "Sapofectosid -Ensuring non-toxic and effective DNA and RNA delivery". International Journal of Pharmaceutics, 534:195-205. Sama, S., E. Woith, W. Walther, G. Jerz, W. Chen, S. Hart, M. F. Melzig, and A. Weng. 2018b. “Targeted suicide gene transfections reveal promising results in nu / nu mice with aggressive neuroblastoma.” Journal of Controlled Release, 275:208-16.

[0159] Sequence - Sequencing number SEQ ID NO:1 Cas9 from Streptococcus pyogenes serovar M1; sequence retrieved from www.uniprot.org / uniprot / Q99ZW2 >sp|Q99ZW2|CAS9_STRP1 CRISPR-associated endonuclease Cas9 / Csn1 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAE ATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSD VDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDLDNLLAQIGDQYADLFLAAKNLSDAI LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELH AILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL SGEQKKAIVDLLFKTTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDRIEMIEERLKTYAHLFDDKVMKQLKRRRYTGWG RLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRER MKRIEEGIGELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL TKAERGGGLSELDKAGFIKRKQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK MIAKSEQEIGKATAKYFFYSNIMNFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVA YSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVE QHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

[0160] SEQ ID NO:2 Cas12a from Francisella tularensis subsp. novicida (strain U112); sequence retrieved from www.uniprot.org / uniprot / A0Q7Q2 >sp|A0Q7Q2|CS12A_FRATN CRISPR-associated endonuclease Cas12a MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQF FIEILSSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFK NLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFK GFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAE ELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGI NEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIA AFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEY ITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILA NFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKL KIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNF ENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYK LLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKF IDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQ GKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKK ITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEI NLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAI EKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVE KQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAG FTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKG KWTIASFFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESD KKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAY HIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN

[0161] SEQ ID NO:3 Cas13a from Leptotrichia buccalis (strain ATCC14201 / DSM1135 / JCM12969 / NCTC10249 / C-1013-b); sequence retrieved from www.uniprot.org / uniprot / C7NBY4 >sp|C7NBY4|CS13A_LEPBD CRISPR-associated endoribonuclease Cas13a MKVTKVGGISHKKYTSEGRLVKSESEENRTDERLSALLNMRLDMYIKNPSSTETKENQKR IGKLKKFFSNKMVYLKDNTLSLKNGKKENIDREYSETDILESDVRDKKNFAVLKKIYLNE NVNSEELEVFRNDIKKKLNKINSLKYSFEKNKANYQKINENNIEKVEGKSKRNIIYDYYR ESAKRDAYVSNVKEAFDKLYKEEDIAKLVLEIENLTKLEKYKIREFYHEIIGRKNDKENF AKIIYEEIQNVNNMKELIEKVPDMSELKKSQVFYKYYLDKEELNDKNIKYAFCHFVEIEM SQLLKNYVYKRLSNISNDKIKRIFEYQNLKKLIENKLLNKLDTYVRNCGKYNYYLQDGEI ATSDFIARNRQNEAFLRNIIGVSSVAYFSLRNILETENENDITGRMRGKTVKNNKGEEKY VSGEVDKIYNENKKNEVKENLKMFYSYDFNMDNKNEIEDFFANIDEAISSIRHGIVHFNL ELEGKDIFAFKNIAPSEISKKMFQNEINEKKLKLKKIFRQLNSANVFRYLEKYKILNYLKR TRFEFVNKNIPFVPSFTKLYSRIDDLKNSLGIYWKTPKTNDDNKTKEIIDAQIYLLKNIY YGEFLNYFMSNNGNFFEISKEIIELNKNDKRNLKTGFYKLQKFEDIQEKIPKEYLANIQS LYMINAGNQDEEEKDTYIDFIQKIFLKGFMTYLANNGRLSLIYIGSDEETNTSLAEKKQE FDKFLKKYEQNNNIKIPYEINEFLREIKLGNILKYTERLNMFYLILKLLNHKELTNLKGS LEKYQSANKEEAFSDQLELINLLNLDNNRVTEDFELEADEIGKFLDFNGNKVKDNKELKK FDTNKIYFDGENIIKHRAFYNIKKYGMLNLLEKIADKAGYKISIIEELKKYSNKKNEIEKN HKMQENLHRKYARPRKDEKFTDEDYESYKQAIENIEEYTHLKNKVEFNELNLLQGLLLRI LHRLVGYTSIWERDLRFRLKGEFPENQYIEEIFNFENKKNVKYKGGQIVEKYIKFYKELH QNDEVKINKYSSANIKVLKQEKKDLYIRNYIAHFNYIPHAEISLLELVLENLRKLLSYDRK LKNAVMKSVVDILKEYGFVATFKIGADKKIGIQTLESEKIVHLKNLKKKLMTDRNSEEL CKLVKIMFEYKMEEKKSEN

Claims

1. Use of a composition comprising saponin in the in vitro delivery of nucleic acid into cells, wherein the saponin is a bisdesmoside-type triterpenoid 12,13-dehydrooleanane-type saponin, and the nucleic acid comprises more than 5.5 kilobase pairs (kbp), for the in vitro delivery of nucleic acid into cells.

2. The saponin is according to formula (I), 【Chemical 1】 wherein, R １ is a xylose residue, an arabinose residue, or a glucose residue, and is bonded to the corresponding xylose residue of formula (I) by its C1 atom, R ２ is, independently of other R ２ residues on the same molecule, H, an acetyl residue, or a xylose residue attached by its C1 atom to the corresponding quinovose residue of formula (I), provided that at least two acetyl residues or at least one acetyl residue and a xylose residue attached to said quinovose residue are present in said saponin, Alternatively, the R of the saponin according to formula (I) １ is a xylose residue, and / or the saponin has exactly two acetyl groups, alternatively, the acetyl group of the saponin according to formula (I) is bonded to the oxygen atoms at the C3 and C4 positions of the corresponding quinovose residue of the saponin, Alternatively, the R of the saponin according to formula (I) ２ One of the residues is a xylose residue, and the R ２ One of the residues is an acetyl group, and the R ２ One of the residues is H, or the xylose residue of the saponin according to formula (I) is bonded to the oxygen atom at the C3 position of the corresponding quinovose residue, and the acetyl group is bonded to the oxygen atom at the C4 position of the corresponding quinovose residue of the saponin Alternatively, the R of the saponin according to formula (I) １ is a xylose residue, and the two Rs of the saponin according to formula (I) ２ groups are acetyl groups, which are attached to the oxygen atoms at the C3 and C4 positions of the corresponding quinovose residues, and the third R ２ group is H, the use according to claim 1.

3. The saponin is GE1741 according to formula (II), 【Chemical 2】 In the formula, R １ is xylose, alternatively, the saponin is SO1861 according to formula (III), [Chemical Formula 3] Use according to claim 2.

4. Use according to claim 1, wherein the nucleic acid is plasmid DNA.

5. Use according to claim 1, wherein the nucleic acid forms part of nanoparticles, which nanoparticles further comprise a nanoparticle-forming compound, or the nanoparticle-forming compound comprises or consists of a polylysine peptide, or the polylysine peptide consists of 5 to 25 lysine residues, or the polylysine peptide consists of 16 lysine residues.

6. Use according to claim 5, wherein the nanoparticles comprise the nanoparticle-forming compound and the nucleic acid in a mass ratio in the range of 3:1 to 15:1, or in the range of 3:1 to 9:

1.

7. Use according to claim 1, wherein the nucleic acid comprises at least a first part of the nucleic acid encoding a CRISPR-associated endonuclease (Cas), wherein the nucleic acid is a gRNA or comprises a gRNA, or the nucleic acid is plasmid DNA encoding Cas, or the nucleic acid is at least plasmid DNA for expressing the Cas, or the nucleic acid is plasmid DNA for expressing the Cas and for expressing the gRNA, or the nucleic acid comprises a second part of the nucleic acid encoding a guide RNA (gRNA).

8. Use according to claim 1, wherein the nucleic acid comprises at least 6 kbp, or at least 7 kbp, or at least 7.5 kbp, or more than at least 8 kbp. **Claim 9**: The use according to claim 4, wherein the plasmid DNA contains at least 6 kbp, or at least 7 kbp, or at least 7.5 kbp, or more than at least 8 kbp. **Claim 10**: The use according to claim 7, wherein the plasmid DNA encoding Cas contains at least 6 kbp, or at least 7 kbp, or at least 7.5 kbp, or more than at least 8 kbp. **Claim 11** The nucleic acid encoding Cas encodes a Cas having at least 90% amino acid residue identity to any one of Cas9 (SEQ ID NO: 1), Cas12a (SEQ ID NO: 2), Cas13a (SEQ ID NO: 3), or Streptococcus pyogenes serotype M1 Cas9, Francisella tularensis subsp. novicida (strain U112) Cas12a, and Leptotrichia buccalis (strain ATCC14201 / DDSM1135 / JCM12969 / NCTC10249 / C-1013-b) Cas13a, and is the use according to claim 7. **Claim 12** **Claim 13**: The use according to claim 1, wherein the cell is a eukaryotic cell. **Claim 13** A method for delivering a nucleic acid containing more than 5.5 kilobase pairs (kbp) into cells in vitro, comprising: (i) providing the nucleic acid; (ii) providing a saponin which is a bisdesmoside-type triterpenoid 12,13-dehydrooleanane-type saponin; (iii) incubating the nucleic acid and the cells in the presence of the saponin. **Claim 14** The saponin is according to formula (I), 【Chemical 4】 wherein R １ is a xylose residue, an arabinose residue, or a glucose residue, and is bonded to the corresponding xylose residue of formula (I) by its C1 atom, R ２ is, independently of the other R ２ residues of the same molecule, H, an acetyl residue, or a xylose residue bonded to the corresponding quinovose residue of formula (I) by its C1 atom, provided that at least two acetyl residues or at least one acetyl residue and a xylose residue bonded to said quinovose residue are present in said saponin, the method according to claim 13. **Claim 15** **Claim 16**: The method according to claim 13, wherein the nucleic acid is plasmid DNA. **Claim 16** **Claim 17**: The nucleic acid forms part of a nanoparticle, the nanoparticle further contains a nanoparticle-forming compound, or the nanoparticle contains a polylysine peptide of the nanoparticle-forming compound, or the polylysine peptide consists of 5 to 25 lysine residues, or the polylysine peptide consists of 16 lysine residues, and is the method according to claim 13. **Claim 18** **Claim 19**: The method according to claim 16, wherein the nanoparticle contains the nanoparticle-forming compound and the nucleic acid in a mass ratio in the range of 3:1 to 15:1, or in the range of 3:1 to 9:

1. **Claim 18** The method according to claim 13, wherein the saponin concentration is 1 μg / mL to 5 μg / mL in step (ii) of the method.

19. The saponin is GE1741 according to formula (II), 【Chemical Formula 5】 wherein, R １ is xylose or the saponin is SO1861 according to formula (III), 【Chemical Formula 6】 The method according to claim 13.

20. The nucleic acid is a nucleic acid encoding a CRISPR / Cas construct comprising at least a first part encoding Cas, the nucleic acid is a gRNA, or the nucleic acid encoding the CRISPR / Cas construct is provided as part of a plasmid DNA, or here, at least the part of the nucleic acid encoding the Cas part of the CRISPR / Cas construct is provided as part of a plasmid DNA, or the plasmid DNA is for expressing the Cas and the gRNA of the CRISPR / Cas construct, or the nucleic acid encodes a gRNA or comprises a second part containing a gRNA, The method according to claim 13.

21. The method according to claim 13, wherein the nucleic acid comprises at least 6 kbp, or at least 7 kbp, or at least 7.5 kbp, or more than at least 8 kbp.

22. The method according to claim 15, wherein the plasmid DNA comprises at least 6 kbp, or at least 7 kbp, or at least 7.5 kbp, or more than at least 8 kbp.

23. The nucleic acid encoding Cas encodes Cas having at least 90% amino acid residue identity to any one of Cas9 (SEQ ID NO: 1), Cas12a (SEQ ID NO: 2), Cas13a (SEQ ID NO: 3), or Streptococcus pyogenes serotype M1, Francisella tularensis subsp. novicida (strain U112) of Cas12a, and Leptotrichia buccalis (strain ATCC14201 / DSM1135 / JCM12969 / NCTC10249 / C-1013-b) of Cas13a, The method according to any one of claims 20.

24. A kit for delivering a nucleic acid containing more than 5.5 kilobase pairs (kbp) into cells in vitro, (a) a first container containing a first plasmid DNA, (c) Polylysine K １６ A third container containing a peptide (d) A fourth container containing a bisdesmoside-type triterpenoid 12,13-dehydrooleanane-type saponin, or one saponin, or at least one saponin selected from GE1741 and SO1861, (e) Instructions for use, comprising or consisting of a first combination, Here, the use at least includes the nanoplex of the polylysine K １６ peptide and the first plasmid DNA, and the preparation of the saponin, or the use at least includes the preparation of the nanoplex of the polylysine K16 peptide and the first plasmid DNA, The first plasmid has a size of at least 5.5 kbp. Or (a) A first container containing a third plasmid DNA, (b) Polylysine K １６ A second container containing a peptide (c) A third container containing a bisdesmoside-type triterpenoid 12,13-dehydrooleanane-type saponin, or one saponin, or at least one saponin selected from GE1741 and SO1861, (d) Instructions for use, comprising or consisting of a second combination, Here, the use includes at least the polylysine K １６ peptide and the nanoplex of the third plasmid DNA, and the preparation of the saponin, or the use includes at least the polylysine K １６ peptide and the preparation of the nanoplex of the third plasmid DNA The third plasmid has a size of at least 5.5 kbp. Or (a) The first container containing the first plasmid DNA, (c) Polylysine K １６ A third container containing a peptide (d) A fourth container containing a bisdesmoside-type triterpenoid 12,13-dehydrooleanane-type saponin, or one saponin, or at least one saponin selected from GE1741 and SO1861, (e) Instructions for use, comprising or consisting of a third combination, Here, the use at least includes the preparation of the nanoplex of the polylysine K １６ peptide and the first plasmid DNA, and the saponin, or the use at least includes the preparation of the nanoplex of the polylysine K 16 peptide and the first plasmid DNA The first plasmid has a size of at least 5.5 kbp. Or, the first plasmid DNA encodes Cas9, or the third plasmid DNA encodes Cas9 and encodes gRNA, a parts kit comprising.

25. The first combination further comprises (b) a second container containing a second plasmid DNA, or The third combination further comprises (b) a second container containing an RNA oligonucleotide, The parts kit according to claim 24.

26. A composition comprising nanoparticles suitable for in vitro delivery of nucleic acids into cells, the nanoparticles comprising (i) The nucleic acid containing more than 5.5 kilobase pairs (kbp), (ii) A polylysine peptide, and (iii) A saponin which is a bisdesmoside-type triterpenoid 12,13-dehydrooleanane-type saponin, comprising or consisting of.

27. The saponin is according to formula (I), 【Chemical Formula 7】 Wherein, R １ is a xylose residue, an arabinose residue, or a glucose residue, and is bonded to the corresponding xylose residue of formula (I) by its C1 atom, R ２ is, independently of other R ２ residues on the same molecule, H, an acetyl residue, or a xylose residue bonded to the corresponding quinovose residue of formula (I) by its C1 atom, provided that at least two acetyl residues or at least one acetyl residue and a xylose residue bonded to the said quinovose residue are present in the said saponin, Alternatively, R of the saponin according to formula (I) １ is a xylose residue and / or the saponin has exactly two acetyl groups, Alternatively, the acetyl group of the saponin according to formula (I) is bonded to the oxygen atoms at the C3 and C4 positions of the corresponding quinovose residue of the saponin, Alternatively, said R of said saponin according to formula (I) ２ One of the residues is a xylose residue, and said R ２ One of the residues is an acetyl group, and said R ２ One of the residues is H, Alternatively, the xylose residue of the saponin according to formula (I) is bonded to the oxygen atom at the C3 position of the corresponding quinovose residue, and the acetyl group is bonded to the oxygen atom at the C4 position of the corresponding quinovose residue of the saponin, Alternatively, the R of the saponin according to formula (I) １ is a xylose residue, and the two Rs of the saponin according to formula (I) ２ groups are acetyl groups, which are bonded to the oxygen atoms at the C3 and C4 positions of the corresponding quinovose residue, and the third R ２ group is H, the composition according to claim 26.

28. The saponin is GE1741 according to formula (II), 【Chemical 8】 wherein, R １ is xylose or the saponin is SO1861 according to formula (III). 【Chemical Formula 9】 The composition according to claim 27.

29. The polylysine peptide consists of 5 to 25 lysine residues, or the polylysine peptide consists of 16 lysine residues, the composition according to claim 26.

30. The nanoparticles contain the polylysine peptide and the nucleic acid in a mass ratio in the range of 3:1 to 15:1, or in the range of 3:1 to 9:1, the composition according to claim 26.

31. The nucleic acid is part of plasmid DNA, the composition according to claim 26.

32. The nucleic acid contains at least the first part of the nucleic acid encoding Cas, the nucleic acid contains or is a gRNA, or the nucleic acid contains the second part of the nucleic acid encoding gRNA, the composition according to claim 26.

33. The nucleic acid contains at least 6 kbp, or at least 7 kbp, or at least 7.5 kbp, or more than at least 8 kbp, the composition according to claim 26.

34. The plasmid DNA contains at least 6 kbp, or at least 7 kbp, or at least 7.5 kbp, or more than at least 8 kbp, the composition according to claim 31.

35. The first part encoding the Cas of the nucleic acid encodes a Cas having at least 90% amino acid residue identity with any one of Cas9 (SEQ ID NO: 1), Cas12a (SEQ ID NO: 2), Cas13a (SEQ ID NO: 3), or Cas9 of Streptococcus pyogenes serotype M1, Francisella tularensis subsp. novicida (strain U112) Cas12a, and Leptotrichia buccalis (strain ATCC 14201 / DDSM 1135 / JCM 12969 / NCTC 10249 / C-1013-b) Cas13a, the composition according to claim 32.

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