Vector-free delivery of gene editing proteins and compositions to cells and tissues

Abstract

To provide a method for vector-free delivery of a gene editing composition across a plasma membrane of a cell.SOLUTION: A method for delivering a gene editing composition across a plasma membrane of a cell, comprises the steps of: providing a population of cells; and contacting the population of cells with a volume of aqueous solution, the aqueous solution comprising the gene editing composition and an alcohol at a concentration of at least about 2%, wherein the volume is a function of: (i) exposed surface area of the population of cells; or (ii) a number of cells in the population of cells.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 273,284, filed December 30, 2015, the entire contents of which are incorporated herein by reference.

[0002] Technical Field The subject matter described herein relates to vector-free delivery of gene editing compounds and complexes to cells and tissues.

[0003] INCORPORATION BY REFERENCE The contents of the text file named "48831_506001WO_ST25.txt", created on December 22, 2016, and measuring 64.2 KB, are incorporated herein by reference in their entirety. [Background technology]

[0004] background The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas genome manipulation system (hereafter referred to as the "CRISPR-Cas system") allows researchers to modify genomic DNA inside cells. The system requires three components: Cas9, which can be derived from Streptococcus pyogenes, is an endonuclease that complexes with CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA) to form RGENs (RNA-guided endonucleases). These RGENs cleave chromosomal DNA inside the cell, generating site-specific double-strand breaks (DSBs). Repair of these DSBs can occur via endogenous high-fidelity homologous recombination (HR) or error-prone non-homologous end joining (NHEJ), leading to targeted mutagenesis and chromosomal rearrangements. The specificity of the RGENs is determined by Watson-Crick base pairing between the crRNA and the target DNA and Cas9 recognition of the NGG-trinucleotide protospacer adjacent motif (PAM). The crRNA and tracrRNA can be fused to form a single guide RNA (gRNA or sgRNA). A major advantage of CRISPR-Cas9 over other genome editing nucleases is that the specificity of Cas9 can be easily reprogrammed by replacing the crRNA, in contrast to zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), whose DNA targeting specificity must be altered by protein engineering strategies.

[0005] CRISPR-Cas systems have been most commonly delivered into cells in the form of plasmid DNA, encoding the three components either as separate plasmids or as a combined plasmid. However, plasmids are associated with off-target editing. Plasmids may randomly integrate within the genome or at sites generated by Cas9. While the latter events can at least be predicted and monitored, the former can be more problematic, as they can be difficult to detect. These foreign sequences can trigger a host immune response, regardless of the integration site, which creates challenges for the use of gene-edited stem or primary cells in clinical applications. Furthermore, cells transfected with plasmids for clinical applications are considered genetically modified by regulatory authorities and are therefore subject to lengthy and costly regulatory procedures. Summary of the Invention

[0006] The devices, methods, and systems described below provide a reliable and efficient system for delivering building blocks of gene editing systems, such as proteins or gene editing complexes, thereby solving many of the problems and shortcomings associated with previous gene editing approaches by avoiding the delivery of plasmid DNA or other vectors that code for such proteins.The systems also offer advantages over other approaches, such as electroporation, magnetofection, liposome-mediated transfection, lentivirus-mediated transfection, or intracellular injection methods.

[0007] While some gene editing systems use DNA plasmid-type Cas9 and guide RNA, the invention described herein delivers RNPs [e.g., gene editing proteins and guide RNA] to achieve more transient gene editing effects, leading to fewer off-target effects. Electroporation can be used to deliver proteins, such as gene editing proteins, to cells, but the methods, systems, and compositions described herein offer several advantages over electroporation. For example, the ethanol-mediated spray delivery system of the present invention delivers gene editing proteins and complexes with greater efficiency, such as 10%, 20%, 50%, 75%, 2-fold or more, compared to electroporation, and with equal or better cell viability after delivery and equal or better gene editing efficiency compared to electroporation. Moreover, cell function, such as stem cell differentiation or immune cell activation, is better preserved using the described ethanol spray method compared to electroporation (e.g., 10%, 20%, 50%, 75%, 2-fold or more). In addition, unlike the system described herein, other methods such as liposome-mediated transfection or electroporation require the latter approach to detach adherent cells from their substratum in order to transfect them, for example using an electroporator, and such an extra manipulation step compromises both the sterility and biological properties / functions of the cells. Moreover, using other methods, for example electroporation, multiple administrations of delivery cargo (gene delivery proteins and / or gene delivery complexes) are not possible due to the high level of cell damage associated with such treatments. The compositions and methods described herein allow for 2, 3, 4, 5 or more administrations (delivery spray, followed by a stop solution), a process that increases the efficiency of delivery of cargo to cells and the amount of gene editing composition to the target cells. Target cells include primary cells (e.g., cells cultured directly from the source organ tissue or obtained from blood) and immortalized cells, for example cell lines, as well as adherent cells and suspension cells, for example free-floating cells.Primary human cells are cultured directly from their source organ tissue or blood cells.

[0008] Electroporation is a widely used vector-free / carrier-free method, which may be efficient for the delivery of nucleic acid and / or protein to some cell types, but may be highly toxic, especially to primary cells.Therefore, alternative membrane disruption methods are required.As mentioned above, the device, method and system described herein are advantageous over such other techniques, especially over electroporation.

[0009] An aspect of the subject matter provides a method for delivering a gene editing composition through the plasma membrane of a cell. In a preferred embodiment, the method does not include a vector, such as a lentiviral vector, a liposome, or electroporation. In some embodiments, the method includes contacting or delivering a cell with a plasmid DNA encoding a gene editing protein; in other embodiments, the method does not include contacting or delivering a cell with a plasmid DNA encoding a gene editing protein. In some embodiments, the method includes contacting or delivering a cell with an RNA encoding a gene editing protein; in other embodiments, the method does not include contacting or delivering a cell with an RNA encoding a gene editing protein. The method described herein includes providing a cell population and contacting the cell population with a volume of an aqueous solution, for example, comprising a gene editing protein and / or complex. The aqueous solution may include a gene editing composition and an alcohol concentration of at least about 2%. In various embodiments, the volume is a function of (i) the exposed surface area of ​​the cell population or (ii) the number of cells in the cell population.

[0010] Preferably, the solution is delivered to the cells in the form of a spray or mist, for example, as a plurality of aqueous particles, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more per cell.For example, the cells are coated with the spray, but are not immersed or submerged in the solution containing the delivery compound.The spray or mist includes the liquid blown or sprayed by air in the form of droplets.

[0011] Examples of conditions that achieve coating of the coated cell population include delivery of a fine particle spray, e.g., conditions that exclude dripping or pipetting a large amount of solution onto the cells so that the substantial cell population is immersed or submerged in the volume of liquid. Thus, the mist or spray includes a certain ratio of the volume of liquid to the volume of cells. Alternatively, the conditions include a certain ratio of the volume of the mist or spray to the exposed cell area, e.g., the area of ​​the exposed cell membrane when the cells are present as a confluent or substantially confluent layer on a substantially flat surface such as the bottom of a well of a tissue culture vessel, e.g., a tissue culture plate, e.g., a microtiter tissue culture plate.

[0012] Thus, there is a need to provide a vector-free, e.g., viral vector-free, approach for delivering a biologically relevant payload, e.g., a compound or composition, across the plasma membrane into a cell. "Cargo" or "payload" are terms used to describe a compound or composition that is delivered to the interior of a cell across the cell plasma membrane by an aqueous solution.

[0013] In some examples, the aqueous solution comprises an alcohol concentration of about 2% to about 50%; at least about 2% and less than about 20, 19, 18, 17, 16, or 15%; or at least about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7.7.5, 8.8.5, 9.9.5, 10, 11, 12, 13, 14, or 15%. In some aspects, the alcohol is at a concentration of less than about 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2%. In some aspects, the alcohol, e.g., ethanol, concentration is not more than 50%. In some aspects, the alcohol, e.g., ethanol, concentration is not more than 70%. In some examples, the alcohol concentration is about 25%. The alcohol concentration can vary depending on the cell type, e.g., the alcohol concentration can be 25%, 27% or 33%. For example, certain target cells, e.g., T cells such as primary T cells, are characterized by a higher tolerance to ethanol and maintain viability and functionality following treatment with a delivery buffer containing greater than 25%, e.g., 27%, 30%, 33%, 35% or more ethanol. NK cells, e.g., primary NK cells, may also have such a tolerance level with respect to the presence / concentration of ethanol in the cargo delivery buffer.

[0014] One or more of the following features may be included in any workable combination: The volume of solution delivered to the cells is a plurality of units, e.g., a spray of aqueous particles, e.g., a plurality of droplets. The volume is stated relative to an individual cell, or relative to the exposed surface area of ​​a confluent or substantially confluent (e.g., at least 75%, at least 80%, e.g., 85%, 90%, 95%, 97%, 98%, 100% confluent) cell population. For example, the volume may be 6.0×10 -7 Microliters / cell ~7.4 x 10 -4 The volume can be 4.9 x 10 microliters / cell. -6Microliters / cell ~2.2 x 10 -3 The volume is 9.3 x 10 -6 Microliters / cell ~2.8 x 10 -5 The volume can be approximately 1.9×10 microliters per cell. -5 The volume can be 6.0 x 10 microliters per cell, within about 10 percent. -7 Microliters / cell ~2.2 x 10 -3 The volume is 2.6 x 10 9 Microliters / square micrometer exposed surface area ~1.1 x 10 -6 The volume can be 5.3 x 10 microliters per square micrometer of exposed surface area. -8 Microliters / square micrometer exposed surface area ~1.6×10 -7 The volume can be approximately 1.1×10 microliters per square micrometer of exposed surface area. -7 It can be in microliters per square micrometer of exposed surface area. The term "about" can be, for example, within ten percent of the stated unit or amount.

[0015] Cell confluency refers to the cells that are in contact with each other on a surface. For example, confluency can be expressed as an estimated (or counted) percentage, for example, 10% confluency means that 10% of a surface, such as the surface of a tissue culture vessel, is covered with cells, and 100% means that the surface is completely covered. For example, adherent cells grow two-dimensionally on the surface of a tissue culture well, plate or flask. Non-adherent cells can be removed by centrifugation, pulling down from the top of the cell mass by vacuum or aspiration of tissue culture medium, or by aspiration or vacuum removal from the bottom of the vessel.

[0016] The step of contacting the cell population with a volume of aqueous solution can be performed by a gas that propels the aqueous solution to form a spray. The gas can include nitrogen, air, or an inert gas. The spray can include discrete volume units ranging in size from 10 nm to 100 μm in diameter, for example 30 to 100 μm. The spray includes discrete volume units having a diameter of about 30 to 50 μm. A total volume of 20 μl of aqueous solution can be sprayed to a cell-occupied area of ​​about 1.82 cm. 2 For example, a single well of a 24-well culture plate can be delivered to a total volume of 10 μl of aqueous solution, which corresponds to a cell-occupied area of ​​approximately 0.2–2 cm. 2 , for example, about 0.95 cm 2 For example, 10 μl of spray is delivered to one well of a 96-well culture plate (area may vary depending on the multi-well plate manufacturer). For example, 10 μl of spray covers a cell-occupied area of ​​approximately 1.82 cm for a 24-well plate. 2 0.64 cm for a 48-well plate 2 and 0.29 cm for 96-well plates 2 Spray delivery volumes are adjusted for microtiter plates, which have more wells and / or a smaller approximate cell growth / cell footprint per well for multi-well plates (additional examples are provided below). As noted above, well sizes can vary by plastics supplier. Suppliers average: 24 wells = approx. 2.0 cm 2 ;Delivery 20μl 48 wells = approx. 0.8 cm 2 ;Delivery 5μl 96 wells = approx. 0.3 cm 2 ;Delivery 2.5μl It is.

[0017] In this example, the volume of solution in each size well was measured. Volume to surface area or volume to cell number ratios are commonly used.

[0018] In another aspect, the culture plate may include sample wells selected from 1, 6, 9, 12, 24, 48, 96, 384, and 1536 wells, and in a particular example, the culture plate has 96 sample wells. The diameter of the sample wells may range from 0.1 mm to 100 mm, for example, a 24-well culture plate may have a diameter of about 15.6 mm, a 48-well culture plate may have a diameter of about 11 mm, and a 96-well culture plate may have a diameter of about 6.4 mm. Approximate well dimensions and spray delivery volumes are shown below.

[0019] TIFF2025010591000002.tif69128*Square wells; **2.5 μl as determined by the volume of solution placed in each size well (see above)

[0020] Typically, the aqueous solution contains a payload to be delivered across the cell membrane into the cells, and the second volume is a buffer or culture medium without a payload. Alternatively, the second volume (buffer or medium) can also contain a payload. In some embodiments, the aqueous solution contains a payload and an alcohol, and the second volume does not contain alcohol (and optionally does not contain a payload). The cell population can be contacted with the aqueous solution for 0.01 to 10 minutes, e.g., 0.1 to 10 minutes, before adding a second volume of buffer or culture medium to submerge or suspend the cell population. The buffer or culture medium can be phosphate buffered saline (PBS). The cell population can be contacted with the aqueous solution for 2 seconds to 5 minutes, before adding a second volume of buffer or culture medium without a payload to submerge or suspend the cell population. The cell population can be contacted with an aqueous solution containing a payload, e.g., for 30 seconds to 2 minutes, before adding a second volume of buffer or culture medium without a payload to submerge or suspend the cell population. The cell population can be contacted with the sprayed solution for about 1-2 minutes before a second volume of buffer or culture medium is added to submerge or suspend the cell population. In some examples, preparation of the aqueous solution is less than about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 minute prior to delivery.

[0021] In some embodiments, the addition of the alcohol to the aqueous solution can be after 5, 10, 15, 20, 25, 30, 45 minutes (e.g., 20-30 minutes) of incubation.

[0022] During the period between spraying of the cells and addition of buffer or culture medium, the cells remain hydrated by a layer of water from the spray volume.

[0023] The gene editing composition may comprise a compound that edits genomic DNA. For example, the gene editing composition may comprise a compound or complex that cuts, nicks, splices, rearranges, translocates, recombines, or otherwise modifies genomic DNA. Alternatively or additionally, the gene editing composition may comprise (i) a compound that is included in a gene editing complex that cuts, nicks, splices, rearranges, translocates, recombines, or otherwise modifies genomic DNA; or (ii) a compound that can be processed or modified to become a compound that is included in a gene editing complex that cuts, nicks, splices, rearranges, translocates, recombines, or otherwise modifies genomic DNA. In various embodiments, the gene editing composition comprises one or more of: (a) a gene editing protein; (b) an RNA molecule; and / or (c) a ribonucleoprotein (RNP).

[0024] In some embodiments, the gene editing composition comprises a gene editing protein, and the gene editing protein is a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a Cas protein, a Cre recombinase, a Hin recombinase, or an Flp recombinase.In additional embodiments, the gene editing protein can be a fusion protein (megaTAL) that combines a homing endonuclease with the modular DNA binding domain of TALEN.For example, megaTAL can be delivered as a protein, or alternatively, the mRNA that codes for megaTAL protein is delivered to cell.

[0025] In various embodiments, the gene editing composition comprises an RNA molecule, wherein the RNA molecule comprises an sgRNA, a crRNA, and / or a tracrRNA.

[0026] In certain embodiments, the gene editing composition comprises an RNP, the RNP comprises a Cas protein and an sgRNA or a crRNA and a tracrRNA. Aspects of the present subject matter are particularly useful for controlling when and how long a particular gene editing compound is present in a cell.

[0027] In various embodiments of the subject matter, the gene editing composition is detectable in the cell population or progeny cells for (a) about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, 48, 60, 72, 0.5-2, 0.5-6, 6-12, or 0.5-72 hours after contacting the cell population with the aqueous solution; or (b) less than about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, 48, 60, 72, 0.5-2, 0.5-6, 6-12, or 0.5-72 hours after contacting the cell population with the aqueous solution.

[0028] In some aspects, the genome of a cell in the cell population or a progeny cell comprises at least one site-specific recombination site for Cre recombinase, Hin recombinase, or Flp recombinase.

[0029] Aspects of the present invention relate to cells that contain one gene editing compound and to inserting another gene editing compound into these cells. For example, one component of RNP can be introduced into a cell that expresses or otherwise already contains another component of RNP. For example, the cells in the cell population or their progeny can contain sgRNA, crRNA, and / or tracrRNA. In some embodiments, the cell population or its progeny expresses sgRNA, crRNA, and / or tracrRNA. Alternatively or additionally, the cells in the cell population or their progeny express Cas protein.

[0030] Various embodiments of the subject matter herein include a Cas protein. In some aspects, the Cas protein is a Cas9 protein or a variant thereof. Exemplary Cas proteins, including non-limiting examples of Cas9 and Cas9 variants, are described herein.

[0031] In various aspects, the concentration of Cas9 protein can be in the range of about 0.1 to about 25 μg. For example, the concentration of Cas9 can be about 1 μg, about 5 μg, about 10 μg, about 15 μg, or about 20 μg. Alternatively, the concentration of Cas9 can be in the range of about 10 ng / μL to about 300 ng / μL; for example, about 10 ng / μL to about 200 ng / μL; or about 10 ng / μL to about 100 ng / μL, or about 10 ng / μL to about 50 ng / μL.

[0032] In certain embodiments, the gene editing composition comprises: (a) a first sgRNA molecule and a second sgRNA molecule, wherein the nucleic acid sequence of the first sgRNA molecule is different from the nucleic acid sequence of the second sgRNA molecule; (b) a first RNP comprising a first sgRNA and a second RNP comprising a second sgRNA, wherein the nucleic acid sequence of the first sgRNA molecule is different from the nucleic acid sequence of the second sgRNA molecule; or (c) a first crRNA molecule and a second crRNA molecule, wherein the nucleic acid sequence of the first crRNA molecule is different from the nucleic acid sequence of the second crRNA molecule. (d) a first crRNA molecule and a second crRNA molecule, wherein the nucleic acid sequence of the first crRNA molecule is different from the nucleic acid sequence of the second crRNA molecule, and further comprises a tracrRNA molecule; or (e) a first RNP comprising a first crRNA and a tracrRNA and a second RNP comprising a second crRNA and a tracrRNA, wherein the nucleic acid sequence of the first crRNA molecule is different from the nucleic acid sequence of the second crRNA molecule.

[0033] In aspects, the ratio of Cas9 protein to guide RNA can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0034] In embodiments, increasing the number of times a cell undergoes a delivery process (or increasing the number of administrations) can increase the editing percentage, and in some embodiments the number of administrations can include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 administrations.

[0035] In various embodiments, the first and second sgRNA or first and second crRNA molecules together comprise a nucleic acid sequence complementary to a target sequence adjacent to a gene, exon, intron, extrachromosomal sequence, or genomic nucleic acid sequence, where the gene, exon, intron, extrachromosomal sequence, or genomic nucleic acid sequence is about, or at least about, 1, 2, 3, 4, 5, 6, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1-100 kilobases in length. In some embodiments, the use of an RNP pair comprising the first and second sgRNA or first and second crRNA molecules can be used to make a polynucleotide molecule comprising a gene, exon, intron, extrachromosomal sequence, or genomic nucleic acid sequence.

[0036] In certain embodiments, the target sequence of the sgRNA or crRNA is about 12 to about 25, or about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 17 to 23, or 18 to 22 nucleotides in length. In some embodiments, the target sequence is 20 nucleotides in length or about 20 nucleotides in length.

[0037] In various embodiments, the first and second sgRNA or the first and second crRNA molecules are complementary to sequences flanking the extrachromosomal sequence in the expression vector.

[0038] Aspects of the present subject matter relate to the delivery of multiple components of a gene editing complex that are not complexed together. In some embodiments, the gene editing composition comprises at least one gene editing protein and at least one nucleic acid, and the gene editing protein and the nucleic acid are not bound or complexed to each other.

[0039] The subject matter allows for high gene editing efficiency while maintaining high cell viability. In some embodiments, at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99%, 1-99% or more of the cell population or its progeny cells become genetically modified after contacting with the aqueous solution. In various embodiments, at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99%, 1-99% or more of the cell population or its progeny cells are viable after contacting with the aqueous solution.

[0040] In certain embodiments, the gene editing composition induces single-stranded or double-stranded breaks in DNA in the cell. In some embodiments, the gene editing composition further comprises a repair template polynucleotide. In various embodiments, the repair template comprises: (a) a first flanking region comprising nucleotides of a sequence complementary to about 40 to about 90 base pairs on one side of the single-stranded or double-stranded break and a second flanking region comprising nucleotides of a sequence complementary to about 40 to about 90 base pairs on the other side of the single-stranded or double-stranded break; or (b) a first flanking region comprising nucleotides of a sequence complementary to at least about 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 90 base pairs on one side of the single-stranded or double-stranded break and a second flanking region comprising nucleotides of a sequence complementary to at least about 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 90 base pairs on the other side of the single-stranded or double-stranded break. A non-limiting description of gene editing using the CRISPR-Cas system (including repair templates) is provided in Ran et al. (2013) Nat Protoc. 2013 Nov; 8(11): 2281-2308, the entire contents of which are incorporated herein by reference. Embodiments that include repair templates are not limited to those that include the CRISPR-Cas system.

[0041] In various embodiments of the present subject matter, the volume of the aqueous solution is delivered to the cell population in the form of a spray. In some aspects, the volume is 6.0×10 -7Microliters / cell ~7.4 x 10 -4 In certain embodiments, the spray contains colloidal particles or subparticles with diameters ranging from 10 nm to 100 μm. In various embodiments, the volume is 2.6×10 -9 Microliters / square micrometer exposed surface area ~1.1 x 10 -6 is in microliters per square micrometer exposed surface area.

[0042] In some embodiments, the RNPs have a size of about 100 Å×100 Å×50 Å or 10 nm×10 nm×5 nm. In various embodiments, the size of the spray particles is adjusted to accommodate at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more RNPs per spray particle.

[0043] For example, contacting the cell population with a volume of aqueous solution can be performed with a gas that propels the aqueous solution to form a spray. In certain embodiments, the cell population is contacted with the aqueous solution for 0.01 to 10 minutes (e.g., 0.1 to 10 minutes) before adding a second volume of buffer or culture medium to submerge or suspend the cell population.

[0044] In some embodiments, the buffer or culture medium comprises phosphate buffered saline (PBS). In various embodiments, the aqueous solution comprises an ethanol concentration of about 5 to about 50%. In non-limiting examples, the aqueous solution comprises one or more of: (a) about 75 to about 98% HO; (b) about 2 to about 50% ethanol; (c) about 6 to about 91 mM sucrose; (d) about 2 to about 35 mM potassium chloride; (e) about 2 to about 35 mM ammonium acetate; (f) about 1 to about 14 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES); (g) about 0.1 to about 10 mM magnesium salt or about 0.5 to about 10 mM magnesium chloride; and / or (h) about 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 mM magnesium salt or magnesium chloride. As shown in FIG. 8, a delivery solution containing 2.5 mM magnesium chloride demonstrated a surprising increase in delivery efficiency compared to a delivery solution without magnesium chloride.

[0045] In various embodiments, the cell population comprises at least one of primary cells or immortalized cells. For example, the cell population may comprise mesenchymal stem cells, lung cells, neural cells, fibroblasts, human umbilical vein (HUVEC) cells, and human embryonic kidney (HEK) cells, primary or immortalized hematopoietic stem cells (HSCs), T cells, natural killer (NK) cells, cytokine-induced killer (CIK) cells, human umbilical cord blood CD34+ cells, B cells. Non-limiting examples of T cells may include CD8+ T cells or CD4+ T cells. In some aspects, a CD8+ subpopulation of CD3+ T cells is used. CD8+ T cells can be purified from a PBMC population by positive isolation using anti-CD8 beads. In some aspects, primary NK cells are isolated from PBMCs, and GFP mRNA can be delivered by platform delivery techniques (i.e., 3% expression and 96% viability in 24 hours). In additional aspects, NK cell lines, such as NK92, can be used.

[0046] Cell types also include cells that have been previously modified to enhance therapeutic efficacy, such as T cells, NK cells, and MSCs. For example, T cells or NK cells expressing chimeric antigen receptors (CAR T cells, CAR NK cells, respectively); T cells expressing modified T cell receptors (TCRs); MSCs that have been virally or non-virally modified to overexpress therapeutic proteins that complement the intrinsic properties of MSCs (e.g., delivery of Epo using lentiviral vectors or BMP-2 using AAV-6) (reviewed in Park et al, Methods, 2015 Aug;84-16); MSCs primed with non-peptide drugs or magnetic nanoparticles for enhanced efficacy and externally controlled targeting, respectively (Park et al., 2015); enzymatic modification (e.g., fucosyltransferase), chemical conjugation (e.g., SLe to MSCs using N-hydroxy-succinimide (NHS) chemistry), and the like. X and MSCs functionalized with targeting moieties to enhance homing of MSCs to the treatment site using either covalent or non-covalent interactions (e.g., engineering the cell surface with palmitated proteins that act as hydrophobic anchors for subsequent conjugation of antibodies) (Park et al., 2015). For example, T cells modified to express a chimeric antigen receptor, e.g., primary T cells or T cell lines (CAR T cells), can be further treated according to the present invention with a complex containing a gene editing protein and / or a guide nucleic acid specific to the sequence encoding the CAR to edit the gene encoding the CAR, thereby reducing or stopping the expression of the CAR in the modified T cells.

[0047] Aspects of the present subject matter also provide a composition for delivering a payload across a cell plasma membrane, the composition comprising a payload and an aqueous solution comprising an alcohol at a concentration of at least 2%. In some embodiments, the composition further comprises a magnesium salt. In certain embodiments, the composition comprises about 0.1 to about 10 mM magnesium salt or about 0.1 to about 10 mM magnesium chloride. The magnesium salt comprises magnesium ions.

[0048] Aspects of the present subject matter further provide a composition for delivering a payload across the plasma membrane of a cell, the composition comprising an aqueous solution comprising a payload, alcohol at a concentration of at least 2%, less than about 46 mM salt lacking magnesium ions, less than about 121 mM sugar, less than 19 mM buffering agent, and about 0.1 to about 10 mM magnesium salt.

[0049] In certain embodiments, the buffer is a weak acid or a weak base effective to adjust or maintain the pH of the aqueous solution at about pH 6.5, 6.75, 7, 7.25, 7.5, 7.75, 8, 8.25, or 8.5. In various embodiments, the alcohol is methanol, ethanol, isopropyl alcohol, butanol, or benzyl alcohol. In some embodiments, the salt lacking magnesium ions includes Na ions or K ions. In some embodiments, the salt is NaCl, KCl, Na2HPO4, C2H3O2NH4, or KH2PO4. In certain embodiments, the sugar includes sucrose. Buffers include, for example, 4-2-(hydroxyethyl)-1-piperazineethanesulfonic acid, 3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]-2-hydroxypropane-1-sulfonic acid, 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid, 3-(N-morpholino)propanesulfonic acid, or piperazine-N,N'-bis(2-ethanesulfonic acid).

[0050] An aspect of the present invention provides an apparatus for delivering a gene editing composition through the plasma membrane of a cell. In various embodiments, the apparatus includes an air pressure generator that generates pressurized gas; a sprayer that is operably connected to the air pressure generator; a reservoir that is configured to contain a volume of an aqueous solution that includes a gene editing composition and an alcohol concentration of more than 2 percent; and a valve between the air pressure generator and the sprayer. In some embodiments, the valve is switchable between a closed position that prevents the pressurized gas from actuating the sprayer and an open position that allows the pressurized gas to actuate the sprayer to generate colloidal droplets from the aqueous solution. In various embodiments, the valve has a switching speed of less than about 250 milliseconds. In certain embodiments, the sprayer is directed to contact the cell population with the aqueous solution, the volume being a function of (i) the exposed surface area of ​​the cell population or (ii) the number of cells in the cell population.

[0051] Additional devices, systems, methods, compositions, and articles described or exemplified herein are also provided.

[0052] In an aspect, delivering a payload across the plasma membrane of a cell includes providing a cell population and contacting the cell population with a volume of an aqueous solution, the aqueous solution comprising the payload and an alcohol content greater than 5 percent. The volume of the aqueous solution can be a function of the exposed surface area of ​​the cell population or a function of the number of cells in the cell population.

[0053] In another aspect, a composition for delivering a payload across the plasma membrane of a cell comprises an aqueous solution comprising a payload, an alcohol concentration greater than 5 percent, less than 46 mM salt, less than 121 mM sugar, and less than 19 mM buffer, e.g., the concentration of alcohol, e.g., ethanol, does not exceed 50%.

[0054] One or more of the following features can be included in any operable combination: The volume of solution delivered to the cells is a plurality of units, e.g., a spray, e.g., a plurality of droplets of aqueous particles. The volume is stated relative to an individual cell, or relative to the exposed surface area of ​​a confluent or substantially confluent (e.g., at least 75%, at least 80%, e.g., 85%, 90%, 95%, 97%, 98%, 100% confluent) cell population. For example, the volume is 6.0×10 -7 Microliters / cell ~7.4 x 10 -4 The volume can be 4.9 x 10 microliters / cell. -6 Microliters / cell ~2.2 x 10 -3 The volume is 9.3 x 10 -6 Microliters / cell ~2.8 x 10 -5 The volume can be approximately 1.9×10 microliters per cell. -5 The volume can be about 6.0 x 10 microliters / cell, within about 10 percent. -7 Microliters / cell ~2.2 x 10 -3 The volume is 2.6 x 10 -9 Microliters / square micrometer exposed surface area ~1.1 x 10 -6 The volume can be 5.3 x 10 microliters per square micrometer of exposed surface area. -8 Microliters / square micrometer exposed surface area ~1.6×10 -7 The volume can be approximately 1.1×10 microliters per square micrometer of exposed surface area. -7 It can be about microliters per square micrometer of exposed surface area. It can be about within 10 percent.

[0055] Cell confluency refers to the cells that are in contact with each other on the surface. For example, confluency can be expressed as an estimated (or counted) percentage, for example, 10% confluency means that 10% of the surface, for example of a tissue culture vessel, is covered with cells, and 100% means that the surface is completely covered. For example, adherent cells grow two-dimensionally on the surface of tissue culture wells, plates or flasks. Non-adherent cells can be removed by centrifugation, pulling from the top of the cell mass by vacuum or aspiration of tissue culture medium, or by aspiration or vacuum removal from the bottom of the vessel.

[0056] The contacting of the cell population with a volume of aqueous solution can be accomplished by a gas that propels the aqueous solution to form a spray. The gas can include nitrogen, air, or an inert gas. The spray can include discrete volume units ranging in size from 10 nm to 100 μm, for example, 30 to 100 μm in diameter. The spray can include discrete volume units having a diameter of about 30 to 50 μm. For example, a total volume of 20 μl of aqueous solution can be sprayed to a cell-occupied area of ​​about 1.9 cm. 2 For example, one well of a 24-well culture plate (see also above regarding volume and well size / area). A total volume of 10 μl of aqueous solution can be delivered to a cell-occupied area of ​​approximately 0.95 cm. 2, for example, into one well of a 48-well culture plate. Typically, the aqueous solution contains the payload to be delivered across the cell membrane into the cells, and the second volume is a buffer or culture medium without the payload. Alternatively, the second volume (buffer or medium) can also contain the payload. In some embodiments, the aqueous solution contains the payload and the alcohol, and the second volume does not contain the alcohol (and optionally does not contain the payload). The cell population can be contacted with the aqueous solution for 0.1 to 10 minutes before adding the second volume of buffer or culture medium to submerge or suspend the cell population. The buffer or culture medium can be phosphate buffered saline (PBS). The cell population can be contacted with the aqueous solution for 2 seconds to 5 minutes before adding the second volume of buffer or culture medium to submerge or suspend the cell population. The cell population can be contacted with the aqueous solution, for example, containing the payload, for 30 seconds to 2 minutes before adding the second volume of buffer or culture medium without the payload to submerge or suspend the cell population. The cell population is allowed to contact the spray for approximately 1-2 minutes before a second volume of buffer or culture medium is added to submerge or suspend the cell population. During the period between the spray of the cells and the addition of the buffer or culture medium, the cells remain hydrated by the layer of water from the spray volume.

[0057] The aqueous solution may include an ethanol concentration of 5-30%. The aqueous solution may include one or more of 75-98% HO, 2-45% ethanol, 6-91 mM sucrose, 2-35 mM KCl, 2-35 mM ammonium acetate, and 1 to 14 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES).

[0058] The cell population can include adherent or non-adherent cells. The adherent cells can include at least one of primary mesenchymal stem cells, fibroblasts, monocytes, macrophages, lung cells, neural cells, fibroblasts, human umbilical vein (HUVEC) cells, Chinese hamster ovary (CHO) cells, and immortalized cells such as human embryonic kidney (HEK) cells or cell lines. The cell population can include non-adherent cells. The non-adherent cells can include at least one of primary hematopoietic stem cells (HSC), T cells, natural killer (NK) cells, cytokine-induced killer (CIK) cells, human umbilical cord blood CD34+ cells, B cells, or cell lines such as Jurkat T cell lines.

[0059] The cell population can be substantially confluent, such as greater than 75 percent confluent. Cell confluency refers to the cells that are in contact with each other on the surface. For example, confluency can be expressed as an estimated (or counted) percentage, such as 10% confluency meaning that 10% of the surface, for example of a tissue culture vessel, is covered with cells, and 100% means that the surface is completely covered. For example, adherent cells grow two-dimensionally on the surface of a tissue culture well, plate, or flask. Non-adherent cells can be removed by centrifugation, pulling from the top of the cell population by vacuum or aspiration of tissue culture medium, or by aspiration or vacuum removal from the bottom of the vessel. The cell population can form a monolayer of cells.

[0060] In some embodiments, the subject compositions or solutions include alcohol; salts; sugars; buffers; and / or ammonium acetate. The alcohols can be selected from, for example, methanol, ethanol, isopropyl alcohol, butanol, and benzyl alcohol. The salts can be selected from, for example, NaCl, KCl, Na2HPO4, KH2PO4, and C2H3O2NH. The sugars can include, for example, sucrose. The buffers can include, for example, 4-2-(hydroxyethyl)-1-piperazineethanesulfonic acid, 3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]-2-hydroxypropane-1-sulfonic acid, 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid, 3-(N-morpholino)propanesulfonic acid, or piperazine-N,N'-bis(2-ethanesulfonic acid). In some examples, the solution or buffer contains magnesium ions, for example, the magnesium ions are optionally provided in the form of a salt, such as MgCl.

[0061] The present subject matter relates to methods for delivering gene editing compounds and complexes across a plasma membrane. The subject methods include the steps of introducing the compound or complex into an aqueous composition to form a matrix, atomizing the matrix into a spray, and contacting the matrix with a plasma membrane.

[0062] Exemplary methods described herein include a payload, which includes an alcohol. The term "alcohol" refers to a polyatomic organic compound that includes a hydroxyl (-OH) functional group attached to at least one carbon atom. The alcohol can be a monohydric alcohol and can include at least one carbon atom, such as methanol. The alcohol can include at least two carbon atoms (e.g., ethanol). In other aspects, the alcohol can include at least three carbons (e.g., isopropyl alcohol). The alcohol can include at least four carbon atoms (e.g., butanol), or at least seven carbon atoms (e.g., benzyl alcohol). Exemplary payloads can include up to 50% (v / v) alcohol, more preferably, the payload includes 2-45% (v / v) alcohol, 5-40% alcohol, and 10-40% alcohol. The payload can include 20-30% (v / v) alcohol.

[0063] Most preferably, the payload comprises 25% (v / v) alcohol. Alternatively, the payload can comprise 2-8% (v / v) alcohol, or 2% alcohol. The alcohol may comprise ethanol, and the payload may comprise 5, 10, 20, 25, 30, 40, or 50% (v / v) ethanol. An exemplary method may comprise methanol as the alcohol, and the payload may comprise 5, 10, 20, 25, 30, 40, or 50% (v / v) methanol. The payload may comprise 2-45% (v / v) methanol, 20-30% (v / v), or 25% (v / v) methanol. Preferably, the payload comprises 20-30% (v / v) methanol. Still alternatively, the alcohol is butanol, and the payload comprises 2, 4, or 8% (v / v) butanol.

[0064] In some aspects of the present subject matter, the payload is in a hypotonic solution or buffer. The payload solution may have an osmolality of 171 mOsm / L. According to an exemplary method, the payload solution has an osmolality of 171 mOsm / L at room temperature.

[0065] According to the present subject matter, the payload may include at least one salt. The salt may be selected from NaCl, KCl, Na2HPO4, C2H302NH4, and KH2PO4. According to an exemplary method, the payload includes each of NaCl, KCl, Na2HPO4, and KH2PO4. The payload may include less than 46 mM salt. Further, the payload includes 2-35 mM salt, or 10-15 mM salt (e.g., 12 mM salt). According to an exemplary method, the salt is KCl, and the payload includes 2.4, 4.8, 7.2, 9.6, 12, 24, 28.8, or 33.6 mM KCl, more preferably 12 mM KCl.

[0066] According to exemplary methods of the present subject matter, the payload may include a sugar (e.g., sucrose, or a disaccharide). According to exemplary methods, the payload includes less than 121 mM sugar, 6-91 mM, or 26-39 mM sugar. Still further, the payload includes 32 mM sugar (e.g., sucrose). Optionally, the sugar is sucrose and the payload includes 6.4, 12.8, 19.2, 25.6, 32, 64, 76.8, or 89.6 mM sucrose.

[0067] According to an exemplary method of the present subject matter, the payload may include a buffering agent (e.g., a weak acid or a weak base). The buffering agent may include a zwitterion. According to an exemplary method, the buffering agent is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. The payload may include less than 19 mM of buffering agent (e.g., 1-15 mm, or 4-6 mM or 5 mM buffering agent). According to an exemplary method, the buffering agent is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and the payload includes 1, 2, 3, 4, 5, 10, 12, 14 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. More preferably, the payload includes 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.

[0068] According to an exemplary method of the present subject matter, the payload comprises ammonium acetate. The payload may comprise less than 46 mM ammonium acetate (e.g., 2-35 mM, 10-15 mM, or 12 mM ammonium acetate). The payload may comprise 2.4, 4.8, 7.2, 9.6, 12, 24, 28.8, or 33.6 mM ammonium acetate.

[0069] The methods described herein include a second aspect of the subject matter, in which a second payload (e.g., an aqueous solution) is provided that includes 68 mM NaCl, 1.4 mM KCl, 5 mM Na2HPO4, and 0.9 mM KH2PO4. The pH of the second payload can be pH 7.4.

[0070] In various aspects, the volume of aqueous solution that is injected with a gas may include, for example, compressed air (e.g., atmospheric air). These and other embodiments may include inert gases, such as helium, neon, and argon.

[0071] In certain aspects of the present subject matter, the cell population may include adherent cells (eg, lung cells, kidney cells, immune cells such as macrophages) or non-adherent cells (eg, suspension cells).

[0072] In certain aspects of the present subject matter, the cell population may be substantially confluent, including substantially greater than 75 percent confluency. In preferred embodiments, the cell population may form a single monolayer.

[0073] Additional non-limiting descriptions of methods, systems, instruments and devices for delivering compounds across cell plasma membranes are described in PCT International Patent Application PCT / US2015 / 057247, filed October 23, 2015, the entire contents of which are incorporated herein by reference.

[0074] The subject matter further relates to devices, systems, techniques and articles for delivering payloads through plasma membranes.The subject matter also relates to devices for delivering payloads such as proteins or protein complexes through plasma membranes.The subject matter may find utility in the field of intracellular delivery, for example, applied to the delivery of gene editing compositions to target sites such as cells, tissues or organs.

[0075] In some embodiments, the device for delivering a payload through the plasma membrane can include a sprayer having at least one spray emitter and a support directed to the sprayer. The method can further include atomizing the payload before contacting the plasma membrane with the payload. In various aspects, the sprayer can be selected from, for example, a mechanical sprayer, an ultrasonic sprayer, an electrospray, a nebulizer, and a venturi tube. The sprayer can be a commercially available sprayer. The sprayer can be an intranasal mucosal spray device. The sprayer can be an intranasal mucosal spray device commercially available from LMA Teleflex (NC, USA). The sprayer can be an intranasal mucosal spray device commercially available from LMA Teleflex (NC, USA) under catalog number MAD300.

[0076] In certain embodiments, the nebulizer can be adapted to provide a colloidal suspension of particles having a diameter of 30-100 μm prior to contacting the plasma membrane with the payload. In various embodiments, the nebulizer can be adapted to provide a colloidal suspension of particles having a diameter of 30-80 μm. For example, the nebulizer can be adapted to provide a colloidal suspension of particles having a diameter of 50-80 μm.

[0077] In some aspects, the sprayer includes a gas reservoir. For example, the sprayer can include a gas reservoir in which a gas is maintained under pressure. In certain aspects, the gas is selected from air, carbon dioxide, and helium. In various aspects, the gas reservoir includes a fixed pressure head generator. In some aspects, the gas reservoir can be in fluid communication with the spray emitter. In various embodiments of the present subject matter, the gas reservoir includes a gas guide that can be in fluid communication with the spray emitter. In some aspects, the gas guide is adapted to pass gas therethrough. The gas guide can include, for example, a hollow body. For example, the gas guide can be a hollow body having an open end. In some aspects, the gas guide includes a hollow body having a first and a second open end. In various aspects, the gas guide is a hollow body having a first and a second opposing open end. In certain aspects, the diameter of the first open end is different from the diameter of the second open end. In various aspects, the diameter of the first open end can be smaller or larger than the diameter of the second open end. In various embodiments, the first open end is in fluid communication with a gas reservoir or the second open end is in fluid communication with a spray emitter.

[0078] In certain embodiments, the device includes a sample reservoir. In a non-limiting example, the sample reservoir is in fluid communication with the sprayer. In various embodiments, the sample reservoir is in fluid communication with the spray emitter. For example, the gas reservoir and the sample reservoir can both be in fluid communication with the spray emitter.

[0079] In some aspects, the instrument includes a sample valve located between the sample reservoir and the gas reservoir. In these and other aspects, the instrument can include a sample valve located between the sample reservoir and the gas guide. In certain aspects, the sample valve can be adapted to regulate the flow of the sample from the sample reservoir. In some examples, the sample valve can be adapted to allow for a continuous or semi-continuous flow of the sample. In various embodiments, the sample valve can be adapted to allow for a semi-continuous flow of a predetermined volume of the sample. For example, the sample valve can be adapted to allow for a semi-continuous flow of 0.5-100 μL or 10 μL of sample. In one non-limiting example, the sample valve has an area of ​​0.065-0.085 cm 2 The tube is adapted to allow semi-continuous flow of 1 μL of sample into the well.

[0080] In various aspects, the sprayer and the support are spaced apart. The support can include a solid support. In some aspects, the support can include a plate including sample wells. For example, the support can include a plate including sample wells selected from 1, 6, 9, 12, 24, 48, 384, and 1536 wells. In certain aspects, the solid support is formed of an inert material. For example, the solid support can be formed of a plastic material, or a metal or metal alloy, or a combination thereof. In some aspects, the support includes a heating element and / or a resistive element. In certain aspects, the support can be reciprocally attached to the instrument. In various embodiments, the support can be reciprocally movable relative to the instrument. For example, the support can be reciprocally movable relative to the sprayer. In various aspects, the support can be reciprocally movable relative to the spray dispenser. In some aspects, the support includes a support actuator to reciprocate the support relative to the sprayer. In various aspects, the support includes a support actuator to reciprocate the support relative to the spray dispenser. In a non-limiting example, the support can include a support actuator for reciprocating the support relative to a longitudinal axis of the spray emitter, hi some aspects, the support can include a support actuator for reciprocating the support laterally relative to a longitudinal axis of the spray emitter.

[0081] In some embodiments, the longitudinal axis of the spray area is coaxial with the longitudinal axis or center point of the support and / or the circular well of the support to which the payload is delivered. In certain embodiments, the longitudinal axis of the spray emitter is coaxial with the longitudinal axis or center point of the support and / or the circular well of the support. In various embodiments, the longitudinal axis of the spray emitter, the longitudinal axis of the support, and the longitudinal axis of the spray area are each coaxial. In certain embodiments, the longitudinal length of the spray area is greater than (may be greater than twice) the diameter of the circular base of the spray area (e.g., the cell area to which the payload is delivered).

[0082] In various embodiments, the device includes a valve located between the gas reservoir and the nebulizer. In some embodiments, the valve is an electromagnetically actuated valve. In certain embodiments, the valve is a solenoid valve or a pneumatic valve. In some embodiments, the valve is located in the gas guide. For example, the valve can be adapted to regulate the gas flow in the gas guide. In various embodiments, the valve is adapted to allow continuous or semi-continuous gas flow. In a non-limiting example, the valve is adapted to allow semi-continuous gas flow at a predetermined time interval, such as a time interval of 1 second. In certain embodiments, the device includes at least one filter. In some embodiments, the filter includes a pore size of less than 10 μm or a pore size of 10 μm. In various embodiments, the filter is located in the gas guide. In some embodiments, the filter is in liquid communication with the gas guide.

[0083] In certain aspects, the instrument includes at least one regulator. The regulator can be, for example, an electrical regulator or a mechanical regulator. In some aspects, the regulator is located in the gas guide. In some aspects, the regulator is in liquid communication with the gas guide. In various aspects, the regulator is an adjustment valve. In some aspects, the pressure in the gas guide is about 1.0-2.0 bar or about 1.5 bar. In a non-limiting example, the pressure in the gas guide is 1.0-2.0 bar and the distance between the sprayer and the support is 31 mm or less. For example, the pressure in the gas guide can be 1.5 bar and the distance between the sprayer and the support can be 31 mm. In various embodiments, the pressure in the gas guide is about 0.05 bar per millimeter of distance between the sprayer and the support. In some aspects, the adjustment valve can be adapted to adjust the pressure in the gas guide to about 1.0-2.0 bar or about 1.5 bar. In certain embodiments, each regulating valve may be adapted to maintain a pressure in the gas guide of about 1.0-2.0 bar or about 1.5 bar.

[0084] In some aspects, the instrument includes two regulators. For example, the instrument can include a first and a second regulator. In various embodiments, the first and second regulators can be located in the gas guide. In some embodiments, the first and second regulators can be in fluid communication with the gas guide. In certain aspects, the first regulator is located between the gas reservoir and the filter. In various aspects, the first regulator can be adapted to adjust the pressure from the gas reservoir in the gas guide to about 2.0 bar or to maintain the pressure in the gas guide at about 2.0 bar. In some aspects, the second regulator can be located between the filter and the valve.

[0085] In various aspects, the spray emitter can be adapted to provide a cone-shaped spray area (e.g., a generally cone-shaped spray area). For example, the spray emitter can be adapted to provide a 30° cone-shaped spray area. In some aspects, the instrument can further include a microprocessor to control any or all parts of the instrument. In various aspects, the microprocessor can be arranged to control any or all of the sample valve, the support actuator, the valve, and the regulator. In some embodiments, the instrument can include a sprayer having at least one spray emitter and a support directed to the sprayer; the sprayer can be selected from a mechanical sprayer, an ultrasonic sprayer, an electrospray, a nebulizer, and a Venturi tube. In certain aspects, the sprayer can be adapted to provide a colloidal suspension of particles having a diameter of 30-100 μm. In a non-limiting example, the instrument can include a sample reservoir and a gas guide, and a sample valve located between the sample reservoir and the gas guide. In some aspects, the sample valve can be adapted to allow a semi-continuous sample flow of 10-100 μL. In various embodiments, the sprayer and the support can be spaced apart to define a generally cone-shaped spray area therebetween; the distance between the sprayer and the support can be about twice the diameter of the circular base of the cell area to which the molecules are delivered; the distance between the sprayer and the support can be 31 mm, and the diameter of the circular base of the cell area to which the molecules are delivered can be 15.5 mm. In certain aspects, the instrument can include a gas guide, and the pressure in the gas guide is 1.0-2.0 bar. In some aspects, the instrument can include at least one filter having a pore size of less than 10 μm.

[0086] Non-limiting examples of variations include: one or more sprayers held on a stationary frame above the moving plate positioner; one or more sprayers held on an adjustable x-y-z-r axis (where r is a rotational range of motion from 0 to 360 degrees); one or more sprayers held on a stationary support above the moving plate positioner; one or more sprayers held on an adjustable x-y axis; one or more sprayers held on a stationary frame above the moving plate positioner; and one or more sprayers held on an adjustable x-axis.

[0087] In some embodiments, one or more sprayers are positioned at the distal end of a catheter or wand for the purpose of addressing a tissue or tissue portion ex vivo or in vivo. For example, aspects of the present subject matter relate to delivery of gene editing payloads to exposed tissue, such as the dermis or ocular tissue of an animal, or to internal tissue, such as tissue that contains a lumen, such as the lung tissue (airway), gastrointestinal tissue (esophagus, intestine), or cardiovascular tissue (artery or vein) of a mammal, such as a human or non-human mammal.

[0088] In various embodiments, one or more sprayers are positioned at the distal end of a catheter or wand for the purpose of addressing a portion or subset of a well plate surface (e.g., a portion of a plate or a particular well of a multi-well plate). In certain embodiments, one or more sprayers are positioned on a stationary support positioned above a continuously moving substrate. The substrate may be compatible with cell culture or may include a plate or well in which cells are cultured.

[0089] In some embodiments, the subject devices or instruments include a visible low power laser and lens arrangement that matches one or more sprayers to indicate where the spray is being or will be delivered. In various embodiments, the laser and lens can be adapted to adopt the same dispersion pattern as the sprayer. In some embodiments, the dispersion pattern is a pyramidal dispersion pattern.

[0090] Various embodiments of the present subject matter include liquid dispensers and pipettes capable of generating microliter, nanoliter or picoliter droplets and positioning the droplets individually onto cells under microscopic control, visualization, manipulation and / or tuning of droplet / particle size. For example, droplets / aqueous particles (e.g., containing one or more RNPs) with volumes in the nanoliter or picoliter range are administered to single cells (alone or as members of a monolayer or mat of contiguous cells) under microscopic guidance. Using this approach, the plasma membrane is not punctured with a needle as in microinjection. Rather, the droplets / particles are dripped or applied onto the cell and enter the cytoplasm without physical manipulation of the plasma membrane as in microinjection. In this way, the droplets / particles can be tailored to a size (e.g., diameter 500 nanometers to 100 microns) or volume (e.g., 1.0×10) for optimal uptake by the cell as a function of the size (diameter) or exposed surface area of ​​the cell. -7 ~10 microliters; 1.0×10 -6 ~10 microliters; 1.0×10 -5 ~1 microliter; 1.0 x 10 -4 ~1.0 microliters; 1.0 x 10 -6 ~1.0×10 -3 Microliter: 6.0 x 10 -7 ~7.4×10 -4 Microliter, 4.9 x 10 -6 ~2.2×10 -3 Microliter, 9.3 x 10 -6 ~2.8×10 -5 Microliter, approximately 1.9 x 10 -5 microliter, or 6.0 x 10 -7 Microliters ~ 2.2 x 10 -3Microliters) are adapted. In some examples, the droplets / particles are about 10 to about 100,000 nanometers in diameter. For example, the droplets / particles can be about 10, 50, 100, 200, 300, 400, 500, 1000, 10,000, or 100,000 nanometers in diameter. In some embodiments, a device or instrument that can be used for microinjection is instead used to deliver the droplets / particles to the cell surface without microinjection puncture of the plasma membrane. In such embodiments, the needle does not puncture the cell, but instead is used to bring the droplets / particles close to the cell and / or to position the droplets / particles on the cell. In certain embodiments involving needles (either with or without a microinjection device or instrument), the size of the needle and / or the pressure at which the solution is forced through the needle can be adjusted to modify the droplet / particle size. In various embodiments, one or more droplets / particles can be delivered to a plurality of cells, such as cultured cells or cells of a living organism. For example, the droplets / particles can be delivered to mucosal cells or ocular cells, or fertilized eggs or zygotes. In one non-limiting example, the droplets are delivered to one or more cells of a mouse, frog, or fish (e.g., zebrafish) zygote. In such an example, the zygote can be briefly removed from the aqueous solution, receive the droplets / particles, and then return to the aqueous solution.

[0091] Aspects of the invention relate to expression vector-free delivery of gene editing compounds and complexes to cells and tissues, such as delivery of Cas-gRNA ribonucleoproteins for genome editing in primary human T cells and hematopoietic stem cells (HSCs).

[0092] Various aspects of CRISPR-Cas system are known in the art.Non-limiting aspects of this system are described in, for example, United States Patent No. 9,023,649 issued on May 5, 2015; United States Patent No. 9,074,199 issued on July 7, 2015; United States Patent No. 8,697,359 issued on April 15, 2014; United States Patent No. 8,932,814 issued on January 13, 2015; PCT International Publication No. 2015 / 071474 published on August 27, 2015; Cho et al., (2013) Nature Biotechnology Vol 31 No 3 pp 230-232 (including supplementary information); and Jinek et al., (2012) Science Vol 337 No 6096 pp 816-821, the entire contents of each of which are incorporated herein by reference.

[0093] Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof. These enzymes are known, for example, the amino acid sequence of the S. pyogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW2 (SEQ ID NO:1) and the NCBI database under accession number Q99ZW2.1. The UniProt database under accession numbers A0A0G4DEU5 and CDJ55032 provide another example of the amino acid sequence of the Cas9 protein (SEQ ID NO:18). Another non-limiting example is the Streptococcus thermophilus Cas9 protein, whose amino acid sequence can be found in the UniProt database under accession number Q03JI6.1 (SEQ ID NO:19). In some embodiments, the unmodified CRISPR enzyme has DNA cleavage activity, like Cas9. In certain embodiments, the CRISPR enzyme is Cas9, and can be Cas9 from Streptococcus pyogenes or S. pneumoniae. In various embodiments, the CRISPR enzyme directs the cleavage of one or both strands at the location of the target sequence, such as within the target sequence and / or within the complement of the target sequence. In some embodiments, the CRISPR enzyme directs the cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500 or more base pairs from the first or last nucleotide of the target sequence.In some embodiments, the vector encodes a CRISPR enzyme that is mutated with respect to the corresponding wild-type enzyme, so that the mutant CRISPR enzyme lacks the ability to cleave one or both strands of the target polynucleotide that contains the target sequence.For example, the aspartic acid to alanine substitution (D10A, where amino acid numbering is shown in SEQ ID NO:1) in the RuvC I catalytic domain of Cas9 from Streptococcus pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cuts a single strand).Other examples of mutations that make Cas9 a nickase include, but are not limited to, H840A, N854A, and N863A (where amino acid numbering is shown in SEQ ID NO:1).In an aspect of the present invention, the nickase can be used for genome editing via homologous recombination.

[0094] In certain embodiments, Cas9 nickases can be used in combination with guide sequences, e.g., two guide sequences that target the sense and antisense strands of a DNA target, respectively, allowing both strands to be nicked and used to induce NHEJ.

[0095] As a further example, two or more catalytic domains of Cas9 (RuvC I, RuvC II, and RuvC III) can be mutated to produce a mutant Cas9 that substantially lacks all DNA cleavage activity. The D10A mutation can be combined with one or more of the H840A, N854A, or N863A mutations to produce a Cas9 enzyme that substantially lacks all DNA cleavage activity. In certain embodiments, a CRISPR enzyme is considered to substantially lack all DNA cleavage activity when the DNA cleavage activity of the mutant enzyme is less than about 25%, 10%, 5%, 1%, 0.1%, 0.01% or less than its non-mutated counterpart. Other mutations may be useful, and when Cas9 or other CRISPR enzymes are from species other than S. pyogenes, mutations may be added to the corresponding amino acids to achieve a similar effect.

[0096] In certain embodiments, the protein to be delivered (e.g., Cas protein or its heterologous) may include an intracellular localization signal. For example, the Cas protein in RNP may include an intracellular localization signal. Depending on the context, for example, a fusion protein comprising Cas9 and a nuclear localization signal may be referred to herein as "Cas9" without specifying the inclusion of the nuclear localization signal. In some embodiments, the payload (e.g., RNP) comprises a fusion protein comprising a localization signal. For example, the fusion protein may comprise a nuclear localization signal, a nucleolus localization signal, or a mitochondrial targeting signal. Such signals are known in the art and non-limiting examples are described in Kalderon et al., (1984) Cell 39 (3 Pt 2): 499-509; Makkerh et al., (1996) Curr Biol. 6 (8):1025-7; Dingwall et al., (1991) Trends in Biochemical Sciences 16 (12): 478-81; Scott et al., (2011) BMC Bioinformatics 12:317 (7 pages); Omura T (1998) J Biochem. 123(6):1010-6; Rapaport D (2003) EMBO Rep. 4(10):948-52; and Brocard & Hartig (2006) Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1763(12):1565-1573, the contents of each of which are hereby incorporated by reference herein. In various embodiments, a Cas protein may contain more than one localization signal, such as two, three, four, five, or more nuclear localization signals. In some embodiments, the localization signal is at the N-terminus of the Cas protein, and in other embodiments, the localization signal is at the C-terminus of the Cas protein.

[0097] Non-limiting examples of nuclear localization signals include: Non-limiting examples of mitochondrial localization signals include: Contains TIFF2025010591000004.tif4128.

[0098] In some embodiments, the enzyme coding sequence encoding the CRISPR enzyme is codon-optimized for expression in a particular cell, such as a eukaryotic cell.The eukaryotic cell can be the cell of a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human primate, or the cell derived therefrom.Generally, codon optimization refers to modifying a nucleic acid sequence for enhanced expression in a host cell by replacing at least one codon (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons, or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) of a natural sequence with the codon that is more frequently or most frequently used in the gene of the host cell of interest, while maintaining the natural amino acid sequence.Various species show a certain bias for a certain codon of a certain amino acid. Codon bias (differences in codon usage among organisms) often correlates with the efficiency of messenger RNA (mRNA) translation, which in turn is thought to depend, among other things, on the properties of the codons being translated and on the availability of specific transfer RNA (tRNA) molecules. The dominance of selected tRNAs in a cell is generally a reflection of the codons most frequently used for peptide synthesis.

[0099] Thus, genes can be adapted for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, from the "Codon Usage Database," and these tables can be adapted in several ways. See Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000). Computer algorithms are also available, such as Gene Forge (Aptagen; Jacobus, Pa.), to codon-optimize a particular sequence for expression in a particular host cell. In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons, or all codons) in the sequence encoding the CRISPR enzyme correspond to the most frequently used codon for a particular amino acid.

[0100] Generally, guide sequence is any polynucleotide sequence that has sufficient complementarity with target polynucleotide sequence to hybridize with target sequence and direct sequence-specific binding of CRISPR complex to target sequence.In some embodiments, the degree of complementarity between guide sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more, or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more, when optimally aligned using a suitable alignment algorithm.In some embodiments, the degree of complementarity is 100%. Optimal alignment can be determined using any algorithm suitable for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transformation (e.g., Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available from soap.genomics.org.cn), and Maq (available from maq.sourceforge.net). In some embodiments, the guide sequence is about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 or more nucleotides in length, or about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 or more nucleotides in length. The length of the guide sequence is more than 5, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 or more nucleotides. In certain embodiments, the length of the guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 or less nucleotides. The ability of the guide sequence to direct the sequence-specific binding of the CRISPR complex to the target sequence can be evaluated by any suitable assay.For example, the components of the CRISPR system sufficient to form a CRISPR complex comprising a test guide sequence can be provided to a host cell having a corresponding target sequence, such as by transfection with a vector encoding the components of the CRISPR sequence, and then the preferential cleavage within the target sequence can be evaluated, such as in the Surveyor assay described herein.Similarly, the cleavage of the target polynucleotide sequence can be examined in vitro by providing the target sequence, the components of the CRISPR complex comprising the test guide sequence, and a control guide sequence that is different from the test guide sequence, and comparing the binding or cleavage rate at the target sequence between the reaction of the test guide sequence and the reaction of the control guide sequence.

[0101] Guide sequences can be selected to target any target sequence. In some embodiments, the target sequence is a sequence in the genome of a cell. Exemplary target sequences include unique sequences in the target genome. For example, for Streptococcus pyogenes Cas9, a unique target sequence in the genome is of the form: It may contain the Cas9 target site of TIFF2025010591000005.tif4128, During the ceremony, TIFF2025010591000006.tif3128 (N is A, G, T, or C; X can be anything) occurs once in the genome. A unique target sequence in the genome is of the form: may contain the Streptococcus pyogenes Cas9 target site in TIFF2025010591000007.tif3128, During the ceremony, TIFF2025010591000008.tif3128 (N is A, G, T, or C; X can be anything) occurs once in the genome. For Streptococcus thermophilus CRISPR1 Cas9, a unique target sequence in the genome is of the form: It may contain the Cas9 target site of TIFF2025010591000009.tif4128, During the ceremony, TIFF2025010591000010.tif3128 (N is A, G, T, or C; X can be anything; W is A or T) occurs once in the genome. A unique target sequence in the genome is of the form: TIFF2025010591000011.tif3128 may contain the S. thermophilus CRISPR1 Cas9 target site, During the ceremony, TIFF2025010591000012.tif3128 (N is A, G, T, or C; X can be anything; W is A or T) occurs once in the genome. For S. pyogenes Cas9, the unique target sequence in the genome is of the form: It may contain the Cas9 target site of TIFF2025010591000013.tif4128, During the ceremony, TIFF2025010591000014.tif3128 (N is A, G, T, or C; X can be anything) occurs once in the genome. A unique target sequence in the genome is of the form: TIFF2025010591000015.tif3128 may contain the Streptococcus pyogenes Cas9 target site, During the ceremony, TIFF2025010591000016.tif3128 (N is A, G, T, or C; X can be anything) occurs once in the genome. In each of these sequences, "M" can be A, G, T, or C, and does not need to be considered when identifying a sequence as unique. In some embodiments, the guide sequence is selected to reduce the degree of secondary structure within the guide sequence. The secondary structure can be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimum Gibbs free energy. An example of one such algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another exemplary folding algorithm is the online web server RNAfold developed at the Institute for Theoretical Chemistry, University of Vienna, using a centroid structure prediction algorithm (see, e.g., AR Gruber et al., 2008, Cell 106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12): 1151-62).

[0102] CRISPR-Cas techniques, which facilitate genome engineering in a wide range of cell types, are rapidly evolving. It has been recently shown that delivery of Cas9-gRNA editing tools in the form of ribonucleoproteins (RNPs) offers several advantages compared to delivery of plasmids encoding Cas9 and gRNAs. Advantages include faster and more efficient editing, fewer off-target effects, and lower toxicity. RNPs have been delivered by lipofection and electroporation, but unresolved limitations of these delivery methods include toxicity and low efficiency, especially for certain clinically relevant cell types. Thus, there is a need to provide a vector-free approach, e.g., a viral vector-free approach, for delivering biologically relevant payloads, e.g., RNPs, across the plasma membrane into cells. "Cargo" or "payload" is a term used to describe a compound or composition that is delivered by aqueous solution across the cell plasma membrane into the cell.

[0103] The subject matter relates to a delivery technique that facilitates the delivery of a wide range of payloads to cells with low toxicity. Genome editing can be achieved by using some aspects of the subject matter to deliver RNP to cells. Cas9 and two Alexa488-labeled gRNAs can be delivered separately to cells, such as cells of A549 human lung cell line. Cas9 can be detected by Western blotting in cells one hour after delivery. After that, the level decreases until Cas9 is no longer detectable. The delivery technique itself does not adversely affect the viability or functionality of Jurkat cells and primary T cells. The subject matter allows gene editing with Cas9 RNP in clinically relevant cell types with minimal toxicity.

[0104] Transient and direct delivery of CRISPR / Cas components such as Cas and / or gRNA has advantages compared to expression vector-mediated delivery. For example, the amount of Cas, gRNA, or RNP can be added at a limited time with more precise timing compared to using expression vectors. Components expressed from vectors can be produced in various amounts and at various times, making it difficult to achieve consistent gene editing without off-target editing. Additionally, preformed complexes (RNPs) of Cas and gRNA cannot be delivered using expression vectors.

[0105] In exemplary embodiments, the aqueous solution may further comprise an additive. Additional non-limiting additives, including suboptimal additives such as glycerol, include glycerol (e.g., at a concentration of 0.01-25% v / v in aqueous solution), NDSB (e.g., at a concentration of about 0.01M to about 1M in aqueous solution), sorbitol such as D-sorbitol (e.g., at a concentration of 0.01-10% w / v in aqueous solution), mannitol such as D-mannitol (e.g., at a concentration of 0.01-10% w / v in aqueous solution), α-crystallin such as α-crystallin purified from bovine ocular lens (e.g., at a concentration of 1-500 μM in aqueous solution; or about 1-400 μM, or about 1-300 μM, or about 1-200 μM, or about 1-100 μM in aqueous solution), and / or α-crystallin purified from bovine ocular lens (e.g., at a concentration of 1-500 μM in aqueous solution; or about 1-400 μM, or about 1-300 μM, or about 1-200 μM, or about 1-100 μM in aqueous solution). The aqueous solution may contain 0.00 μM, or about 1 to 50 μM, or about 1 to 20 μM, or about 1 to 10 μM), gelatin such as gelatin A or gelatin B (e.g., at a concentration of 0.01 to 10% w / v in an aqueous solution), glycine (e.g., at a concentration of about 0.01 M to about 1 M in an aqueous solution), proline (e.g., at a concentration of about 1 mM to about 200 mM in an aqueous solution), Tween-20 (e.g., at a concentration of about 0.01% to about 5% v / v in an aqueous solution), L-histidine (e.g., at a concentration of about 1 mM to about 200 mM in an aqueous solution), myo-inositol (e.g., at a concentration of 0.01 to 10% w / v in an aqueous solution), and trehalose (e.g., at a concentration of about 1 mM to about 200 mM in an aqueous solution).

[0106] In a preferred embodiment, the additive can be α-crystallin. Examples of α-crystallins and associated accession numbers for the amino acid sequences of α-crystallins and human α-crystallins include CAA32891.1, ACP18852.1, AAB22011.1, AAB22010.1, AMM63587.1, EAX09498.1, EAW67163.1, AAI13599.1, AAP35416.1, AMM63591.1, and AAB22009.1.

[0107] In some examples, the amino acid sequence of alpha-crystallin includes the accession number CAA32891.1 (EMBLL X14789 mRNA. Translation: CAA32891.1), which shows the amino acid sequence (SEQ ID NO:23). TIFF2025010591000017.tif11128

[0108] Exemplary regions or fragments include, but are not limited to, amino acids 1-50 and 60-104.

[0109] In some examples, the amino acid sequence of alpha-crystallin includes the accession number ACP18852.1 (EMBL FJ876064 mRNA. Translation: ACP18852.1.), which provides the amino acid sequence (SEQ ID NO:24). TIFF2025010591000018.tif14128

[0110] Exemplary regions or fragments include, but are not limited to, amino acids 1-51, 14-161, or 67-50.

[0111] In some embodiments, commercially available alpha-crystallins can be of two isoforms, cry-aA or cry-aB. In some embodiments, the ratio of cry-aA to cry-aB can be 1:1, 1:2, 1:3, 1:4, 1:5, or 2:1, 3:1, 4:1, or 5:1. In lenses, alpha-crystallins are polydisperse molecules consisting of alpha A and alpha B subunits in a 3:1 ratio. In some examples, alpha-crystallins can be purified from bovine lenses, e.g., bovine alpha-crystallin (Sigma C4163).

[0112] The amino acid sequence of the alpha-crystallin A chain CRYAA (bovine) (SEQ ID NO:25) is shown. TIFF2025010591000019.tif14128

[0113] The amino acid sequence of alpha-crystallin B chain CRYAB (bovine) (SEQ ID NO:26) is shown. TIFF2025010591000020.tif14128

[0114] In some embodiments, human alpha A or B crystallin can be purchased from any suitable supplier, including Cell Sciences. In some examples, alpha crystallin A (product number CRC167) can have the amino acid sequence (SEQ ID NO:27). TIFF2025010591000021.tif14130

[0115] In another example, alpha crystallin B (product number CRC168) can have the amino acid sequence (SEQ ID NO:28). TIFF2025010591000022.tif14130

[0116] In some embodiments, human alpha A or B crystallin may be purchased from any suitable supplier, including Abcam, where alpha A (ab48778) has the amino acid sequence (SEQ ID NO:29). TIFF2025010591000023.tif14130

[0117] In another aspect, alpha crystallin B (ab48779) from Abeam has the amino acid sequence (SEQ ID NO:30). TIFF2025010591000024.tif14130

[0118] In some embodiments, the MgCl2 can be at a concentration of less than about 2.5, 2, 0.5, 0.1, or 0.01 MgCl2 and / or less than about 5, 4, 3, 2, 1, 0.5, 0.1, 0.01% w / v of glycerol.

[0119] Further advantages of the compositions and methods described herein include minimizing or reducing the precipitation of gene editing protein cargo, such as Cas9. Bacterial protein Cas9 has been observed to precipitate out of solution under certain conditions, and this precipitation can reduce the function or activity of Cas9. To reduce, minimize, or eliminate the precipitation of Cas9, the delivery buffer composition optionally includes an excipient or additive that prevents the protein from precipitation. Such protectants include heat shock proteins, such as α-crystallin. Other heat shock protein protectants include Hsp60, Hsp72, and Hsp73 (Whitley et al., 1999, J. of Vascular Surgery 29(4):748-751).

[0120] Other excipients or additives include sugar alcohols, such as sorbitol. The addition of α-crystallin and / or sorbitol improved the nuclease activity of Cas-9:gRNA after exposure to alcohol, such as ethanol-based delivery solutions, thereby overcoming the problems and shortcomings of Cas9 precipitation / activity reduction in certain buffers.

[0121] Thus, the invention includes compositions, such as buffered solutions, for delivering gene editing proteins or gene editing complexes to cells, which contain at least 2% (but less than 70%) alcohol, such as ethanol, and optionally a heat shock protein such as alpha-crystallin and / or a sugar alcohol such as sorbitol. Methods for editing a target gene in a cell include contacting the cells (in which gene editing is desired) with a spray of a mist of droplets comprising the gene editing protein or complex, incubating the cells, e.g., for a period of about 0-10 minutes, 0.1-10 minutes, 1-5 minutes, e.g., about 2 minutes, to destabilize the plasma membrane of the cells and thereby allow entry of the cargo into the cell through the plasma membrane, contacting the cells with a stop solution, e.g., for a period of 0-5 minutes, 0.1-5 minutes, 0.2-4 minutes, 0.3-3 minutes, 0.4-2 minutes, 0.5-1 minutes, e.g., 30 seconds, to reverse the cell membrane permeabilization / destabilization process, and incubating the cells in cell culture medium, e.g., standard cell culture medium, during which gene editing occurs, e.g., the cells may remain in the solution for the duration of the culture period. As mentioned above, a notable advantage of the described method is that this sequence of steps can be optionally repeated several times (2, 3, 4, 5, 6, 8, 10 or more times) to increase the amount of cargo delivered to the cells and / or the efficiency of gene editing. Optionally, the recovery period between administrations (e.g., subsequent delivery sprays) ranges from 1 to 8 hours, such as 2 to 6 hours, e.g., about 4 hours, between cargo delivery events and spray delivery events.

[0122] The method and composition described herein are advantageous over other delivery methods because the time from cell processing to viable gene-edited cells suitable for clinical use is shorter.The delivery rate of cargo using the ethanol spray delivery system described herein is comparable to electroporation, but cell recovery is faster than electroporation.For T cells, proliferation is not delayed by the method of the present invention, but is delayed for 2 days by electroporation.For MSCs, cell differentiation using the technique of the present invention is equivalent to untreated control, but is delayed by electroporation.In terms of viability and functionality, cells are ready for use 4 hours after processing using the spray technique described herein.

[0123] Therefore, the speed of processing cells for subsequent use, e.g., delivery to human or animal subjects for clinical treatment, is advantageous over procedures that require very long periods of time for the processed cells to regain normal health / function. For example, delivery by electroporation impairs the function of some cells, such as MSCs and T cells. This drawback of electroporation is likely due to the large electrical gradient (especially in small cells) and local heating that causes DNA damage and endogenous repair mechanisms (Meaking et al., 1995, Biochimica et Biophysics Acta 1264:357-362 and Branzei et al., 2008, Nature Reviews Molecular Cell Biology 9:297-308). For example, the functional integrity of some cells becomes impaired, e.g., differentiation is reduced and activity is reduced after electroporation, e.g., functional integrity is reduced for up to several weeks after electroporation. In contrast, the viability and functionality of cells treated using the methods and compositions is minimally or not impaired, and cells are ready for use within hours (1, 2, 6, 12, 18, 24 hours after treatment) or days (1, 2, 3, 4, 5, 6, 7 days after treatment). Thus, the overall main advantage of the gene editing techniques described herein is the avoidance of membrane destruction by electric gradients. Moreover, the parameters of electroporation can vary widely depending on the cell type and experimental conditions. In contrast, the methods and systems of the present invention work well for a wide range of cell types, the methods can be completed in minutes, and are performed in cell-friendly conditions. The viability and functionality of cells treated using the spray delivery methods and compositions described herein is minimally or not impaired, and cells are ready for use within hours (1, 2, 6, 12, 18, 24 hours after treatment) or days (1, 2, 3, 4, 5, 6, 7 days after treatment).

[0124] Each embodiment disclosed herein is considered applicable to each of the other disclosed embodiments. In some aspects, the aqueous solution does not include MgCl2 and / or glycerol. Thus, all combinations of the various elements described herein are within the scope of the present invention.

[0125] [The present invention 1001] 1. A method for delivering a gene editing composition across the plasma membrane of a cell, comprising: Providing a cell population; and contacting the cell population with a volume of an aqueous solution comprising the gene editing composition and an alcohol at a concentration of at least about 2%, The volume is a function of (i) the exposed surface area of ​​the cell population or (ii) the number of cells in the cell population. The method. [The present invention 1002] The aqueous solution is (a) About 5% to about 50%; (b) at least about 5% and less than about 50, 40, 30, 20, 19, 18, 17, 16, or 15%; or (c) at least about 2.5, 3, 3.5, 4, 4.5, or 5% The method of claim 1001, further comprising: [The present invention 1003] 1. The gene editing composition, (a) Gene editing protein; (b) an RNA molecule; and / or (c) Ribonucleoproteins (RNPs) The method of the present invention 1001, comprising one or more of the following: [The present invention 1004] The method of claim 1003, wherein the gene editing composition comprises a gene editing protein, and the gene editing protein is a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a MegaTAL, a Cas protein, a Cre recombinase, a Hin recombinase, or an Flp recombinase. [The present invention 1005] The method of claim 1003, wherein the gene editing composition comprises an RNA molecule, and the RNA molecule comprises an sgRNA, a crRNA, and / or a tracrRNA. [The present invention 1006] The method of claim 1003, wherein the gene editing composition comprises an RNP, the RNP comprising a Cas protein, and a gRNA or crRNA and a tracrRNA. [The present invention 1007] The gene editing composition is administered to the cell population or its progeny cells, (a) about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, 48, 60, 72, 0.5 to 2, 0.5 to 6, 6 to 12, or 0.5 to 72 hours after contacting the cell population with the aqueous solution, or (b) less than about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, 48, 60, 72, 0.5 to 2, 0.5 to 6, 6 to 12, or 0.5 to 72 hours after contacting the cell population with the aqueous solution; The method of the present invention 1001, which is detectable. [The present invention 1008] The method of claim 1004, wherein the genome of the cells in the cell population or progeny cells comprises at least one site-specific recombination site for Cre recombinase, Hin recombinase, or FLP recombinase. [The present invention 1009] The method of any one of claims 10 to 15, wherein the cells or progeny cells in the cell population comprise sgRNA, crRNA, and / or tracrRNA. [The present invention 1010] The method of any one of claims 10 to 15, wherein the cell population or its progeny expresses sgRNA, crRNA, and / or tracrRNA. [The present invention 1011] The method of claim 1005, wherein the cells in the cell population or their progeny express a Cas protein. [The present invention 1012] The method of claim 1004, wherein the Cas protein is a Cas9 protein or a variant thereof. [The present invention 1013] 1. The gene editing composition, (a) a first sgRNA molecule and a second sgRNA molecule, wherein the nucleic acid sequence of the first sgRNA molecule differs from the nucleic acid sequence of the second sgRNA molecule; (b) a first RNP comprising a first sgRNA and a second RNP comprising a second sgRNA, wherein the nucleic acid sequence of the first sgRNA molecule differs from the nucleic acid sequence of the second sgRNA molecule; (c) a first crRNA molecule and a second crRNA molecule, wherein the nucleic acid sequence of the first crRNA molecule differs from the nucleic acid sequence of the second crRNA molecule; (d) a first crRNA molecule and a second crRNA molecule, wherein the nucleic acid sequence of the first crRNA molecule differs from the nucleic acid sequence of the second crRNA molecule, further comprising a tracrRNA molecule; or (e) a first RNP comprising a first crRNA and a tracrRNA and a second RNP comprising a second crRNA and a tracrRNA, wherein the nucleic acid sequence of the first crRNA molecule differs from the nucleic acid sequence of the second crRNA molecule; The method of the present invention 1003, comprising: [The present invention 1014] The method of the invention 1013, wherein the first and second sgRNA or the first and second crRNA molecules together comprise a nucleic acid sequence complementary to a target sequence adjacent to a gene, exon, intron, extrachromosomal sequence, or genomic nucleic acid sequence, and the gene, exon, intron, extrachromosomal sequence, or genomic nucleic acid sequence is about 1, 2, 3, 4, 5, 6, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1-100 kilobases in length, or is at least about 1, 2, 3, 4, 5, 6, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1-100 kilobases in length. [The present invention 1015] The method of any one of claims 10 to 14, wherein the first and second sgRNA or the first or second crRNA molecule are complementary to sequences adjacent to an extrachromosomal sequence present in an expression vector. [The present invention 1016] The method of claim 1001, wherein the gene editing composition comprises at least one gene editing protein and at least one nucleic acid, and the gene editing protein and the nucleic acid are not bound or complexed to each other. [The present invention 1017] 1001. The method of claim 1001, wherein at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99%, 1-99%, or more of the cell population or its progeny cells become genetically modified after contact with said aqueous solution. [The present invention 1018] 1001. The method of claim 1001, wherein at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99%, 1-99%, or more of the cell population or its progeny cells are viable after contact with said aqueous solution. [The present invention 1019] The method of claim 1001, wherein the gene editing composition induces single-stranded or double-stranded breaks in DNA in the cell. [The present invention 1020] The method of claim 1001, wherein the gene editing composition further comprises a repair template polynucleotide. [The present invention 1021] The repair template is (a) a first flanking region that includes nucleotides whose sequence is complementary to about 40 to about 90 base pairs on one side of the single-stranded or double-stranded break, and a second flanking region that includes nucleotides whose sequence is complementary to about 40 to about 90 base pairs on the other side of the single-stranded or double-stranded break; or (b) a first flanking region comprising nucleotides of a sequence complementary to at least about 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 90 base pairs on one side of the single-stranded or double-stranded break, and a second flanking region comprising nucleotides of a sequence complementary to at least about 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 90 base pairs on the other side of the single-stranded or double-stranded break. The method of the present invention 1020, comprising: [The present invention 1022] The method of claim 10, wherein said volume of aqueous solution is delivered to the cell population in the form of a spray of aqueous particles. [The present invention 1023] (a) The volume of the aqueous particle spray is 6.0 × 10 -7 Microliters / cell ~7.4 x 10 -4 microliters per cell; and / or (b) the aqueous particles have discrete volume units in the size range of 10 nm to 100 μm, 30 to 100 μm, 50 to 80 μm, or 30 to 50 μm in diameter; The method of the present invention 1022. [The present invention 1024] The method of claim 1021, wherein the spray comprises colloidal particles or subparticles having diameters between 10 nm and 100 μm. [The present invention 1025] The volume is 2.6×10 -9 Microliters / square micrometer exposed surface area ~1.1 x 10 -6 The method of the present invention 1001, wherein the surface area is exposed to a temperature of 100° C. or less. [The present invention 1026] The method of claim 1001, wherein the step of contacting the cell population with a volume of an aqueous solution is performed by a gas that propels the aqueous solution to form a spray. [The present invention 1027] The method of claim 10, wherein the cell population is contacted with said aqueous solution for 0.1 to 10 minutes and then a second volume of buffer or culture medium is added to submerge or suspend the cell population. [The present invention 1028] The method of claim 1022, wherein the buffer or culture medium comprises phosphate buffered saline (PBS). [The present invention 1029] 1001. The method of claim 1001, wherein said aqueous solution contains an ethanol concentration of about 2 to about 20%. [The present invention 1030] The aqueous solution is (a) Approximately 75 to 98% H2O; (b) about 2–20% ethanol; (c) about 6 to about 91 mM sucrose; (d) about 2 to about 35 mM potassium chloride; (e) about 2 to about 35 mM ammonium acetate; (f) about 1 to about 14 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); and / or (g) about 0.1 to about 10 mM magnesium salt; and / or (h) about 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 mM magnesium salt The method of the present invention 1001, comprising one or more of the following: [The present invention 1031] The method of claim 10, wherein the aqueous solution contains about 0.1 to about 10 mM magnesium chloride. [The present invention 1032] The method of claim 1030, wherein said aqueous solution comprises at least about 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 mM magnesium chloride. [The present invention 1033] The method of claim 1001, wherein the cell population comprises primary cells or immortalized cells. [The present invention 1034] The method of the present invention 1033, wherein the cell population comprises at least one of primary or immortalized mesenchymal stem cells, lung cells, neural cells, fibroblasts, human umbilical vein (HUVEC) cells, and human embryonic kidney (HEK) cells, primary or immortalized hematopoietic stem cells (HSC), T cells, natural killer (NK) cells, cytokine-induced killer (CIK) cells, human umbilical cord blood CD34+ cells, B cells, A549 cells, Beas-2B cells, Jurkat cells, or peripheral blood mononuclear cells (PBMCs). [The present invention 1035] 1. A composition for delivering a payload across the plasma membrane of a cell, the composition comprising an aqueous solution comprising a payload and an alcohol at a concentration of at least 2%, the payload comprising a gene editing composition. [The present invention 1036] The composition of the present invention 1035 further comprising a magnesium salt. [The present invention 1037] The composition of the present invention 1035, comprising about 0.1 to about 10 mM of a magnesium salt. [The present invention 1038] 1037. A composition of the invention comprising at least about 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 mM magnesium chloride. [The present invention 1039] The composition of the present invention 1035, wherein the alcohol comprises methanol, ethanol, isopropyl alcohol, butanol, or benzyl alcohol. [The present invention 1040] The composition of the present invention 1039 comprising about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50% ethanol. [The present invention 1041] The composition of the present invention 1035 having a pH of about 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. [The present invention 1042] 1. A composition for delivering a payload across the plasma membrane of a cell, the composition comprising an aqueous solution comprising the payload, an alcohol at a concentration of at least 2%, less than about 46 mM salt lacking magnesium ions, less than about 121 mM sugar, less than 19 mM buffering agent, and about 0.1 to about 10 mM magnesium salt, wherein the payload comprises a gene editing composition. [The present invention 1043] The composition of claim 1042, wherein the buffer is a weak acid or a weak base effective to adjust or maintain the pH of said aqueous solution at about pH 6.5, 6.75, 7, 7.25, 7.5, 7.75, 8, 8.25, or 8.5. [The present invention 1044] The composition of claim 1042, wherein the alcohol comprises methanol, ethanol, isopropyl alcohol, butanol, or benzyl alcohol. [The present invention 1045] The composition of claim 1042, wherein the salt lacking magnesium ions contains Na ions or K ions. [The present invention 1046] The composition of claim 1042, wherein the salt lacking magnesium ions comprises NaCl, KCl, Na2HPO4, C2H3O2NH4, or KH2PO4. [The present invention 1047] The composition of claim 1042, wherein the sugar comprises sucrose. [The present invention 1048] The composition of the invention 1042, wherein the buffer comprises 4-2-(hydroxyethyl)-1-piperazineethanesulfonic acid, 3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]-2-hydroxypropane-1-sulfonic acid, 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid, 3-(N-morpholino)propanesulfonic acid, or piperazine-N,N'-bis(2-ethanesulfonic acid). [The present invention 1049] A composition of the present invention 1042 comprising about 32 mM sucrose, about 12 mM potassium chloride, about 12 mM ammonium acetate, about 5 mM HEPES, about 15% ethanol, and about 2.5 mM MgCl2. [The present invention 1050] A composition of the present invention 1049 comprising 32 mM sucrose, 12 mM potassium chloride, 12 mM ammonium acetate, 5 mM HEPES, 2.5 mM MgCl2, 15% ethanol, and 85% H2O. [The present invention 1051] The composition of claim 10, wherein the pH of the solution is adjusted to about pH 7.4 before adding ethanol. [The present invention 1052] 1. An apparatus for delivering a gene editing composition across the plasma membrane of a cell, comprising: An air pressure generating device that produces pressurized gas; a sprayer operably connected to said air pressure generating device; a reservoir configured to contain a volume of an aqueous solution comprising the gene editing composition and a concentration of alcohol greater than 2 percent; a valve between the air pressure generator and the nebulizer switchable between a closed position to prevent actuation of the nebulizer by pressurized gas and an open position to allow actuation of the nebulizer by pressurized gas to generate colloidal droplets from the aqueous solution, the valve having a switching speed of less than 250 milliseconds. Including, the sprayer is oriented to contact the cell population with the aqueous solution, the volume being a function of (i) the exposed surface area of ​​the cell population or (ii) the number of cells in the cell population; The said instrument. [The present invention 1053] The method of claim 10, wherein the aqueous solution further comprises an additive comprising a heat shock protein or a sugar alcohol. [The present invention 1054] The method of any one of claims 1 to 3, wherein the heat shock protein comprises alpha-crystallin. [The present invention 1055] The method of claim 1054, wherein the α-crystallin is present at a concentration of about 1 to about 500 μM. [The present invention 1056] The method of claim 1053, wherein the sugar alcohol comprises sorbitol at a concentration of about 1 to about 10% weight / volume. [The present invention 1057] 10. The method of claim 10, wherein the aqueous solution comprises an alcohol concentration of less than about 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5%. [The present invention 1058] The method of any one of claims 10 to 12, wherein the cells comprise stem cells. [The present invention 1059] The method of any one of claims 10 to 58, wherein the stem cells comprise mesenchymal stem cells (MSCs). [The present invention 1060] The method of claim 1059, wherein the MSCs comprise bone marrow-derived MSCs. [The present invention 1061] The method of claim 1001, wherein the aqueous solution does not contain glycerol or MgCl2. Details of one or more variations of the subject matter described herein are set forth in the accompanying drawings and the following description. Other features and advantages of the subject matter described herein will be apparent from the description and drawings, and from the claims. All references cited herein, including the contents of the accession number citations for protein or nucleic acid sequences, are incorporated by reference. [Brief description of the drawings]

[0126] [Figure 1] 1 is a diagram showing a scheme for editing a target gene in A549 cells. Guide RNA is delivered in the form of sgRNA. Dual RNA is chemically synthesized. sgRNA1 is in vitro transcribed. Guide RNA is incubated with Cas9. Cas9:gRNA complex is delivered into A549 cells using a delivery platform. After cleavage and non-homologous end joining (NHEJ) with Cas9, DNA is recovered from the cells and edits are detected using droplet digital PCR (ddPCR) and drop-off probe assays. [Diagram 2] Electrophoresis gel images demonstrating the functionality of Cas9 protein in an in vitro editing assay. The functionality of recombinant Cas9 purchased from Toolgen was confirmed in an in vitro editing assay. For a positive control, DNA, gRNA and Cas9 were supplied in an Agilent kit and these reagents were used to generate the expected DNA fragments 1 and 2. Cas9 sourced from Toolgen (ToolGen, Inc., Seoul, South Korea) also successfully generated DNA fragments 1 and 2 when using control DNA and gRNA. [Diagram 3]Electrophoretic gel images demonstrating the functionality of Cas9 protein in delivery solution in an in vitro editing assay. The Agilent in vitro assay was performed in either the buffer provided in the Agilent kit or in ethanol-free delivery solution (S buffer). The expected DNA fragments 1 and 2 were produced when Agilent buffer was used but not S buffer. [Figure 4] Electrophoretic gel images demonstrating the function of Cas9 protein in various ratios of delivery solution to kit buffer in an in vitro editing assay. Using the Agilent in vitro assay, various ratios of "ethanol-free delivery solution (S buffer)":"Agilent kit buffer" were tested. Ratios are shown in Table 2. The expected DNA fragments 1 and 2 were produced in the presence of all ratio combinations except when Agilent kit buffer was absent (lane 6). [Diagram 5] This is an electrophoretic gel image showing the effect of MgCl2 concentration in delivery solution on the function of Cas9 RNP in in vitro editing assay.The presence of MgCl2 in delivery solution allows Cas9 to function in delivery solution in the absence of ethanol.Increasing MgCl2 concentration has neither adverse nor positive effect on activity. [Figure 6] 1 is a graphic depiction of the methods described herein, illustrating the delivery mechanism. [Figure 7] 1 is a bar graph illustrating the percent expression of GFP mRNA in NK cells using the methods and delivery platforms described herein. [Figure 8] 1 is a set of images validating the effectiveness of a delivery solution containing 15% EtOH and 2.5 mM MgCl2. When the delivery solution was modified to contain 15% ethanol and 2.5 mM MgCl2, successful delivery of the reporter protein, ovalbumin-FITC (OVA-FITC), to A549 cells was observed. Delivery appeared to be enhanced in the presence of 2.5 mM MgCl2. [Figure 9]A set of Western blots showing that delivery of Cas9 protein results in rapid and transient protein expression compared to lipofected plasmid DNA. Cas9 protein was delivered into A549 cells and detected by Western blotting of cell lysates. When using the delivery platform, Cas9 was detected 1 hour after delivery and levels decreased thereafter. GAPDH was used as a loading control. In contrast, when a plasmid encoding Cas9 was delivered into A549 cells by lipofection, expression was indiscernible over a period of 6 hours. [Figure 10] Figures 10A and B are Western blots showing the dose delivery response of Cas9 protein. When increasing doses of Cas9 protein were delivered, increasing levels of Cas9 were detected in (Figure 10A) A549 cells and (Figure 10B) Jurkat cells. [Figure 11] Figures 11A-E are images showing the intracellular localization of Cas9 protein after delivery in A549 cells. (Figure 11A) Cas9-Cy3 protein (red) was delivered to cells and analyzed by fluorescence microscopy 1 hour after delivery. DAPI nuclear counterstain (blue) indicates nuclei. Addition of MgCl2 to the delivery solution (+MgCl2) led to increased delivery efficiency of Cas9. (Figure 11B) Cas9 protein was delivered and cells were fixed with methanol 1 hour after delivery. Expression of Cas9 was visualized by immunofluorescence using anti-Cas9 antibody (green). DAPI nuclear counterstain (blue) indicates nuclei. (Figure 11C) Dual RNA1-FAM (green) was delivered to cells and cells were analyzed by fluorescence microscopy 1 hour after delivery. DAPI nuclear counterstain (blue) indicates nuclei. Addition of MgCl2 to the delivery solution (+MgCl2) resulted in increased delivery efficiency of Dual-RNA1-FAM. (Figure 11D) RNP containing Cas9:Dual-RNA1-FAM (green) was delivered to cells, and cells were analyzed by fluorescence microscopy 1 hour after delivery. DAPI nuclear counterstain (blue) indicates nuclei. (Figure 11E) RNP containing Cas9:Dual-RNA1-FAM was delivered, and cells were fixed with methanol 1 hour after delivery. RNP expression was visualized by immunofluorescence using anti-Cas9 antibody (red). DAPI nuclear counterstain (blue) indicates nuclei. [Figure 12A] 12A-C are a set of graphs showing the effect of the delivery platform technique on the viability of Jurkat cells (FIG. 12A), human peripheral blood mononuclear cells (PBMCs) (FIG. 12B), and HSCs (FIG. 12C). Delivery solution without payload (SBO) was delivered to Jurkat cells, human PBMCs, or HSCs. Annexin V and propidium iodide assays demonstrated minimal reduction in viability in each cell type. UT=untreated. [Figure 12B] See legend to Figure 12A. [Figure 12C] See legend to Figure 12A. [Figure 13A] 13 is a set of graphs comparing viability and efficiency using the Neon electroporator and delivery platform. (FIG. 13A) Comparing viability of Jurkat cells after delivery using the delivery platform vs. Neon. (FIG. 13B) Efficiency of delivery of BSA-FITC to Jurkat cells using the delivery platform vs. the Neon electroporator. [Figure 13B] See legend to Figure 13A. [Figure 14A] Figures 14A and B are a set of graphs showing the effect of delivery platform technology on Jurkat cell activation. Delivery solution without payload (SBO) was delivered to Jurkat cells. Control cells were untreated (UT). Expression of CD69 (Figure 14A) and CD25 (Figure 14B) was analyzed 4 or 24 hours after stimulation with PHA, respectively. [Figure 14B] See legend to Figure 14A. [Figure 15] 1 is the amino acid sequence of an exemplary Cas9 protein (SEQ ID NO:4) that includes, from N-terminus to C-terminus, a Streptococcus pyogenes protein (SEQ ID NO:1), followed by a nuclear localization signal (SEQ ID NO:2), followed by a human influenza hemagglutinin (HA) epitope tag (SEQ ID NO:3). The nuclear localization signal and the HA epitope tag are underlined. [Figure 16]1 is a set of Western blots showing delivery of Cas9 protein into PBMCs and Beas-2B cells and detected by Western blotting of cell lysates. [Figure 17] Electrophoretic agarose gel image showing the effect of delivery solution on the function of gene editing tools. Lane 1 is Cas9 600ng / μl in RNase-free water (positive control); Lane 2 is Cas9 600ng / μl in sprayed RNase-free water; Lane 3 is Cas9 600ng / μl in delivery solution; Lane 4 is Cas9 600ng / μl in sprayed delivery solution; Lane 5 is complexed Cas9 / gRNA in delivery solution; Lane 6 is complexed Cas9 / gRNA in sprayed delivery solution; Lane 7 is complexed Cas9 / gRNA in delivery solution to which EtOH is added immediately; Lane 8 is Cas9 in delivery solution containing 4 times more S buffer; Lane 9 is labeled Alexa 488 Cas9 in delivery solution; Lane 10 is negative control (Cas9 in delivery solution, no gRNA added); In the negative control, DNA is not cleaved. Editing was observed in all samples except sample 7, where EtOH was added together with all other components (without a preceding 10 min incubation). For all other samples, a band of fragmented DNA appeared near the uncut DNA. The results showed that neither the delivery solution nor the spray interfered with the cleavage activity of Cas9 and RNP. The agarose gel was a 1.0% gel containing cybersafe dye and run at 100V for 60 min with gel loading dye. [Figure 18] 1 is an image showing purification of Cas9, where the larger tagged Cas9 separated from the unbound tag and eluted from the column first. [Figure 19] Figure 1 shows the elution of labeled Cas9 after separation on a Sephadex column. Four aliquots were viewed on a transilluminator. The intensity of the second eluent (from the left) shows the most concentrated, and the free dye (far right) was used as a reference. [Figure 20]Images depicting uptake of Alexa 488-labeled Cas9 delivered into A549 cells by the delivery platform technology (green dots). Cell nuclei (blue) were stained with DAPI to facilitate cell visualization. [Figure 21] Image showing 500 ng / μl unlabeled Cas9 in 2 μl of delivery solution (0.5 μl 10 μg / μl Cas9, 2.5 μl S buffer, 2.5 μl ethanol, plus 10 μl MGW) on a glass microscopy slide viewed under a light microscope. Clusters visible in the picture (arrows) represent Cas9 protein aggregates that precipitated out of solution immediately after the addition of 25% ethanol. [Figure 22] 1A-1C are images showing the effect of ethanol concentration in the delivery solution on Cas9 precipitation. The top of the three images shows 93 ng / μl of labeled Cas9 in the delivery solution (25% S buffer, 25% ethanol, water plus 10 μl) at 10× magnification, and the bottom of the three images shows 20× magnification. 2 μl of sample on a glass slide was observed under a fluorescent microscope. No aggregation or precipitation was observed for 1%, 5% and 10%. Small aggregates formed and precipitated at 15% ethanol (arrows), and large clusters of protein formed and separated from the solution for 20 and 25% ethanol (arrows). Images were recorded 10 seconds after adding ethanol to a solution containing Cas9 and all components of the delivery solution except ethanol. [Figure 23] 1 is an image showing the effect of ethanol concentration in the delivery solution on Cas9 precipitation. 93 ng / μl labeled Cas9 in delivery solution (25% S buffer, 25% ethanol, water plus 10 μl) is shown at 20× magnification. 12% ethanol was the lowest concentration that induced protein precipitation. [Figure 24] Figure 1 shows images of ethanol and protein-free Cas9 eluted from the column. Eluted Cas9 and Cas9 in S buffer (40x magnification) are shown to show that there was no significant aggregation or precipitation prior to the addition of ethanol and to show that S buffer did not have a detrimental effect on protein solubility. [Diagram 25]Figure 1 shows the image of electrophoretic agarose gel illustrating the Cas9 concentration for the RNP complexes prepared in the delivery solution containing different amounts of ethanol. Samples correspond to the samples reported in Table 9. Sample 10 represents a negative control in which the RNP tool was not present, and thus the DNA target was intact. Editing was confirmed by the presence of two front bands in all other samples, which represent the two fragments of the target DNA cut by the RNP tool. The agarose gel was a 1.5% gel run at 100V. [Figure 26A] Figures 26A-26C are images showing the effect of NDSB-201 on the precipitation of Cas9. Figure 26A is an image showing 2 μl of 500 ng / μl unlabeled Cas9 in delivery solution (0.5 μl of 10 μg / μl Cas9, 2.5 μl of S buffer, 2.5 μl of ethanol, 10 μl of MGW added) on a glass slide for microscopy observed under an optical microscope. Clusters visible in the picture represented Cas9 protein aggregates that precipitated out of solution immediately after the addition of 25% ethanol; Figure 26B is an image showing RNP (3:1 gRNA / Cas9) in a delivery solution containing 0.5 M NDSB, where the precipitation phenomenon was significantly reduced; Figure 26C is an image showing RNP (3:1 gRNA / Cas9) in a delivery solution containing 20% ​​glycerol, where no visible precipitation was observed. [Figure 26B] See legend to Figure 26A. [Figure 26C] See legend to Figure 26A. [Figure 27]Gel showing the editing efficiency of Cas9:gRNA on HPRT DNA (editing efficiency = band 2 / (band 1 + band 2)). Editing efficiency was normalized to 0% ethanol sample. Two replicates are shown for each condition. Samples were as follows: lane 1: 0% ethanol, lane 2: 25% ethanol, lane 3: sorbitol (4% w / v), lane 4: D-mannitol (2% w / v), lane 5: α-crystallin (100 μm), lane 6: α-crystallin (220 μm), lane 7: NDSB (0.2M), lane 8: trehalose (26.4 mm), lane 9: trehalose (26.4 mm) / α-crystallin (100 μm), lane 10: TF-ethanol (0.7%) used instead of ethanol, lane 11: uncut HPRT DNA. [Figure 28] FIG. 13 is a gel showing the editing efficiency of Cas9:gRNA on HPRT DNA (editing efficiency=band 2 / (band 1+band 2)). Editing efficiency was normalized to the 0% ethanol sample. Two replicates are shown for each condition. The samples were as follows: lane 1: 1% ethanol, lane 2: 25% ethanol, lane 3: D-sorbitol (4% w / v) in S buffer, lane 4: D-sorbitol, lane 5: α-crystallin (220 μM), lane 6: α-crystallin (220 μM) / D-sorbitol (4% w / v) in S buffer, lane 7: gelatin A (1% w / v), lane 8: gelatin B (1% w / v), lane 9: glycine (0.25 M), lane 10: proline (75 mM), lane 11: L-arginine (50 mM), lane 12: histidine (12.8 mM), lane 13: myo-inositol (1% w / v), lane 14: Tween-20 (0.1% v / v), lane 15: uncut HPRT DNA. [Figure 29]Figures 29A and 29B are bar graphs showing the editing efficiency of Cas9:gRNA on HPRT DNA (editing efficiency = band 2 / (band 1 + band 2)). Editing efficiency was normalized to the 0% ethanol sample. Two replicates are shown for each condition. Figure 29A shows a bar graph showing the effect of additives including 0% EtOH, 25% EtOH, D-sorbitol, D-mannitol, α-crystallin (100 μM), α-crystallin (221 μM), NDSB, trehalose, α-crystallin / trehalose, 7% TF-EtOH, and a negative control. FIG. 29B shows a bar graph illustrating the effect of additives including 0% EtOH, 25% EtOH, D-sorbitol (buffer), D-sorbitol (additive), α-crystallin, α-crystallin / sorbitol (buffer), gelatin A, gelatin B, glycine, proline, L-arginine, L-histidine, myo-inositol, Tween-20, and a negative control. [Diagram 30] 1 is a bar graph showing the percentage of CRISPR-induced hprt editing in MSC cells in the presence and absence of additives. [Diagram 31] 1 is a bar graph showing the percentage of CRISPR-induced hprt editing in U2OS cells in the presence and absence of additives (2 hits). [Diagram 32] 1 is a bar graph showing editing efficiency in smaller wells (24-well, 48-well, and 96-well plates). Editing efficiency increased for cells seeded in smaller wells. [Diagram 33] FIG. 13 shows the effect of increasing Cas9 concentrations on editing efficiency. Increasing amounts of Cas9 increased the editing percentage in MSCs. [Diagram 34] 1 is a bar graph showing the effect of Cas9:gRNA ratio on editing efficiency. Changing the ratio of Cas9 to guide RNA did not affect editing efficiency. [Diagram 35] 1 is a bar graph showing the effect of delivered RNP dose on editing efficiency. Increasing doses increased the editing percentage in MSCs. [Diagram 36] FIG. 1 is a bar graph showing the effect of MgCl2 on editing efficiency. Addition of MgCl2 did not improve editing efficiency. [Figure 37] 1 is a bar graph showing a comparison of editing efficiency between the delivery platform technique and electroporation. Editing efficiency was greater when Cas9 RNP was delivered by the delivery platform technique in MSCs compared to electroporation. [Figure 38] 1 is a bar graph showing a comparison of cell viability following RNP delivery using the delivery platform technique compared to electroporation. Comparable viability was observed in cells treated with either the delivery platform technique or electroporation. [Figure 39] Figure 1 shows a comparison of cell functionality after RNP delivery by the delivery platform technique compared to electroporation. The functionality of MSCs was not affected by the delivery platform technique. Three representative images of MSCs differentiated from untreated group (untreated), Cas9 RNP group delivered by delivery platform technique (delivery platform), and Cas9 RNP group delivered by electroporation (electroporation) are shown. There was no difference between untreated cells and delivery platform treated cells. However, differentiation was inhibited in cells treated by electroporation. [Diagram 40] 1 is a bar graph showing editing efficiency in adherent and suspension cell lines, and primary cells. Editing efficiency data for adherent (MSC, U2OS) and suspension (T cells, Jurkat) cell lines (U2OS, Jurkat) and primary cells (MSC, T cells). Successful editing was observed in both cell lines and primary human cells. [Diagram 41] 1 is a bar graph showing the editing efficiency using Cas9 mRNA. Delivery of Cas9 mRNA and guide RNA by delivery platform technology induces editing in MSCs. Similar reference symbols in various figures indicate similar elements. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0127] Detailed Description Currently, the CRISPR-Cas9 system is most commonly delivered to cells in the form of plasmid DNA that encodes the three components either as separate plasmids or as a combined plasmid. mRNA encoding Cas9 has also been used (Liang et al., (2015) J Biotechnol 20;208:44-53). The Cas9 protein has been used as an alternative to the plasmid or mRNA encoding Cas9. Interestingly, Cas9 ribonucleoproteins (Cas9-containing RNPs), in which purified Cas9 protein is complexed with gRNA prior to delivery to cells, have recently been reported to offer several advantages over plasmid and mRNA approaches, as described, for example, in: Cho SW, Lee J, Carroll D, Kim JS, Lee J. Heritable gene knockout in Caenorhabditis elegans by direct injection of Cas9-sgRNA ribonucleoproteins. Genetics. 2013. 195(3):1177-80; Sung YH, Kim JM, Kim HT, Lee J, Jeon J, Jin Y, Choi JH, Ban YH, Ha SJ, Kim CH, Lee HW, Kim JS. Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases. Genome Res. 2014. 24(1):125-131; Kim S, Kim D, Cho SW, Kim J, Kim JS. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 2014. 24(6):1012-9 (hereinafter "Kim et al."); Lin S, Staahl BT, Alla RK, Doudna JA.Enhanced homology-directed human genome engineering by controlled timing of CRISPR / Cas9 delivery. eLife. 2014. 15;3:e04766; Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL, Hu JH, Maeder ML, Joung JK, Chen ZY, Liu DR. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol. 2015. 33(1):73-80; Liang X, Potter J, Kumar S, Zou Y, Quintanilla R, Sridharan M, Carte J, Chen W, Roark N, Ranganathan S, Ravinder N, Chesnut JD. Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J Biotechnol. 2015. 20;208:44-53 (hereafter Lin et al."); and Schumann K, Lin S, Boyer E, Simeonov DR, Subramaniam M, Gate RE, Haliburton GE, Ye CJ, Bluestone JA, Doudna JA, Marson A. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. Proc Natl Acad Sci US A. 2015. 18; 112(33):10437-42, the entire contents of each of which are incorporated herein by reference.

[0128] Gene editing nucleases, including ZFNs, are described in Bhakta, M. et al., Genome Research 23:530-538; 2013, and Beerli, R. et al., Proc. Natl. Acad. Sci v.95 pp 14628-14633; 1998, and TALs are described in Cermak, T. et al., Nucleic Acids Research 2011, v.39, no. 12, Miller, J. et al., Nature Biotechnology vol. 29 no. 2; 2011, Christian, M. et al., Genetics 186:757-761; 2010, Deng, D. et al, Science 2012: v.335 p.720, and Boch, J. et al., Science 2009: v.326. p. 1509, the entire contents of each of which are incorporated herein by reference. Additionally, Cre is described in Chevalier, B. et al., Nucleic Acids Research 2001, v.29 no. 18, the entire contents of which are incorporated herein by reference. MegaTal is described in Sather, B. et al Sci Transl Med 7(307) 2015; Ibarra, G. et al., Molecular Therapy-Nucleic Acids (2016) 5,e352; Osborn, M. et al., Molecular Therapyv. 24 no. 3, 570-581 (2016); Wang, Y. et al., Nucleic Acid Research 2014; v.42, 6463-6475; and Gaj, T. et al., Cold Spring Harbor Perspectives in Biology 2015, each of which is incorporated herein by reference.Cas9 is described in Wiedenheft, Bl. et al., Nature 2012; v.482; 331, Fineran, P. et al., Virology 2012; 433; 202-209, and Hsu, P. et al., Cell 2014; v.157; 1262-1278, the entire contents of each of which are incorporated herein by reference.

[0129] Some cell types can silence plasmids and mRNAs, while others may be refractory to DNA transfection. Several studies have shown that RNPs produce editing that is both more rapid and more efficient than plasmid transfection. This may be due to the direct introduction of fully formed complexes that can function immediately in the cell and do not require transcription, translation, and assembly as in the case of plasmids. RNPs have also been reported to be less toxic than plasmid DNA. This may be because the exogenous DNA itself may be toxic to some cells, or because the half-life of RNPs is significantly shorter than that of plasmids, resulting in longer-term expression of these foreign mRNAs and proteins. Importantly, the shorter expression time of RNPs is also associated with less off-target editing compared to plasmids, likely due to reduced persistence of activity of the Cas9 protein. Plasmids may also be randomly integrated into the genome or at the site of Cas9 generation. The latter event can at least be predicted and monitored, whereas the former may be more problematic as it may be difficult to detect. Regardless of the site of integration, these foreign sequences can trigger a host immune response, which poses a challenge for the use of gene-edited stem or primary cells in clinical applications. Furthermore, cells transfected with plasmids for clinical applications are considered genetically modified by regulatory authorities, thus requiring time-consuming and costly regulatory procedures.

[0130] The subject matter provides for vector-free (e.g., non-viral vector) delivery of a payload across the plasma membrane. In some embodiments, the delivery does not include an expression vector. In particular, it has been discovered that intracellular delivery of a substance can be achieved by contacting a cell (and / or cell population) with an aqueous solution that includes an alcohol and a delivery substance (e.g., a payload). The alcohol acts to permeabilize the membrane, allowing the payload to translocate across the membrane. However, if the volume of the aqueous solution in contact with the cell is too large and / or the exposure occurs for too long a time, permanent or severe (e.g., irreversible) damage to the cell can occur (adversely affecting cell viability). Conversely, if the volume of the aqueous solution in contact with the cell is too small and / or the exposure occurs for too short a time, intracellular delivery of the substance is not achieved. Thus, an appropriate volume of the aqueous solution can be applied and / or the length of exposure can be controlled to achieve delivery of the payload across the plasma membrane while maintaining cell viability.

[0131] The appropriate volume of the aqueous solution to be contacted with the cell population can vary based on the intended application, for example, based on (e.g., is a function of) the number of cells in the population, the exposed cell surface area, the size of the cells, the composition of the aqueous solution, the payload, the technique for contacting the aqueous solution with the cell population, etc.

[0132] The delivery platform is used to deliver the Cas9 RNP to cells so that genome editing is achieved.

[0133] Vector-free genome editing To determine whether genome editing can be achieved by delivering Cas9 RNP using the delivery platform technique, a strategy is devised to delete a region from a target gene in the A549 human lung cell line (Figure 1). Cas9-mediated cleavage is followed by non-homologous end joining (NHEJ). PCR primers flanking the target region and internal probes acting as reference and drop-off probes are designed. If editing is successful, the ddPCR assay results in the separation of droplets indicating where in the edited gene the drop-off probe cannot bind and only the reference probe binds.

[0134] A two-guide RNA approach is employed using two-part guide RNA and single-guide RNA. For the two-part guide RNA approach, a 42 bp CRISPR RNA (crRNA) molecule is designed to target a selected region within the target gene. A 69 bp trans-activating crRNA (tracrRNA or trRNA) is also designed. The crRNA and tracrRNA molecules are chemically synthesized, and the tracrRNA molecule is labeled with FAM. For the experiment, the tracrRNA is incubated with the crRNA to form a two-part guide RNA molecule. For the single-guide RNA approach, an sgRNA corresponding to the target sequence is in vitro transcribed from a DNA template.

[0135] Cas9 RNPs are formed by incubating the Cas9 protein with either a bipartite guide RNA or a single guide RNA.

[0136] General definitions and general techniques Unless specifically defined otherwise, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, and biochemistry).

[0137] In the above description and in the claims, phrases such as "at least one of" or "one or more of" may be present before a conjunctive listing of elements or features. The term "and / or" may also be present in a listing of more than one element or feature. Unless otherwise implied or explicitly contradicted by the context in which it is used, such phrases are intended to mean any of the listed elements or features individually, or any of the listed elements or features in combination with any of the other listed elements or features. For example, the phrases "at least one of A and B," "one or more of A and B," and "A and / or B" are intended to mean "A alone, B alone, or A and B together," respectively. Similar interpretations are also intended for listings containing more than two items. For example, the phrases "at least one of A, B, and C," "one or more of A, B, and C," and "A, B, and / or C" are intended to mean "A alone, B alone, C alone, A and B together, A and C together, B and C together, or A and B and C together," respectively. Additionally, use of the term "based on" above and in the claims is intended to mean "based at least in part on," allowing for unrecited features or elements.

[0138] The term "about" as used herein in connection with a numerical value or numerical range means ±10% of the stated or claimed numerical value or numerical range, unless the context requires a more limited range.

[0139] When a range for a parameter is provided, it is understood that all integers and tenths thereof within that range are also provided by the present invention. For example, "0.2-5 mg" is a disclosure of 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, etc. up to 5.0 mg or less. Guide RNA (gRNA) can be used in two formats: crRNA and tracrRNA can be used in the form of duplex RNA (or "dual RNA") or single guide RNA (sgRNA) molecules can be made. "gRNA" as used herein refers to the guide RNA of the CRISPR-Cas system. The term "gRNA" can refer to a combination of crRNA and tracrRNA (dual RNA) or a single RNA molecule (sgRNA) that includes both crRNA and tracrRNA sequences.

[0140] As used herein, complex refers to the association of molecules by non-covalent interaction.For example, complex can comprise two or more molecules that are hydrostatically associated with each other, for example, by hydrogen bond.A non-limiting example of complex is RNP that comprises Cas protein and gRNA.

[0141] As used herein, a "gene editing protein" is a protein that cleaves the sugar phosphate backbone of a DNA molecule or binds to target DNA with sufficient affinity to reduce gene expression. A non-limiting example of a protein that binds to target DNA with sufficient affinity to reduce gene expression is nuclease-inactivated Cas9 (dCas9), i.e., Cas9 that cannot cleave DNA. An example of dCas9 is the D10A and H840A double mutant of Cas9 protein having the amino acid sequence shown in SEQ ID NO:1.

[0142] As used herein, an "expression vector" is a DNA or RNA vector capable of causing expression of one or more polynucleotides. Preferably, the expression vector is also capable of replicating in a host cell. The expression vector can be either prokaryotic or eukaryotic, and is typically a plasmid. The expression vector of the present invention includes any vector that functions (i.e., directs gene expression) in a host cell of the present invention, including one of the prokaryotic or eukaryotic cells described herein, such as a gram-positive, gram-negative, pathogenic, non-pathogenic, symbiotic, spherical, rod-shaped, or spiral bacterial cell; an archaeal cell; or a protozoan, algae, fungus, yeast, plant, animal, vertebrate, invertebrate, arthropod, mammalian, rodent, primate, or human cell. The expression vector of the present invention includes regulatory sequences, such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the host cell and control the expression of the polynucleotide. In particular, the expression vector of the present invention includes a transcription control sequence. A transcription control sequence is a sequence that controls the initiation, elongation, and termination of transcription. Particularly, important transcription control sequences are the sequences that control transcription initiation, such as promoter, enhancer, operator and repressor sequences.Suitable transcription control sequences include any transcription control sequence that can function in at least one of the cells of the present invention.Various such transcription control sequences are known to those skilled in the art.In a preferred embodiment, the method does not include the use of viral vectors such as adenovirus to deliver nucleic acid molecules or constructs.

[0143] Payload compositions such as polynucleotides, polypeptides, complexes, or other agents may be purified and / or isolated. Specifically, as used herein, an "isolated" or "purified" compound, nucleic acid molecule, polynucleotide, polypeptide, or protein, or a complex comprising, for example, a protein and a polynucleotide, is substantially free of other cellular material with which it naturally occurs, or culture medium if produced by recombinant techniques, or chemical precursors or other chemicals if chemically synthesized. A purified compound is at least 60% by weight (dry weight) of the compound of interest. Preferably, the preparation is at least 75% by weight, more preferably at least 90% by weight, and most preferably at least 99% by weight of the compound of interest. For example, a purified compound is a compound that is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w / w) of the desired compound. Purity is measured by any appropriate standard method, such as column chromatography, thin layer chromatography, or high performance liquid chromatography (HPLC) analysis. A purified or isolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) is free of genes or sequences that flank it in its natural state. Examples of isolated or purified nucleic acid molecules include: (a) DNA that is part of a naturally occurring genomic DNA molecule, but is not flanked on either side by nucleic acid sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid that is incorporated into a prokaryotic or eukaryotic vector or genomic DNA in such a way that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) another molecule, such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene that encodes a fusion protein. Isolated nucleic acid molecules according to the present invention further include synthetically produced molecules as well as any nucleic acid that has been chemically modified and / or has a modified backbone. EXAMPLES

[0144] Example 1: The following materials and methods were used to generate the data described herein. Cells and cell culture A549 cells (A549 European Collection of Cell Cultures (ECACC) were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA) (Cat No 86012804) and Beas-2B (Sigma-Aldrich Corp Cat. No. 95102433) cells were routinely cultured in DMEM (Gibco) supplemented with 5% fetal bovine serum and 2 mM L-glutamine (Gibco). Jurkat cells (European Collection of Authenticated Cell Cultures; ECACC) were grown and maintained at 37 °C and 5% CO2 in RPMI 1640 (Gibco) supplemented with 10% (v / v) FBS (Sigma), and 1% (v / v) penicillin / streptomycin. Primary human PBMCs were freshly isolated and cultured in 10% (v / v) heat-inactivated FBS, 0.01% (v / v) They were maintained in RPMI 1640 (Gibco) supplemented with 2-β-mercaptoethanol (lifesciences) and 1% (v / v) L-glutamine at 37°C and 5% CO2. CD34+ bone marrow-derived cells (HSCs) were purchased from Lonza and expanded and maintained in HPMG hematopoietic growth medium (Lonza) supplemented with growth factors SCF (25 ng / ml), TPO (50 ng / ml) and FLT-3 (50 ng / ml). Cultures were maintained at 37°C and 5% CO2 for 1 week, with medium changed every 3 days.

[0145] Delivery using a delivery platform The delivery system includes a delivery tool, device, or instrument and a permeabilization delivery solution (Figure 6 shows a graphical representation of the delivery mechanism). The delivery device includes a sprayer held on an adjustable xyz axis above a plate positioner platform. The sprayer is connected to a 5 bar compressor via tubing. A positioning collar allows centering of the spray over the wells, and the plate positioner platform allows for good reproducible positioning of the wells below the sprayer. The payload is pipetted into a delivery port located on the top surface of the sprayer, and the spray is generated using a spray actuator button. A preferred delivery solution includes 32 mM sucrose, 12 mM potassium chloride, 12 mM ammonium acetate, 5 mM HEPES, and 25% ethanol) in molecular research water (Sigma-Aldrich), and optionally 2.5 mM MgCl2. Prior to the addition of ethanol, the solution was adjusted to pH 7.4 and filter sterilized. For various payload volumes, the water volume was adjusted accordingly.

[0146] For delivery to adherent cells, cells were seeded into 48-well or 96-well culture plates (Nunc) at a density that would result in cells reaching 70-95% confluency at the time of delivery (alternatively, culture plates with sample wells selected from 1, 6, 9, 12, 24, 48, 96, 384, and 1536 may be used). The supernatant was removed from the target wells, the plate was placed on the plate positioner platform, and the xyz axes were used to position the sprayer at a distance of 31 mm from the spray head facing the cell monolayer. 10 μl of delivery solution was sprayed onto the cells. After 2 min at room temperature, 100 μl of 0.5× PBS was added onto the cells using a micropipette, and the cells were incubated at room temperature for 30 s (for 48-well culture plates); if using 96-well plates, 50 μl of 0.5× PBS was added onto the cells. The 0.5× PBS solution was removed and 100 μl of fresh culture medium was added. After 3 hours, 100 U / ml penicillin and 100 μg / ml streptomycin were added to each well. For double spray, the procedure was repeated after 1-5 hours (e.g., 2 hours or preferably 4 hours) of incubation from the first spray treatment. The cells were then incubated for 3 hours, after which antibiotics were added.

[0147] For delivery to suspension cells, 1.0 x 10 cells 6 The cells were placed into 0.4 μm polyester membrane inserts (Corning). The inserts were placed in the in-house vacuum equipment and a vacuum of -0.5 bar to -0.68 bar was applied to remove the culture medium. The inserts were then placed into a 12-well plate and positioned under a nasal spray head. The spray procedure was performed as described for adherent cells, and the cells were transferred to a new culture plate when fresh culture medium was added.

[0148] In vitro transcription of sgRNA In vitro transcription of sgRNA was performed using the Agilent (Santa Clara, CA) Sure Guide in vitro transcription kit according to the manufacturer's guidelines.

[0149] Cas9 RNP assembly and delivery Recombinant Cas9 protein was purchased from ToolGen, Inc and IDT-DNA. Guide RNA was generated using the SureGuide IVT kit (Agilent) following the manufacturer's guidelines. pCas9-GFP-382a2-384 plasmid was used as a positive control in the assay. This plasmid contained CAS9-GFP and two gRNAs. Cas9 protein (1ug-20ug) was premixed with in vitro transcribed gRNA (1ug-20ug) and the complex was incubated at room temperature for 10 minutes. RNP complex was added to delivery solution (1x S buffer 25% EtOH) and delivered into cells using the delivery platform. For plasmid-mediated expression of RGEN, Lipofectamine 2000 was used to infect 1x10 cells. 5 were transfected with pCas9-GFP-382a2-384.

[0150] Western blotting Cells were detached and placed in ice-cold 1x PBS solution and centrifuged at 2200 rpm for 5 min. The pellet was dissolved in radioimmunoprecipitation (RIPA) buffer (Sigma, Dublin) and 25 mg of protein was loaded onto a 4–20% polyacrylamide gel. After gel electrophoresis, proteins were transferred onto nitrocellulose membranes and blocked in 5% milk / 1x TBS 0.1% Tween-20 (Sigma, Dublin). The membrane was probed with primary antibodies overnight at 4°C, followed by application of secondary antibodies linked to horseradish peroxidase for 1 h at room temperature. Visualization of the gel was performed using the ECL detection system (Gbox, Syngene, Cambridge UK). Primary antibodies used included anti-cas9 (Abcam, Cambridge UK), GAPDH (Sigma, Dublin).

[0151] Immunofluorescence Immunofluorescence was performed using anti-Cas9 antibody from Millipore at a dilution of 1 / 200.

[0152] PCR assay for editing The reaction mixture used for PCR was 30.5 μl H2O, 10 μl 5× buffer, 5 μl 25 mM MgCl2, 1 μl 10 mM dNTPs, 1 μl 10 μM primer F, 1 μl 10 μM primer R, 0.5 μl Taq polymerase, 1 μl DNA, total volume 50 μl. 35 cycles were used. 95℃: 120 seconds (s) ------------ 95℃:30s 55℃:30s x 35 72℃:45s ----------- 72℃: 5 minutes

[0153] Viability assay Jurkat cells were analyzed for viability and the presence of apoptotic markers using Annexin V and Propidium Iodide (PI) Apoptosis Detection Kit (eBioscience) according to the manufacturer's protocol. Cells were washed once in 1× binding buffer and diluted with 1× binding buffer at cell concentrations of 1–5×10 6 Cells were resuspended at 100 μl / ml. APC-conjugated Annexin V was added to 100 μl of cell suspension. Cells were incubated with the fluorescent dye for 15 min at room temperature in the dark. After incubation, cells were washed with 1× binding buffer and resuspended in 100 μl of 1× binding buffer. 5 μl of PI was added to the cells. Cells were analyzed by flow cytometry within 4 hours of staining. The percentage of viable and apoptotic cells was calculated and graphed.

[0154] Analysis of activation markers Jurkat cells in 24-well plates were stimulated with 5 μg / ml PHA for 4 hr and 24 hr. Expression of surface markers CD25 and CD69 activation markers was analyzed by flow cytometry. Cells were harvested, washed twice with PBS, and diluted with 1 × 10 cells in FACs buffer. 6Cells were resuspended at a concentration of 100 μl / ml. 100 μl of cell suspension was stained with CD25 APC (eBioscience) and CD69 PE (eBioscience) or isotype control for 15 min at 4°C in the dark. Cells were washed by adding 500 μl and resuspended in 100 μl FACs buffer and analyzed on a BD Accuri C6 flow cytometer.

[0155] Effect of delivery solution composition on the activity of Cas9 RNP in in vitro editing assays The effect of the delivery solution composition on the activity of the Cas9 protein was first analyzed in an in vitro assay. Cas9 protein (Toolgen; Seoul, South Korea) was incubated with the control gRNA and control plasmid provided in the Agilent kit together with the kit buffer (solution A, Table 1). Cleavage products were observed (Figure 2).

[0156] Next, the effect of replacing the kit buffer with delivery solution (without ethanol) was determined (Solution B, Table 1). No delivery product was observed (Figure 3).

[0157] Table 1. Composition of buffers for in vitro editing TIFF2025010591000025.tif52128

[0158] Experiments were performed to determine the effect of the delivery solution. A variety of different delivery solution:kit buffers were used. It was found that cleavage occurred at certain delivery solution ratios up to 1.5 μl S buffer:0.5 μl kit buffer (Table 2 and Figure 4). Therefore, the effect of adding MgCl2 to the delivery solution / reaction buffer was examined (Solution C, Table 1). Addition of 1.25 mM MgCl2 to solution B caused the cleavage reaction to occur (Figure 5). Some cleavage can occur in the absence of MgCl2, but this appears to be suboptimal and inconsistent. Therefore, 2.5 mM MgCl2 was included in the delivery solution from this point on. The desired delivery solution contains 32 mM sucrose, 12 mM KCl, 12 mM ammonium acetate, 5 mM HEPES, 2.5 mM MgCl2, 15% ethanol, and 85% H2O.

[0159] Table 2. Buffer composition for in vitro editing when Agilent kit buffer is mixed with delivery solution (10x S buffer) in various ratios. TIFF2025010591000026.tif58128

[0160] We investigated the efficiency of a delivery platform solution containing 15% ethanol and supplemented with 2.5 mM MgCl2 to deliver the reporter payload, Ovalbumin-FITC (OVA-FITC). Surprisingly, delivery of OVA-FITC appeared to be enhanced in a delivery solution containing 2.5 mM MgCl2 compared to a delivery solution without MgCl2 (Figure 8).

[0161] Therefore, for subsequent studies, Cas9 RNP was delivered in a delivery solution containing 15% ethanol supplemented with 2.5 mM MgCl2.

[0162] Time course of Cas9 protein delivery and expression Cas9 is usually delivered in the form of DNA encoding the Cas9 protein. However, the DNA can remain in the cells for days to months, which can result in off-target effects where the enzyme cleaves non-specific sites in the cells. It has been suggested that delivering Cas9 in protein form leads to a reduced duration of expression of the enzyme and therefore reduced off-target effects. We investigated whether purified recombinant Cas9 protein without gRNA could be delivered to A549 cells using the delivery platform. The duration of expression compared to plasmid-mediated expression was also examined. Cas9 protein (5 μg) was delivered to A549 cells and expression was examined by Western blot analysis of cell lysates. Cas9 protein was detected from cells 1 hour after delivery, and the levels decreased thereafter (Figure 9). In contrast, when cells were lipofected with a plasmid encoding Cas9, no expression was observed before 6 hours, and strong expression was observed at 24 hours (Figure 9).

[0163] Cas9 protein was also successfully delivered to BEAS-2B cells, Jurkat cells, human PBMCs and HSCs (Table 3).

[0164] Table 3. Cell types in which Cas9 protein was successfully delivered TIFF2025010591000027.tif55138

[0165] Cas9 protein delivery With viral delivery of CRISPR / Cas9, it can be difficult to control the dose of the editing tool delivered, therefore we investigated whether our delivery platform technology could deliver increasing doses of Cas9 protein.

[0166] A dose response was observed when increasing concentrations of Cas9 protein were delivered to A549 and Jurkat cells (Figures 10A and B).

[0167] Cas9 protein and RNP delivery – subcellular localization Because Cas9 RNP functions in the nucleus, we determined the subcellular localization of Cas9 protein and Cas9 RNP after delivery using our delivery platform. We used Cy3-labeled Cas9 and FAM-labeled dual RNA to examine the localization of Cas9 and RNP by fluorescence microscopy. In addition, we detected Cas9 by immunofluorescence using an anti-Cas9 antibody.

[0168] The delivery platform was used to deliver 1 ug of Cas9-Cy3 protein to A549 cells, and 1 hour after delivery, Cas9 was observed throughout the cytoplasm and within the nucleus of the cells (Figure 11A).

[0169] The delivery platform was used to deliver 10ug of unlabeled Cas9 protein into A549 cells, and Cas9 was detected by immunofluorescence using an anti-Cas9 antibody. One hour after delivery, Cas9 was observed throughout the cytoplasm and in the nucleus of the cells (Figure 11B). The staining pattern was similar to that observed for Cas9-Cy3. The delivery platform was used to deliver 100uM sgRNA1-FAM into A549 cells. One hour after delivery, sgRNA1-FAM was observed throughout the cytoplasm and in the nucleus of the cells (Figure 11C).

[0170] Next, we investigated the localization of RNPs after delivery into A549 cells. Unlabeled Cas9 was complexed with sgRNA1-FAM and delivered using the delivery platform. One hour after delivery, sgRNA1-FAM was observed throughout the cytoplasm and in the nucleus of the cells (Figure 11D). The staining pattern appeared to form more "speckles" than the pattern of sgRNA1-FAM shown in Figure 11C, indicating that the sgRNA was indeed complexed with Cas9.

[0171] We next investigated the localization of RNPs by immunofluorescence using anti-Cas9 antibodies. Unlabeled Cas9 was complexed with sgRNA1-FAM and delivered using the delivery platform. One hour after delivery, Cas9 was observed throughout the cytoplasm and in the nucleus of the cells (Figure 11E). The staining pattern was similar to that shown in Figure 11D, as expected.

[0172] Effect of delivery platform-mediated delivery on Jurkat cells, PMBCs and HSCs For clinical applications, it is important that a high rate of viability and functionality is maintained by the cells following the delivery and transfection protocols. The effect of the delivery platform technique on the viability of Jurkat cells, PBMCs and HSCs was examined. For analysis of the effect of platform-mediated delivery, a delivery solution without payload was delivered to Jurkat cells, human PBMCs and HSCs. Cell viability after delivery was investigated by Annexin V / Propidium Iodide (PI) staining. Cell viability was minimally reduced compared to untreated controls (Figure 12). Additionally, GFP mRNA was delivered to primary NK cells (Figure 7) and the cells had a viability of 96.6%.

[0173] Comparing viability and efficiency: Ethanol spray delivery compared to the Neon electroporator Electroporation has been used to deliver Cas9 RNP to cells in vitro (Kim et al., (2014) Genome Res 24(6):1012-9; Lin et al., (2014) eLife 15;3:e04766; Liang et al., (2015) J Biotechnol 20;208:44-53; Schumann et al., (2015) Proc Natl Acad Sci USA 18;112(33):10437-42). However, electroporation is known to affect the viability and function of clinically relevant cells, including T cells and stem cells. Therefore, the effect of platform-mediated delivery was compared to that of electroporation. We used Neon electroporation, which has been used by other groups (Liang et al., (2015) J Biotechnol 20;208:44-53; Schumann et al., (2015) Proc Natl Acad Sci USA 18;112(33):10437-42).

[0174] Bovine serum albumin-FITC (BSA-FITC) was delivered to Jurkat cells and viability was determined 24 hours after delivery. When using the delivery platform, viability decreased from 81% in untreated controls to 65% in cells that received BSA-FITC (Figure 13A). In contrast, when BSA-FITC was delivered to cells by electroporation, viability levels dropped to 8%. A similar decrease in viability was seen when buffer alone (no BSA-FITC) was delivered.

[0175] The delivery efficiency of BSA-FITC was investigated by flow cytometry 24 hours after spraying. When using the delivery platform, 34% of 65% of viable cells were BSA-FITC positive (Figure 13B). When using electroporation, 62% of 8% of viable cells were BSA-FITC positive. Thus, of the original starting population, BSA-FITC was present in 22% of cells after delivery via the platform and in 5% of cells after electroporation.

[0176] Effect of delivery via a delivery platform technology on the functionality of primary Jurkat cells Next, we investigated the effect of platform-mediated delivery on the functionality of Jurkat cells. We used the delivery platform to deliver spray buffer only to cells. CD69 and CD25 are early and late cell surface markers of T cell activation, respectively. We used the delivery platform to deliver a delivery solution without payload (spray buffer only, SBO) to cells. We stimulated cells with PHA and measured the expression of CD69 and CD25 by flow cytometry 4 hours and 24 hours after stimulation, respectively. We observed upregulation of CD69 and CD25 in treated cells (Figure 14).

[0177] Example 2: Establishment of an in vitro editing assay to test the function of gene editing tools and the effect of delivery solutions on the function of gene editing tools The Crispr Cas9 system of gene editing has been widely adopted. However, most users utilize a DNA plasmid form of Cas9 in combination with a guide RNA. This was of interest since delivering an RNP version of the system would be more transient and therefore should result in fewer off-target effects. The essential tools (recombinant Cas9 nuclease, single-stranded guide, bipartite guide) have recently become commercially available. We designed a bespoke guide RNA sequence based on the corresponding plasmid sequence reported in the literature. We performed a validation assay to check the quality of the reagents. To evaluate the gene editing tool, we developed an assay based on the in vitro editing test of the Agilent Sure Guide Cas9 Programmable Nuclease Kit. In this assay, Cas9 protein was incubated with a guide (single-stranded or bipartite) to form an RNP. This RNP was then incubated with a purified length of DNA (~250-750 bp) that contained the target site. If the editing is successful, two strands of DNA are generated as a result of the editing, which is evident after separation on an agarose DNA gel. This assay serves two purposes: first, it serves as a quality control assay to check the functionality of the gene editing tool, and second, it serves to determine the effect of the delivery solution on the gene editing tool, in particular on the Cas9 / RNP activity. For the latter purpose, a solution of Cas9 or RNP was prepared in the delivery solution and sprayed, and then an aliquot of the sprayed solution was collected and used for the editing assay.

[0178] material Agilent Sure Guide Cas9 Nuclease Kit: The kit provided Cas9 nuclease, control DNA target, control gRNA, 10x Cas9 digestion buffer (Agilent) and RNase-free water. Optimized volumes for the control reactions are listed in Table 4. Various gRNA and DNA targets were synthesized or provided by IDT-DNA Cas9 (IDT-DNA). Delivery solutions are described herein.

[0179] In vitro editing (IVE) assay: testing the functionality of gene editing tools The assay was based on the Agilent Sure Guide Cas9 Nuclease Kit. The optimized volumes for each component are listed in Table 4. To validate the procedure, DNA target, gRNA and Cas9 were provided in the kit. All components kept on ice were added in the ratios listed in Table 4 to a test tube suitable for a thermocycler. The tubes were then transferred to a thermocycler preheated to 30°C. The following program was run: 30 minutes at 30℃ 15 minutes at 65°C Maintain at 4℃

[0180] The digested samples were then analyzed by gel electrophoresis, and successful editing was confirmed by the presence of additional DNA bands corresponding to the expected size DNA fragments. This assay confirmed that the recombinant Cas9 protein and the various guide RNAs used in these studies were functional.

[0181] Table 4. Volumes of components for IVE reaction* TIFF2025010591000028.tif64128

[0182] All gRNAs and corresponding target DNAs tested are listed here: sun sgGFP RNA with target DNA pCMV EGFR, sgRNA EGFP4 with target DNA EGFP, sgHPRT1 with HRT1 DNA as target, sgAAVS1 T1, sgAAVS1 T2 and AAVS1 T3 with AAVS1 as target DNA.

[0183] Testing the effect of delivery solutions on functional gene editing tools In this assay, an IVE protocol was employed to study the effects of the delivery solution and spraying procedure on the functionality of Cas9 and RNPs. To do so, Cas9 alone or as RNPs (Cas9 complexed with gRNA) was first prepared in a delivery solution, which was then either used for IVE or sprayed and then collected for IVE. Editing occurred, confirming that the components of the RNPs were able to cleave the target DNA sequence and therefore were functional despite the presence of ethanol and other chemicals in the delivery solution.

[0184] Solutions of Cas9 or RNP in delivery solution were made as reported in Table 5. RNP was formed by incubating Cas9 with gRNA for 15 min before adding S buffer and water. Additionally, corresponding samples were made in water for comparison purposes. A molar ratio of Cas9 / gRNA of 1:1 was used to make the complex. For Cas9-only solutions, gRNA was provided when the IVE reaction was performed (see Table 6).

[0185] Table 5. Composition of Cas9 solution or RNP solution TIFF2025010591000029.tif38157

[0186] All components except ethanol were added to the tube and incubated for 10 minutes. Ethanol was then added. For each solution, an aliquot of 1 μl (Cas9 solution) or 2 μl (RNP) was transferred to a tube for IVE testing according to Tables 6 and 7. To test the effect of the spraying process on RNP activity, another aliquot of 10 μl was sprayed into an empty well of a 48-well plate, and 1 μl or 2 μl of the solution was collected after spraying and transferred to a PCR tube. For IVE, see Tables 6 and 7. The samples were digested as described above and then analyzed by agarose gel electrophoresis. A typical image of the gel is provided in Figure 17.

[0187] Table 6. Volumes of components per reaction, Agilent Cas9 buffer TIFF2025010591000030.tif64128

[0188] Table 7. Volumes of components per reaction, Agilent Csa9 buffer TIFF2025010591000031.tif62128

[0189] Example 3: Conjugation of Cas9 with a fluorescent label for visualization of intracellular delivery To analyze whether Cas9 had entered cells after delivery using the delivery platform technique, we explored conjugating the protein with a fluorescent label. Cas9 labeling methods targeting amine groups must be avoided because they lead to precipitation of Cas9 due to their physicochemical properties. Therefore, Cas9 was labeled with the fluorescent tag Alexa-488 maleimide via the thiol groups of cysteine ​​(two cysteine ​​residues per protein molecule). This reaction did not change the overall charge of the protein at physiological pH. The subsequent experimental protocol was based on previous work (Zuris, JA et al., Nat Biotechnol 2015; 33(1) 73-80).

[0190] material Cas9 was purchased from either Toolgen Genome Engineering (South Korea) or IDT Integrated DNA Technologies (USA). TCEP (Tris(2-carboxyethyl)phosphine), Hepes, glycerol and KCl were analytical grade and purchased from Sigma Aldrich. Alexa 488 maleimide (Mw 720.66 g / mol) was purchased from Thermo Fisher Scientific and received as 1 mg of powder. A 10 mM stock solution was prepared by adding 139 μl of DMSO to the powder. The stock solution was stored at -20°C. To avoid protein precipitation, Alexa 488 maleimide solution must be added to the Cas9 solution such that the final concentration of DMSO is 10% or less. For 12.5 μM Cas9, a 20-fold molar excess corresponds to 250 μM Alexa dye, which corresponds to a volume of 6.25 μl of 10 mM stock solution in 250 μl. To use all 250 μl of the protein solution produced, 6.25 μl of dye stock solution was added to 250 μl of Cas9 solution to a final volume of 256.25 μl (DMSO was 2.5%, so well below the 10% limit that induces protein precipitation). Sephadex G-25 (Sigma-Aldrich) was utilized for size-exclusion gel chromatography. The elution buffer contained 250 mM KCl, 20 mM Hepes (pH 7.8).

[0191] Procedure for labeling Cas9 A 2 mg / ml Cas9 solution (12.5 μM) was made by dissolving 0.5 mg of Cas9 in 197 μl of labeling buffer (20 mM Hepes (pH 7.8); 250 mM KCl; 50 μl of glycerol (63.1 mg); 3 μl of 10 mM TCEP (10-fold molar excess)). The solution was deoxygenated with argon. A 20 molar excess of maleimide in DMSO was added dropwise while mixing by pipetting up and down. The mixture was incubated overnight at 4 °C in an Ar atmosphere (Zuris et al. Nat Bitoechnol 2015 33(1): 73-80). NOTE: The KCl concentration will be 200 mM in a final volume of 250 μl, the salts KCl and glycerol help keep Cas9 in solution. TCEP is a reducing agent frequently used in biochemistry and molecular biology applications. TCEP can protect cysteines from forming disulfide bonds and, unlike dithiothreitol and β-mercaptoethanol, does not react as readily with maleimide residues.

[0192] The next day, after checking that there was no visible precipitate in the vial, the protein solution was loaded onto a Sephadex G25 column to elute the labeled Cas9 from the free label, thus purifying and separating it. A weak band separated almost immediately, indicating successful labeling (Figure 18). The eluted protein was significantly diluted; however, the fluorescence was visible to the naked eye. Fluorescence was confirmed using a transilluminator for green fluorescence (see Figure 19). The bicinchoninic acid assay was used to check the Cas9 concentration after purification. A more concentrated aliquot was used for RNP studies. The concentration ranged from 500 to 700 ng / μl for a collected volume of approximately 1 ml.

[0193] Example 4: Approaches to overcome the Cas9 precipitation problem During the labeling process, Cas9 protein was prone to aggregation and precipitation. Others have also reported solubility issues with Cas9 in various solutions (Burger, A et al. 2016. Development 2016 143(11): 2025-37). Precipitation of Cas9 was also observed when the protein was added to the delivery solution. Since ethanol can promote protein precipitation, we hypothesized that one of the main factors contributing to Cas9 precipitation was the presence of ethanol in the delivery solution. The effect of increasing ethanol concentrations on Cas9 precipitation was evaluated.

[0194] From the studies described below, ethanol above 15% induced rapid precipitation of the protein. With this information, we decided to increase the solubility of Cas9 by 1) decreasing the ethanol concentration in the delivery solution and 2) protecting the protein with excipients / chaperones.

[0195] Effect of ethanol concentration on Cas9 precipitation A. Effect of ethanol concentration in the delivery solution on Cas9 precipitation When the labeled Cas9 was observed under a fluorescent microscope, the solution exhibited a green tint with small green "speckles." When the delivery solution (25% ethanol) was added to the Cas9, those speckles greatly increased in size and number. To ensure that the precipitation was not caused by the presence of the fluorescent probe on the protein, an unlabeled Cas9 solution in a delivery solution containing 25% ethanol was observed under a microscope. A photograph is displayed in FIG. 21, where protein aggregates were visible. While viewing the glass slide under the microscope, protein aggregation and precipitation from the solution occurred simultaneously.

[0196] After the above preliminary steps, fluorescent dye-labeled Cas9 solutions containing increasing concentrations of ethanol were prepared and analyzed by fluorescence microscopy immediately after the addition of ethanol. 133ng / μl Alexa 488 Cas9, labeled and purified in-house, was added to S buffer and water as listed in Table 8. Ethanol was added to the tubes immediately prior to the microscopic analysis of the samples.

[0197] Immediately after the addition of ethanol, 5 μl of the solution was transferred to a glass slide and observed under a fluorescent microscope. Samples 1-6 were tested first, and the results are shown in Figure 22. After the first result determination, samples 7, 8, and 9 were added (Figure 23). Due to the low concentration of Alexa 488 Cas9, the maximum amount that could be delivered in 10 μl was 931 ng, about 5-fold less than the amount usually delivered for editing experiments (5 μg).

[0198] Table 8. Volume of Cas9 and ethanol per 10 μl reaction TIFF2025010591000032.tif85147 *1xS buffer: 130mM sucrose, 50mM KCl, 50mM ammonium acetate, 20mM Hepes (pH 7.4); 10xS buffer: 1.3M sucrose, 0.5M KCl, 0.5M ammonium acetate, 200mM Hepes (pH 7.4)

[0199] It is clear from Figure 22 that aggregation appeared in samples containing 15% ethanol, and this phenomenon becomes more pronounced at 20 and 25%. No observable aggregation was observed for 1, 5 and 10% ethanol. For comparison, Cas9 without ethanol and protein as it elutes from the column is reported in Figure 24.

[0200] Since ethanol contents below 10% lead to poor uptake of the payload into the cells and ethanol contents above 15% lead to significant precipitation of proteins, a thorough study was performed in the range of 10% to 15% ethanol, Figure 23. When ethanol reached 12%, aggregation was observed at the edge of the droplet on the glass slide, indicating that 10% ethanol was the best compromise to promote payload uptake and avoid protein precipitation.

[0201] Finally, it was important to emphasize that over time (1-2 h), protein aggregation was observed in all samples (except for 1% ethanol, which was further investigated after 2 h). These observations indicated that solutions containing Cas9 and ethanol must be used within minutes of preparation.

[0202] Of note, solubility tests were performed with Alexa 488 Cas9 made from commercially available Cas9, so the concentration of Cas9 was lower than that typically used for editing experiments. The labeled protein undergoes significant dilution after it is purified. The delivery solution for these assays was intended to maintain a ratio of 25% ethanol and 25% S buffer in a volume of 10 μl used for one delivery, which meant that the volume available for the addition of Cas9 was a maximum of 5 μl. The labeled protein was more diluted than the parent protein, making it difficult to deliver 5 μg in the available volume of 5 μl.

[0203] The most suitable ethanol content for the delivery solution was 10%. At such a content, permeabilization of cell membranes was still possible, and precipitation of Cas9 was avoided when the solution was prepared and used within a few minutes. However, a decrease in the efficiency of delivery of large payloads such as mRNA occurred at ethanol concentrations below 25%, and therefore it was preferable to maintain a 25% ethanol concentration and either avoid or reduce precipitation of Cas9.

[0204] B. Effect of ethanol concentration and Cas9 concentration in the delivery solution on in vitro editing efficiency In vitro editing tests were performed to investigate the effect of Cas9 precipitation on the editing efficiency of gene editing tools. Several solutions containing different amounts of Cas9 for different ethanol contents were made, as summarized in Table 9. RNP complexes were made by adding all the reagents (Cas9 and gRNA) and the components of the delivery solution (S buffer, MGW) except ethanol. After 10 minutes of incubation and before spraying, ethanol was added. The ratio Cas9 / gRNA was 1:1 molar ratio. Cas9 was added to gRNA (384b, 5.3 μM, 223.7 ng / μl stock solution) in a 1:1 molar ratio and incubated for 15 minutes, then S buffer and MGW were added and the mixture was incubated for 10 minutes. Finally, ethanol was added just before spraying.

[0205] Table 9. Cas9 concentrations for RNP complexes made in delivery solutions containing different amounts of ethanol. TIFF2025010591000033.tif79128

[0206] Immediately after adding the ethanol, 10 μl of the solution was sprayed into an empty well of a 48-well plate. Then, 2 μl of the sprayed solution was transferred to a test tube suitable for a thermocycler and used for IVE testing as reported in Table 10 to evaluate the functionality of the complex. The components kept in ice were added to a reaction tube suitable for a thermocycler, also kept in ice. The tube was then transferred to a thermocycler preheated to 30° C. The following program was run: 30 minutes at 30℃ 15 minutes at 65°C -Keep at 4℃ The digested samples were then analyzed by gel electrophoresis using a 1.5% gel.

[0207] Table 10. Component volumes per reaction for IVE of Cas9 / gRNA complexes. TIFF2025010591000034.tif55128

[0208] As shown in Figure 25, all DNA targets were edited to some extent. Samples 1-5 showed no difference, suggesting that the amount of Cas9 protein used was in large excess to that required for editing, and therefore, despite precipitation, the Cas9 remaining in solution was sufficient to edit the target. The effect of ethanol was only evident at the lowest Cas9 concentration. In sample 7 (50 ng / μl Cas9, 20% ethanol), the edited band was weaker and the signal of uncut DNA was stronger. This indicated that ethanol (the highest concentration of ethanol) inducing precipitation of the lowest concentration of Cas9 deprived the system of functional Cas9 available for editing. At higher concentrations, the protein remaining in solution was likely sufficient to cleave the target DNA.

[0209] C. Use of excipients to prevent Cas9 precipitation i) Effect of high salt concentration on Cas9 precipitation Cas9 requires a relatively high salt concentration to stay in solution, with most important publications using KCl at a concentration of approximately 200-250 mM. In an attempt to prevent precipitation occurring after the addition of ethanol, Cas9 or RNP solutions were made in a delivery solution in which water was replaced by 0.5 M KCl or 1 M KCl. As previously described, all components except ethanol were added, then the solution was incubated at room temperature for 15 min, and finally ethanol was added. In all cases, substantial precipitation of the protein or complex was observed.

[0210] ii) Effect of glycerol on Cas9 precipitation Glycerol is known to increase the solubility of proteins. In this experiment, 5, 20 or 20% glycerol were added to the delivery solution formulation. After 15 minutes of incubation at room temperature, ethanol was again added to the rest of the components. In the sample containing Cas9, only the addition of ethanol still resulted in precipitation, but no large aggregates formed. Conversely, for RNPs in the delivery solution containing 20% ​​glycerol, no precipitation was observed even after the addition of ethanol (Figure 25). For RNPs in the delivery solutions containing 5% and 10% glycerol, it was difficult to establish whether a precipitate was formed, since the visible particles looked like a background stain present on some glass slides. Indeed, the typical large clusters of proteins did not appear (Figures 26A-26C). However, cell damage was observed when the glycerol-containing solution was sprayed on the cells. Thus, glycerol was not a suitable excipient to use in this situation.

[0211] iii) Effect of NDSB-201 (a non-surfactant sulfobetaine, a zwitterionic compound) on Cas9 precipitation NDSB-201 is known to improve protein solubility. Cas9 alone or with RNP was made into delivery solutions containing 0.05 (D'Astolfo DS et al., Cell 161(3): 674-90), 0.2 and 0.5M NDSB. Again, all components except ethanol were added together, the solution was incubated for 15 minutes, and then ethanol was added. When ethanol was added, precipitates were formed for all samples containing only Cas9. No precipitation was observed for RNP (ribonucleoprotein, a complex of Cas9 and guide RNA) when 0.5M NDSB was used (Figures 26A-26C). For RNP with 0.05 and 0.2M NDSB, little or no precipitation was observed in both cases (difficult to distinguish due to background noise).

[0212] iv) Effect of other excipients on Cas9 precipitation Considering the above results, we designed a series of experiments to evaluate the ability of known protectants / chaperones / excipients to "protect" Cas9 protein from ethanol-induced precipitation. Heat shock proteins (HSPs) are essential for the correct folding and maturation of a wide variety of client proteins, and for protecting proteins from stress-induced unfolding and aggregation (Morimoto, RI, 2008: Genes & Development, v. 22, p. 1427-1438; Richter, K et al. 2010: MolCell, v. 40, p. 253-266). Small HSPs (sHSPs) are the major "holding" chaperones that prevent irreversible aggregation of proteins by holding unfolded proteins in a conformation suitable for subsequent refolding (Eyles, SJ, and LM Gierasch, 2010: PNAS, v. 107, p. 2727-2728; Stengel, F et al, 2010: PNAS, v. 107, p. 2007-2012). α-Crystallins are important members of the small heat shock protein family and are critical structural components of the lens (Reddy, et al 2006: Iubmb Life, v. 58, p. 632-641).

[0213] The following stock concentrations of components were used: Cas9 (10 μg / μl) (IDT, 1074182), Cas9 digestion buffer (Sureguide kit, Agilent), gRNA for HPRT (IDT) (21.8 μM), D-sorbitol (Sigma, S3889), D-mannitol (Sigma, M4125), α-crystallin from bovine ocular lens (Sigma, C4163), NDSB (Sigma, 82804), gelatin A (Sigma, G1890), gelatin B (Sigma, G9391), glycine (Sigma, G7126), proline (Sigma, P5607), Tween-20 (Sigma, P9416), L-histidine (Sigma, H8000), myo-inositol (Sigma, 15125), and trehalose (Sigma, 90210).

[0214] Additives were selected based on current literature on protein stabilization and prevention of aggregation. The final list of selected additives is shown in Table 11. Additives were tested to see if they could preserve the functionality of Cas-9 nuclease in the presence of ethanol. Cas9 stock solution was diluted with nuclease-free H2O to obtain a 1 μg / μl solution. Cas9 and sgRNA were added at a molar ratio of 1:3. They were then incubated at room temperature for 10 minutes to allow the formation of Cas9:sgRNA ribonucleoproteins (RNPs). Additives were added to the concentrations shown in Table 12. Finally, ethanol was added and the solution was incubated at room temperature for 2 minutes. The solution was then added directly to the in vitro editing reaction as summarized in Table 13. Samples were mixed and incubated in a thermocycler with the following program according to the manufacturer's specifications: 30°C for 30 minutes, 65°C for 15 minutes, and maintained at 4°C. Samples were placed on ice and 4 μl of 6× loading dye (NEB, reduced) was added. Samples were loaded onto a 2% agarose gel stained with Sybrsafe according to the manufacturer's instructions and run at 100V for 1 hour.

[0215] The Genetools program (Syngene) was used to measure the editing efficiency of Cas9:gRNA after exposure to various delivery solutions with or without additives. The intensity of band 2 (edited HPRT) relative to band 1 was obtained and expressed as a percentage. All values ​​were normalized to the ethanol-free sample.

[0216] To test the effect of these protective agents on gene editing in cells, Cas9 RNP was delivered into cells together with some of the above protective agents by a delivery platform technique. Bone marrow-derived mesenchymal stem cells (MSCs) were cultured at 9 × 10 3 Cells were seeded at a density of 10000 cells / well and cultured overnight at 37 °C. The next morning, 5 μg of Cas9 and guide RNA (HPRT; 1:6 ratio) were complexed and added to delivery S buffer containing α-crystallin (220 μM), NDSB (0.5 M) or sorbitol (final 4%) instead of sucrose. Ethanol (25% v / v) was then added and delivered to the cells by spraying. The process was repeated after 4 h, and the cells were then cultured for 72 h, after which editing was analyzed by droplet digital PCR.

[0217] Table 11. Additives used in this study TIFF2025010591000035.tif220160

[0218] Table 12. Components of the in vitro editing reaction. Final concentration of Cas9 was 75 ng / μl. TIFF2025010591000036.tif39128

[0219] Table 13. Components of the in vitro editing reaction. Final concentration of Cas9 was 75 ng / μl. TIFF2025010591000037.tif41128

[0220] In vitro editing experiments showed that additives could preserve the function of Cas9 nuclease in the presence of ethanol (Figure 28 and Figures 19A-29B). In particular, the addition of α-crystallin and sorbitol improved the nuclease activity of Cas-9:gRNA after exposure to the delivery solution. The in vitro editing efficiency of Cas-9:gRNA improved, on average, from 49% in the control without additives to 98% efficiency in the sorbitol sample and 103% efficiency in the α-crystallin sample, respectively. To improve CRISPR-Cas9 editing of hprt in MSCs, α-crystallin and NDSB were added to the delivery solution (Figure 30). The addition of α-crystallin at a final concentration of 220 μM increased the editing of hprt from 11.09% (control without additives) to 16.34%. The addition of NDSB to the delivery solution had no effect on hprt editing in MSCs. Alpha-crystallin is also a better additive than sorbitol in maximizing hprt editing in U2OS cells (Figure 31).

[0221] Ethanol is known to displace water at protein sites and alter protein structure (Dwyer, 1999 Biopolymers 49(7): 635-45). This may result in exposure of hydrophobic cores that may lead to protein aggregation. Recently, a study by Ferns et al. (Ferns, 2012: Neurochemical Research, v. 37, p. 244-252) found that ethanol-induced aggregation of GAPDH was prevented by α-crystallin. Thus, α-crystallin may prevent the aggregation of Cas-9.

[0222] Example 5: Optimization of delivery protocols to achieve higher editing efficiency The ability of the delivery platform technique to affect editing in adherent cells is affected by the size of the target cell monolayer, the concentration of Cas9, the ratio of protein to guide RNA and the number of times the cells are treated with this technique. There may be an optimal ratio of Cas9 protein to guide RNA, which may be both cell-specific and gene target-specific.

[0223] Materials used included U2OS cells (osteosarcoma cell line - ECACC), bone marrow derived mesenchymal stem cells (MSC; Lonza), Jurkat E6.1 cells (T cell line - ECACC), DMEM low glucose (Sigma-Aldrich), FBS (Sigma), RPMI-1640 (Life Technologies), Trypsin / EDTA (Gibco), Cas9 nuclease (IDT-DNA), crRNA (IDT-DNA), trRNA (IDT-DNA), Delivery S buffer, α-crystallin (Sigma), ethanol, Hex-labeled drop-off probe (IDT-DNA), FAM-labeled reference probe (IDT-DNA), and ddPCR Supermix (BioRad).

[0224] cell culture 5 x 10 for 24-well plate 4 3 x 10 for 48-well plate 4 and 1 x 10 for 96-well plates 4 U2OS cells were seeded and cultured overnight at 37 °C. The following morning, 5 μg of Cas9 (HPRT guide; 1:3 ratio) was added to the delivery S buffer, along with α-crystallin (220 μM) and ethanol (25% v / v). This was then delivered to the cells in the form of a spray. The cells were allowed to recover for 4 hours before being sprayed again with the same payload. The cells were then cultured for 72 hours before being harvested and analyzed. In a separate set of experiments, bone marrow-derived mesenchymal stem cells (MSCs) were sprayed at a density of 9 × 10 3 The cells were seeded at 100 cells / well in a 96-well plate and cultured overnight at 37° C. The following morning, Cas9 5rig was added to the delivery S buffer, and α-crystallin (220 riM) and ethanol (25% v / v) were also added.

[0225] In the first experiment, the effect of MgCl2 addition was examined. Cas9 RNP (5rig; HPRT targeting 1:6) was delivered to MSC cells with or without 2.5 mM MgCl2. Cells were allowed to recover for 4 hours before the process was repeated, and then cells were cultured for 72 hours before editing analysis. In the next experiment, different amounts of guide were also added (crRNA:trRNA was at a molar ratio of 1:1), thereby achieving molar ratios of protein to guide RNA of 1:1, 1:3, and 1:6 in a final volume of 10 ril. The guides used in these experiments targeted sites within the HPRT gene and sequences adopted from the literature (Liang et al., 2015 J Biotechnol 208L44-53). The medium was removed from the cells and the payload in the delivery solution was added to the spray nozzle. After spraying the cells and incubating the cells for 2 minutes, 150 μl of 0.5× PBS (stop solution) was added. The cells were incubated for an additional 30 seconds, the stop solution was removed, and 150 μl of regular medium was added. The cells were cultured for 72 hours before being harvested for editing analysis.

[0226] In the second experiment, the amount of Cas9 was varied from 0.1 μg to 24 μg at a protein:guide ratio of 1:1. The payload was delivered and the cells were treated as described above. In the third experiment, the cells were repeatedly processed. 5 μg of Cas9 was delivered to the cells as described above at a guide ratio of 1:6. The cells were then trypsinized and reseeded in the same wells and then cultured overnight. The following morning, the cells were processed again under the same conditions. The cells were left in medium for 4 hours before being processed a third time under the same conditions. The cells were again cultured overnight. The following morning, the cells were processed a fourth time under the same conditions and left for 4 hours before being trypsinized, seeded into wells of a 48-well plate and cultured for 72 hours. The cells were then harvested and prepared for gene editing analysis by droplet digital PCR and drop-off assay.

[0227] Edit Analysis The presence of edits was analyzed using droplet digital PCR (ddPCR), a method for performing digital PCR based on water-oil emulsion droplet technology. The sample was divided into 20,000 droplets, and PCR amplification of template molecules was performed in each individual droplet. In this method, this technique was used to detect the presence of edits in the HPRT gene due to Cas9 RNP action on the genome. The assay was set up to specifically address non-homologous end joining repair of double-strand breaks in DNA. The forward and reverse primers generated an amplicon of approximately 150 bp. Two internal probes were used: one probe that acts as a reference probe and should bind to all amplified DNA strands, and a second probe that is located across the Cas9 cut site and is bound if editing does not occur, but cannot be bound if editing does occur.

[0228] In this experiment, DNA was extracted from samples using the MagNA Pure Compact System. ddPCR components were prepared according to the manufacturer's guidelines. Components were transferred to a DG8 cartridge and droplets were generated using a QX200 droplet generator. Droplets were then transferred to a 96-well PCR plate and sealed. PCR was performed and the droplets were analyzed using a QX200 droplet reader.

[0229] i) Effect of cell monolayer size on editing efficiency Editing efficiency increased when the diameter of the cell monolayer was reduced by seeding cells into smaller wells, thereby better matching the diameter of the monolayer to the diameter of the spray (Figure 32).

[0230] U2OS cells were seeded in either 24, 48, or 96 well plates and cultured overnight. The next day, two doses of Cas9 RNP (5 μg; HPRT gRNA at 1:6 ratio) were delivered by the delivery platform technique and cells were cultured for an additional 72 h before harvesting. Editing efficiency was analyzed by ddPCR, showing that cells seeded in the smallest wells produced the greatest editing efficiency.

[0231] ii) The effect of increasing Cas9 concentrations on editing efficiency The percentage of editing achieved by delivering Cas9 at a 1:1 ratio with guide RNA increased with increasing concentrations of Cas9, reaching a plateau at approximately 5 μg Cas9 (Figure 33).

[0232] MSCs were seeded in 96-well plates and cultured overnight. The next day, various concentrations of Cas9 RNP (0.1, 0.5, 1.0, 5.0, 10.0, 15.0, 24.0 μg) were delivered by the delivery platform technique at a 1:1 molar ratio with HPRT guide RNA and cells were cultured for another 72 h before harvesting. Editing efficiency was analyzed by ddPCR, which showed that editing efficiency increased with increasing amounts of Cas9 protein, reaching a plateau at approximately 5 μg Cas9.

[0233] iii) Effect of Cas9:gRNA ratio on editing efficiency Delivering 5ug of Cas9 together with guide RNA at various ratios (1:1, 1:3 or 1:6) did not alter the editing efficiency in MSC cells (Figure 34).

[0234] MSCs were seeded in 96-well plates and cultured overnight. The next day, Cas9 RNP (5 μg) was delivered with HPRT guide RNA at either 1:1, 1:3 or 1:6 molar ratios by the delivery platform technique and cells were cultured for an additional 72 hours before harvesting. Editing efficiency was analyzed by ddPCR, which showed that with these cells and specific target and guide RNAs, there was no difference in editing efficiency at the protein:guide RNA ratios tested.

[0235] iv) Effect of the number of doses of delivered RNP on editing efficiency Increasing the number of times the cells underwent the delivery step (5 μg Cas9, ratio 1:6) increased the percentage of editing detected in the HPRT gene (Figure 35).

[0236] MSCs were seeded in 96-well plates and cultured overnight. Over the next 3 days, 1, 2, 3 or 4 doses of Cas9 RNP (5 μg; HPRT gRNA at 1:6 ratio) were delivered by the delivery platform technique, and after the final dose was delivered, cells were cultured for an additional 72 hours before harvesting. Editing efficiency was analyzed by ddPCR and showed that it increased with the number of doses of Cas9 RNP that cells received. *p<0.05 by Student's t-test for independent means.

[0237] As revealed herein, platform delivery techniques can deliver a series of molecules to cells while maintaining cell viability and functionality. This property allows for a multiple administration regime of the same or different molecules at different times (Figure 35). This is different from electroporation, which cannot achieve multiple administrations due to adverse effects on cells that result in much lower viability. Therefore, a potential way out for delivering a plasmid form of Cas9 is to allow a period of time for this protein to be expressed by cells, and then deliver a guide RNA that will affect editing when combined.

[0238] v) Effect of MgCl2 on editing efficiency Based on previous in vitro editing studies, it was observed that the addition of MgCl2 was necessary to affect editing. In this study, the impact of adding it to cells together with Cas9 RNP was evaluated. It was demonstrated that the addition of MgCl2 did not improve editing efficiency, and subsequent studies did not include the addition of MgCl2 (Figure 25). MSC cells were seeded in 96-well plates and Cas9 RNP (5ug; HPRT 1:6 ratio) was delivered by the delivery platform technique in the presence or absence of MgCl2 (2.5mM).

[0239] Example 6: Comparison with electroporation Comparison with electroporation, the industry standard for protein delivery, was evaluated for delivery efficiency, viability and functionality.

[0240] A. Comparing editing efficiency: delivery platforms with electroporation In MSCs where Cas9 was delivered by the delivery technique provided herein, editing efficiency was greater compared to electroporation (Figure 37).

[0241] MSCs were seeded in 96-well plates and cultured overnight. Over the next 3 days, 4 doses of Cas9 RNP (5 μg; HPRT gRNA at 1:6 ratio) were delivered by the delivery platform technique, and after the final dose was delivered, cells were cultured for an additional 72 hours before harvesting. For comparison with electroporation, MSCs were prepared in buffer and processed according to the manufacturer's instructions. The same amount of Cas9 and guide was delivered per delivery platform-treated cell. Cells were harvested and editing efficiency was analyzed by ddPCR. Successful editing was observed when Cas9 was delivered by either method, but the efficiency was significantly greater in cells where Cas9 RNP was delivered by the delivery platform technique. *p<0.05 by Student's t-test for independent means.

[0242] B. Comparison of cell viability following RNP delivery: delivery platforms compared to electroporation Cell viability was similar between cells edited with Cas9 RNP delivered by either the delivery platform technique or electroporation (Figure 38). 24 hours after delivery of the final round of Cas9 RNP, cells were analyzed for viability by EBAO counting. No significant difference in viability was observed between these two groups.

[0243] C. Comparing Cellular Functionality Following Delivery of RNPs: Delivery Platforms Compared to Electroporation The functionality of MSCs is not affected by the delivery platform technique (Figure 39). Three representative images of differentiated MSCs from an untreated group (untreated), a group of Cas9 RNPs delivered by the delivery platform technique (delivery platform), and a group of Cas9 RNPs delivered by electroporation (electroporation) were examined. One day after delivery of Cas9 RNPs (5rig; HPRT gRNA at a ratio of 1:6), 1x10 cells were cultured at 1x10 cells / mL. 5The cells were seeded in culture medium in 6-well plates and cultured for 5 days or until the cells were 90-100% confluent. The medium was then removed and 2 ml of adipogenic differentiation medium was added to the cells. The medium was changed every 3 days until differentiation was observed (usually 2-3 weeks). Oil droplets (white) could be observed within the cells, indicating differentiation. There was no difference between untreated and delivery platform-treated cells. However, differentiation seemed to be inhibited in cells treated by electroporation. Both adherent and suspension cells, and both primary cells and cell lines were evaluated for editing following delivery of Cas9 RNP by the delivery platform technique.

[0244] Example 7: Editing efficiency in adherent and suspension cell lines and primary cells MSCs and U2OS cells were seeded in 96-well plates and cultured overnight. Over the next 3 days, 4 doses of Cas9 RNP (5rig; HPRT gRNA at 1:6 ratio) were delivered by the delivery platform technique and the cells were cultured for an additional 72 hours. For suspension cells, 1.5 × 10 6 Isolated human CD3+ T cells or Jurkat cells were seeded in 96-well filter plates (1.2 rim). Cas9 RNP (5rig; CXCR4 gRNA at 1:6 ratio) was delivered by the delivery platform technique and cells were cultured for an additional 72 hours. Cells were harvested and editing efficiency was analyzed by ddPCR. Successful editing was observed in both cell lines and primary human cells (Figure 40).

[0245] Example 8: Editing efficiency using Cas9 mRNA The ability to affect editing in cells after delivery of Cas9 mRNA was evaluated. Gene editing was detected in cells in which Cas9 mRNA and guide RNA were delivered in the same payload (Figure 41). MSCs were seeded in 96-well plates and cultured overnight. The next day, Cas9 mRNA (4 μg) and guide RNA (equivalent to a 1:6 ratio; HPRT gRNA) were delivered by the delivery technique and cultured for another 72 hours before harvesting the cells. Editing efficiency was analyzed by ddPCR and editing was detected in these cells.

[0246] Vector-free delivery of gene-editing proteins to cells and tissues The Crispr Cas9 system has rapidly become prominent in the field of genome editing. Crispr Cas9 recognizes specific sequences in the genome with the help of guide RNA and induces double-strand breaks in DNA. Cells can repair this break using an error-prone non-homologous end joining process to edit genes. The ability of the delivery platform technology provided herein to deliver gene editing tools to cells and affect editing was evaluated.

[0247] To evaluate whether the tools (recombinant Cas9 protein and commercially available guide RNA) induced cleavage in DNA targets, an in vitro editing assay was established based on the Agilent SureGuideCas9 Programmable Nuclease Kit. The effect of the delivery platform delivery solution on the function of Cas9 and guide RNA was also evaluated using this non-limiting assay. It was found that the ability of Cas9 to edit was not counteracted by the exemplary delivery solution. Furthermore, the activity of Cas9 was preserved when the Cas9 protein was added to the delivery solution, sprayed into the well, collected, and functionally evaluated in an in vitro editing test to repeat the complete delivery process. To confirm the uptake of Cas9 into target cells after delivery, the Cas9 protein was labeled with the fluorescent tag Alexa-488 maleimide via the thiol group of the cysteine ​​amino acid (two cysteine ​​residues per protein molecule). The successful labeling of the protein allowed for the intracellular visualization of the protein.

[0248] Cas9 is a bacterial protein that does not naturally occur in mammalian cells. Cas9 has a tendency to precipitate out of solution under certain conditions, which affects its function. For most cell types, optimal delivery of cargo to cells using the platform delivery technique was achieved at ethanol concentrations up to 25% (v / v), and it was observed that Cas9 was prone to precipitation when in contact with levels of ethanol above 15%. To reduce ethanol-induced precipitation of Cas9, a series of excipients were evaluated after delivery to cells using in vitro editing assays. While some excipients, such as glycerol and non-detergent sulfobetaine (NDSB), prevented ethanol-induced precipitation, these excipients caused some degree of cell damage when included in spray delivery experiments. However, the addition of the heat shock protein α-crystallin to the delivery solution appeared to promote the editing ability of Cas9 in the in vitro editing assay and was also compatible with delivery to cells. When Cas9 RNP was delivered to cells in the presence of α-crystallin, effective genome editing of the cells was detected by droplet digital PCR. By adding this excipient to delivery solution, the protocol for Cas9 RNP genome editing via delivery platform technology has been optimized in both adherent and suspended cells, as well as in primary cells and cell lines.As an example, this optimization has led to gene editing in 50% of primary human MSCs.Furthermore, it has been demonstrated that the level of editing via delivery platform technology in MSCs is greater than that achieved by electroporation, and the viability and functionality of edited MSCs are superior with the delivery technology provided herein compared to electroporation.

[0249] Other Aspects All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are incorporated by reference. All other published references, documents, manuscripts, accession numbers, and scientific literature cited herein are incorporated by reference.

[0250] The subject matter described herein may be embodied in a system, an apparatus, a method, and / or an article depending on the desired configuration. The embodiments shown in the above description do not represent all embodiments consistent with the subject matter described herein. Instead, they are merely some examples consistent with aspects related to the described subject matter. Although a few variations have been described in detail above, other modifications or additions are possible. In particular, in addition to the features and / or variations shown herein, further features and / or variations may be provided. For example, the above embodiments may be directed to various combinations and subcombinations of the disclosed features and / or combinations and subcombinations of some further features disclosed above. In addition, the logic flow shown in the accompanying drawings and / or described herein does not necessarily require the particular order shown, or sequential order, to achieve the desired results. Other embodiments may be within the scope of the following claims.

[0251] Sequence information SEQUENCE LISTING <110> Avectas Limited <120> Vector-Free Delivery of Gene Editing Proteins and Compositions to Cells and Tissues <150> US 62,273,284 <151> 2015-12-30 <160> 30 <170> PatentIn version 3.5 <210> 1 <211> 1368 <212> PRT <213> Streptococcus pyogenes serotype M1 <400> 1 Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915,920,925 Lys Tyr Asp Ser Arg Met Asn Thr Lys Tyr Asp 930,935,940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965,970,975 Glu Ile Asn Asn Tyr His Ala His Asp Ala Tyr Leu Asn Ala Val 980,985,990 Val Gly Thr Ala Leu Ile Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu With Arg Lys Arg Pro Leu Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Light Light Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 <210> 2 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> GGSGPPKKKRKV <400> 2 Gly Gly Ser Gly Pro Pro Lys Lys Lys Arg Lys Val 1 5 10 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Human influenza hemagglutinin (HA) epitope tag <400> 3 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5 <210> 4 <211> 1389 <212> PRT <213> Artificial Sequence <220> <223> Cas9 followed by Nuclear Localization Signal followed by HA Tag <400> 4 Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975 Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990 Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Light Light Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 Gly Gly Ser Gly Pro Pro Lys Lys Lys Arg Lys Val Tyr Pro Tyr 1370 1375 1380 Asp Val Pro Asp Tyr Ala 1385 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> nuclear localization signal <400> 5 Pro Lys Lys Lys Arg Lys Val 1 5 <210> 6 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> nuclear localization signal <400> 6 Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys 1 5 10 15 <210> 7 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> nuclear localization signal <220> <221> misc_feature <222> (3)..(12) <223> Xaa can be any naturally occurring amino acid <400> 7 Lys Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys Lys Lys Lys 1 5 10 15 <210> 8 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> nuclear localization signal <220> <221> misc_feature <222> (3)..(3) <223> Xaa can be any naturally occurring amino acid <400> 8 Lys Lys Xaa Lys 1 <210> 9 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> nuclear localization signal <220> <221> misc_feature <222> (3)..(3) <223> Xaa can be any naturally occurring amino acid <400> 9 Lys Arg Xaa Lys 1 <210> 10 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> nuclear localization signal <220> <221> misc_feature <222> (3)..(3) <223> Xaa can be any naturally occurring amino acid <400> 10 Lys Lys Xaa Arg 1 <210> 11 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> nuclear localization signal <220> <221> misc_feature <222> (3)..(3) <223> Xaa can be any naturally occurring amino acid <400> 11 Lys Arg Xaa Arg 1 <210> 12 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> nuclear localization signal <400> 12 Ala Val Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys 1 5 10 15 Lys Lys Leu Asp 20 <210> 13 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> nuclear localization signal <400> 13 Met Ser Arg Arg Arg Lys Ala Asn Pro Thr Lys Leu Ser Glu Asn Ala 1 5 10 15 Lys Lys Leu Ala Lys Glu Val Glu Asn 20 25 <210> 14 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> nuclear localization signal <400> 14 Pro Ala Ala Lys Arg Val Lys Leu Asp 1 5 <210> 15 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> nuclear localization signal <400> 15 Pro Pro Lys Lys Lys Arg Lys Val 1 5 <210> 16 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> nuclear localization signal <400> 16 Lys Leu Lys Ile Lys Arg Pro Val Lys 1 5 <210> 17 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> nuclear localization signal <400> 17 Met Leu Ser Leu Arg Gln Ser Ile Arg Phe Phe Lys Pro Ala Thr Arg 1 5 10 15 Thr Leu Cys Ser Ser Arg Tyr Leu Leu 20 25 <210> 18 <211> 1368 <212> PRT <213> Streptococcus pyogenes <400> 18 Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Asp Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Ala Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Arg Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Arg Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Ser Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Ala Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Glu Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Asp Ile Leu Lys Glu Tyr Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Ile Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Lys Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915,920,925 Lys Tyr Asp Ser Arg Met Asn Thr Lys Tyr Asp 930,935,940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965,970,975 Glu Ile Asn Asn Tyr His Ala His Asp Ala Tyr Leu Asn Ala Val 980,985,990 Val Gly Thr Ala Leu Ile Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu With Arg Lys Arg Pro Leu Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Light Light Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Arg Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Phe Gly Ala Pro Ala Ala 1310 1315 1320 Phe Ile Tyr Phe Asp Thr Thr Ile Gly Arg Asn Arg Tyr Lys Ser 1325 1330 1335 Ile Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 <210> 19 <211> 1388 <212> PRT <213> Streptococcus thermophilus <400> 19 Met Thr Lys Pro Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Thr Thr Asp Asn Tyr Lys Val Pro Ser Lys Lys Met 20 25 30 Lys Val Leu Gly Asn Thr Ser Lys Lys Tyr Ile Lys Lys Asn Leu Leu 35 40 45 Gly Val Leu Leu Phe Asp Ser Gly Ile Thr Ala Glu Gly Arg Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Arg Asn Arg Ile Leu 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Thr Glu Met Ala Thr Leu Asp Asp Ala 85 90 95 Phe Phe Gln Arg Leu Asp Asp Ser Phe Leu Val Pro Asp Asp Lys Arg 100 105 110 Asp Ser Lys Tyr Pro Ile Phe Gly Asn Leu Val Glu Glu Lys Ala Tyr 115 120 125 His Asp Glu Phe Pro Thr Ile Tyr His Leu Arg Lys Tyr Leu Ala Asp 130 135 140 Ser Thr Lys Lys Ala Asp Leu Arg Leu Val Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Tyr Arg Gly His Phe Leu Ile Glu Gly Glu Phe Asn Ser 165 170 175 Lys Asn Asn With Gln Lys Asn Phe Gln Asp Phe Leu Asp Thr Tyr 180 185 190 Asn Ala Ile Phe Glu Ser Asp Leu Ser Leu Glu Asn Ser Gln Leu 195 200 205 Glu Glu Ile Val Lys Asp Lys Ile Ser Lys Leu Glu Lys Lys Asp Arg 210 215 220 Ile Leu Lys Leu Phe Pro Gly Glu Lys Asn Ser Gly Ile Phe Ser Glu 225 230 235 240 Phe Leu Lys Leu Ile Val Gly Asn Gln Ala Asp Phe Arg Lys Cys Phe 245 250 255 Asn Leu Asp Glu Lys Ala Ser Leu His Phe Server Lys Glu Ser Tyr Asp 260 265 270 Glu Asp Leu Glu Thr Leu Leu Gly Tyr Ile Gly Asp Tyr Ser Asp 275 280 285 Val Phe Leu Lys Ala Lys Lys Leu Tyr Asp Ala Ile Leu Leu Ser Gly 290,295,300 Phe Leu Thr Val Thr Asp Asn Glu Thr Glu Ala Pro Leu Ser Ser Ala 305 310 315 320 Met Ile Lys Arg Tyr Asn Glu His Lys Glu Asp Leu Ala Leu Leu Lys 325 330 335 Glu Tyr and Arg Asn Served Leu Lys Thr Tyr Asn Glu Val Phe Lys 340 345 350 Asp Asp Thr Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Lys Thr Asn 355 360 365 Gln Glu Asp Phe Tyr Val Tyr Leu Lys Lys Leu Leu Ala Glu Phe Glu 370 375 380 Gly Ala Asp Tyr Phe Leu Glu Lys Ile Asp Arg Glu Asp Phe Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro Tyr Gln Ile His Leu 405 410 415 Gln Glu Met Arg Ala Ile Leu Asp Lys Gln Ala Lys Phe Tyr Pro Phe 420 425 430 Leu Ala Lys Asn Lys Glu Arg Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Asp Phe Ala Trp 450 455 460 Served with Arg Lys Arg Asn Glu Lys Thr Ile Pro Trp Asn Phe Glu Asp 465 470 475 480 Val Ile Asp Lys Glu Ser Ser Ala Glu Ala Phe Ile Asn Arg Met Thr 485,490,495 Ser Phe Asp Leu Tyr Leu Pro Glu Glu Lys Val Leu Pro Lys Ser 500 505 510 Leu Tyr Glu Thr Phe Asn Val Tyr Asn Glu Leu Thr Lys Val Arg 515,520,525 Phe Ile Ala Glu Ser Met Arg Asp Tyr Gln Phe Leu Asp Ser Met Lys Gln 530 535 540 Lys Asp Ile Val Arg Leu Tyr Phe Lys Asp Lys Arg Lys Val Thr 545 550 555 560 Asp Lys Asp Ile Ile Glu Tyr Leu His Area Ile Ile Tyr Gly Tyr Asp Gly 565,570,575 Glu Leu Lys Gly and Glu Lys Gln Phe Asn Ser Leu Ser Thr 580,585,590 Tyr His Asp Leu Asn Ile Ile Asn Asp Lys Glu Phe Leu Asp Asp 595,600,605 Ser Ser Asn Glu Ala Ile Ile Glu Glu Ile Ile His Thr Leu Thr Ile 610 615 620 Phe Glu Asp Arg Glu Met Ile Lys Gln Arg Leu Ser Lys Phe Glu Asn 625 630 635 640 Ile Phe Asp Lys Ser Val Leu Lys Lys Leu Ser Arg Arg His Tyr Thr 645 650 655 Gly Trp Gly Lys Leu Ser Ala Lys Leu Ile Asn Gly Ile Arg Asp Glu 660 665 670 Lys Ser Gly Asn Thr Ile Leu Asp Tyr Leu Ile Asp Asp Gly Ile Ser 675 680 685 Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ala Leu Ser Phe Lys 690 695 700 Lys Lys Ile Gln Lys Ala Gln Ile Ile Gly Asp Glu Asp Lys Gly Asn 705 710 715 720 Ile Lys Glu Val Val Lys Ser Leu Pro Gly Ser Pro Ala Ile Lys Lys 725 730 735 Gly Ile Leu Gln Ser Ile Lys Ile Val Asp Glu Leu Val Lys Val Met 740 745 750 Gly Gly Arg Lys Pro Glu Ser Ile Val Val Glu Met Ala Arg Glu Asn 755 760 765 Gln Tyr Thr Asn Gln Gly Lys Ser Asn Ser Gln Gln Arg Leu Lys Arg 770 775 780 Leu Glu Lys Ser Leu Lys Glu Leu Gly Ser Lys Ile Leu Lys Glu Asn 785 790 795 800 Ile Pro Ala Lys Leu Ser Lys Ile Asp Asn Asn Ala Leu Gln Asn Asp 805 810 815 Arg Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Lys Asp Met Tyr Thr Gly 820 825 830 Asp Asp Leu Asp Ile Asp Arg Leu Ser Asn Tyr Asp Ile Asp His Ile 835 840 845 Ile Pro Gln Ala Phe Leu Lys Asp Asn Ser Ile Asp Asn Lys Val Leu 850 855 860 Val Ser Ser Ala Ser Asn Arg Gly Lys Ser Asp Asp Val Pro Ser Leu 865 870 875 880 Glu Val Val Lys Lys Arg Lys Thr Phe Trp Tyr Gln Leu Leu Lys Ser 885 890 895 Lys Leu Ile Ser Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg 900 905 910 Gly Gly Leu Ser Pro Glu Asp Lys Ala Gly Phe Ile Gln Arg Gln Leu 915,920,925 Glu Thr Arg Gln With Thr Lys His Val Has Arg With Asp Glu 930,935,940 Lys Phe Asn Asn Lys Lys Asp Glu Asn Asn Arg Ala Val Arg Thr Val 945 950 955 960 Lys Ile Thr Leu Lys Ser Thr Leu Val Ser Gln Phe Arg Lys Asp 965,970,975 Phe Glu Leu Tyr Lys Val Arg Glu Ile Asn Asp Phe His His Ala His 980,985,990 Asp Ala Tyr Leu Asn Ala Val Val Ser Ala Leu Lys Lys Tyr 995 1000 1005 Pro Lys Leu Glu Pro Glu Phe Val Tyr Gly Asp Tyr Pro Lys Tyr 1010 1015 1020 Asn Ser Phe Arg Glu Arg Lys Ser Ala Thr Glu Lys Val Tyr Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Ile Phe Lys Lys Ser Ile Ser Leu Ala 1040 1045 1050 Asp Gly Arg Val Is Glu Arg Pro Leu Is Glu Val Asn Glu Glu 1055 1060 1065 Thr Gly Glu Ser Val Trp Asn Lys Glu Ser Asp Leu Ala Thr Val 1070 1075 1080 Arg Arg Val Leu Ser Tyr Pro Gln Val Asn Val Val Lys Lys Val 1085 1090 1095 Glu Glu Gln Asn His Gly Leu Asp Arg Gly Lys Pro Lys Gly Leu 1100 1105 1110 Phe Asn Ala Asn Leu Ser Ser Lys Pro Lys Pro Asn Ser Asn Glu 1115 1120 1125 Asn Leu Val Gly Ala Lys Glu Tyr Leu Asp Pro Lys Lys Tyr Gly 1130 1135 1140 Gly Tyr Ala Gly Ile Ser Asn Ser Phe Thr Val Leu Val Lys Gly 1145 1150 1155 Thr Ile Glu Lys Gly Ala Lys Lys Lys Ile Thr Asn Val Leu Glu 1160 1165 1170 Phe Gln Gly Ile Ser Ile Leu Asp Arg Ile Asn Tyr Arg Lys Asp 1175 1180 1185 Lys Leu Asn Phe Leu Leu Glu Lys Gly Tyr Lys Asp Ile Glu Leu 1190 1195 1200 Ile Ile Glu Leu Pro Lys Tyr Ser Leu Phe Glu Leu Ser Asp Gly 1205 1210 1215 Ser Arg Arg Met Leu Ala Ser Ile Leu Ser Thr Asn Asn Lys Arg 1220 1225 1230 Gly Glu Ile His Lys Gly Asn Gln Ile Phe Leu Ser Gln Lys Phe 1235 1240 1245 Val Lys Leu Leu Tyr His Ala Lys Arg Ile Ser Asn Thr Ile Asn 1250 1255 1260 Glu Asn His Arg Lys Tyr Val Glu Asn His Lys Lys Glu Phe Glu 1265 1270 1275 Glu Leu Phe Tyr Tyr Ile Leu Glu Phe Asn Glu Asn Tyr Val Gly 1280 1285 1290 Ala Lys Lys Asn Gly Lys Leu Leu Asn Ser Ala Phe Gln Ser Trp 1295 1300 1305 Gln Asn His Ser Ile Asp Glu Leu Cys Ser Ser Phe Ile Gly Pro 1310 1315 1320 Thr Gly Ser Glu Arg Lys Gly Leu Phe Glu Leu Thr Ser Arg Gly 1325 1330 1335 Ser Ala Ala Asp Phe Glu Phe Leu Gly Val Lys Ile Pro Arg Tyr 1340 1345 1350 Arg Asp Tyr Thr Pro Ser Ser Leu Leu Lys Asp Ala Thr Leu Ile 1355 1360 1365 His Gln Ser Val Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ala 1370 1375 1380 Lys Leu Gly Glu Gly 1385 <210> 20 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Cas9 target site <220> <221> N <222> (9)..(20) <223> N is A, G, T, or C; and X can be anything <220> <221> misc_feature <222> (21)..(21) <223> Xaa can be any naturally occurring amino acid <400> 20 Met Met Met Met Met Met Met Met Asn Asn Asn Asn Asn Asn Asn Asn 1 5 10 15 Asn Asn Asn Asn Xaa Gly Gly 20 <210> 21 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> Cas9 target site <220> <221> N <222> (8)..(20) <223> N is A, G, T, or C; and X can be anything <220> <221> misc_feature <222> (21)..(22) <223> Xaa can be any naturally occurring amino acid <400> 21 Met Met Met Met Met Met Met Met Asn Asn Asn Asn Asn Asn Asn Asn 1 5 10 15 Asn Asn Asn Asn Xaa Xaa Ala Gly Ala Ala Trp 20 25 <210> 22 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Cas9 target site <220> <221> N <222> (9)..(20) <223> N is A, G, T, or C; and X can be anything <220> <221> misc_feature <222> (21)..(21) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (24)..(24) <223> Xaa can be any naturally occurring amino acid <400> 22 Met Met Met Met Met Met Met Met Asn Asn Asn Asn Asn Asn Asn Asn 1 5 10 15 Asn Asn Asn Asn Xaa Gly Gly Xaa Gly 20 25 <210> 23 <211> 104 <212> PRT <213> Homo sapiens <400> 23 Met Asp Val Thr Ile Gln His Pro Trp Phe Lys Arg Thr Leu Gly Pro 1 5 10 15 Phe Tyr Pro Ser Arg Leu Phe Asp Gln Phe Phe Gly Glu Gly Leu Phe 20 25 30 Glu Tyr Asp Leu Leu Pro Phe Leu Ser Ser Thr Ile Ser Pro Tyr Tyr 35 40 45 Arg Gln Ser Leu Phe Arg Thr Val Leu Asp Ser Gly Ile Ser Glu Val 50 55 60 Arg Ser Asp Arg Asp Lys Phe Val Ile Phe Leu Asp Val Lys His Phe 65 70 75 80 Ser Pro Glu Asp Leu Thr Val Lys Val Gln Asp Asp Phe Val Glu Ile 85 90 95 His Gly Lys His Asn Glu Arg Gln 100 <210> 24 <211> 175 <212> PRT <213> Homo sapiens <400> 24 Met Asp Ile Ala Ile His His Pro Trp Ile His Arg Pro Phe Phe Pro 1 5 10 15 Phe His Ser Pro Ser Arg Leu Phe Asp Gln Phe Phe Gly Glu His Leu 20 25 30 Leu Glu Ser Asp Leu Phe Pro Thr Ser Thr Ser Leu Ser Pro Phe Tyr 35 40 45 Leu Arg Pro Pro Ser Phe Leu Arg Ala Pro Ser Trp Phe Asp Thr Gly 50 55 60 Leu Ser Glu Met Arg Leu Glu Lys Asp Arg Phe Ser Val Asn Leu Asp 65 70 75 80 Val Lys His Phe Ser Pro Glu Glu Leu Lys Val Lys Val Leu Gly Asp 85 90 95 Val Ile Glu Val His Gly Lys His Glu Glu Arg Gln Asp Glu His Gly 100 105 110 Phe Ile Ser Arg Glu Phe His Arg Lys Tyr Arg Ile Pro Ala Asp Val 115 120 125 Asp Pro Leu Thr Ile Thr Ser Ser Leu Ser Ser Asp Gly Val Leu Thr 130 135 140 Val Asn Gly Pro Arg Lys Gln Val Ser Gly Pro Glu Arg Thr Ile Pro 145 150 155 160 Ile Thr Arg Glu Glu Lys Pro Ala Val Thr Ala Ala Pro Lys Lys 165 170 175 <210> 25 <211> 173 <212> PRT <213> Artificial Sequence <220> <223> Alpha-crystallin A chain CRYAA <400> 25 Met Asp Ile Ala Ile Gln His Pro Trp Phe Lys Arg Thr Leu Gly Pro 1 5 10 15 Phe Tyr Pro Ser Arg Leu Phe Asp Gln Phe Phe Gly Glu Gly Leu Phe 20 25 30 Glu Tyr Asp Leu Leu Pro Phe Leu Ser Ser Thr Ile Ser Pro Tyr Tyr 35 40 45 Arg Gln Ser Leu Phe Arg Thr Val Leu Asp Ser Gly Ile Ser Glu Val 50 55 60 Arg Ser Asp Arg Asp Lys Phe Val Ile Phe Leu Asp Val Lys His Phe 65 70 75 80 Ser Pro Glu Asp Leu Thr Val Lys Val Gln Glu Asp Phe Val Glu Ile 85 90 95 His Gly Lys His Asn Glu Arg Gln Asp Asp His Gly Tyr Ile Ser Arg 100 105 110 Glu Phe His Arg Arg Tyr Arg Leu Pro Ser Asn Val Asp Gln Ser Ala 115 120 125 Leu Ser Cys Ser Leu Ser Ala Asp Gly Met Leu Thr Phe Ser Gly Pro 130 135 140 Lys Ile Pro Ser Gly Val Asp Ala Gly His Ser Glu Arg Ala Ile Pro 145 150 155 160 Val Ser Arg Glu Glu Lys Pro Ser Ser Ala Pro Ser Ser 165 170 <210> 26 <211> 175 <212> PRT <213> Artificial Sequence <220> <223> Alpha-crystalline B chain CRYAB <400> 26 Met Asp Ile Ala Ile His His Pro Trp Ile Arg Arg Pro Phe Phe Pro 1 5 10 15 Phe His Ser Pro Ser Arg Leu Phe Asp Gln Phe Phe Gly Glu His Leu 20 25 30 Leu Glu Ser Asp Leu Phe Pro Ala Ser Thr Ser Leu Ser Pro Phe Tyr 35 40 45 Leu Arg Pro Pro Ser Phe Leu Arg Ala Pro Ser Trp Ile Asp Thr Gly 50 55 60 Leu Ser Glu Met Arg Leu Glu Lys Asp Arg Phe Ser Val Asn Leu Asp 65 70 75 80 Val Lys His Phe Ser Pro Glu Glu Leu Lys Val Lys Val Leu Gly Asp 85 90 95 Val Ile Glu Val His Gly Lys His Glu Glu Arg Gln Asp Glu His Gly 100 105 110 Phe Ile Ser Arg Glu Phe His Arg Lys Tyr Arg Ile Pro Ala Asp Val 115 120 125 Asp Pro Leu Ala Ile Thr Ser Ser Leu Ser Ser Asp Gly Val Leu Thr 130 135 140 Val Asn Gly Pro Arg Lys Gln Ala Ser Gly Pro Glu Arg Thr Ile Pro 145 150 155 160 Ile Thr Arg Glu Glu Lys Pro Ala Val Thr Ala Ala Pro Lys Lys 165 170 175 <210> 27 <211> 173 <212> PRT <213> Artificial Sequence <220> <223> Alpha crystallin A <400> 27 Met Asp Val Thr Ile Gln His Pro Trp Phe Lys Arg Thr Leu Gly Pro 1 5 10 15 Phe Tyr Pro Ser Arg Leu Phe Asp Gln Phe Phe Gly Glu Gly Leu Phe 20 25 30 Glu Tyr Asp Leu Leu Pro Phe Leu Ser Ser Thr Ile Ser Pro Tyr Tyr 35 40 45 Arg Gln Ser Leu Phe Arg Thr Val Leu Asp Ser Gly Ile Ser Glu Val 50 55 60 Arg Ser Asp Arg Asp Lys Phe Val Ile Phe Leu Asp Val Lys His Phe 65 70 75 80 Ser Pro Glu Asp Leu Thr Val Lys Val Gln Asp Asp Phe Val Glu Ile 85 90 95 His Gly Lys His Asn Glu Arg Gln Asp Asp His Gly Tyr Ile Ser Arg 100 105 110 Glu Phe His Arg Arg Tyr Arg Leu Pro Ser Asn Val Asp Gln Ser Ala 115 120 125 Leu Ser Cys Ser Leu Ser Ala Asp Gly Met Leu Thr Phe Cys Gly Pro 130 135 140 Lys Ile Gln Thr Gly Leu Asp Ala Thr His Ala Glu Arg Ala Ile Pro 145 150 155 160 Val Ser Arg Glu Glu Lys Pro Thr Ser Ala Pro Ser Ser 165 170 <210> 28 <211> 175 <212> PRT <213> Artificial Sequence <220> <223> Alpha crystallin B <400> 28 Met Asp Ile Ala Ile His His Pro Trp Ile Arg Arg Pro Phe Phe Pro 1 5 10 15 Phe His Ser Pro Ser Arg Leu Phe Asp Gln Phe Phe Gly Glu His Leu 20 25 30 Leu Glu Ser Asp Leu Phe Pro Thr Ser Thr Ser Leu Ser Pro Phe Tyr 35 40 45 Leu Arg Pro Pro Ser Phe Leu Arg Ala Pro Ser Trp Phe Asp Thr Gly 50 55 60 Leu Ser Glu Met Arg Leu Glu Lys Asp Arg Phe Ser Val Asn Leu Asp 65 70 75 80 Val Lys His Phe Ser Pro Glu Glu Leu Lys Val Lys Val Leu Gly Asp 85 90 95 Val Ile Glu Val His Gly Lys His Glu Glu Arg Gln Asp Glu His Gly 100 105 110 Phe Ile Ser Arg Glu Phe His Arg Lys Tyr Arg Ile Pro Ala Asp Val 115 120 125 Asp Pro Leu Thr Ile Thr Ser Ser Leu Ser Ser Asp Gly Val Leu Thr 130 135 140 Val Asn Gly Pro Arg Lys Gln Val Ser Gly Pro Glu Arg Thr Ile Pro 145 150 155 160 Ile Thr Arg Glu Glu Lys Pro Ala Val Thr Ala Ala Pro Lys Lys 165 170 175 <210> 29 <211> 173 <212> PRT <213> Artificial Sequence <220> <223> Alpha A <400> 29 Met Asp Val Thr Ile Gln His Pro Trp Phe Lys Arg Thr Leu Gly Pro 1 5 10 15 Phe Tyr Pro Ser Arg Leu Phe Asp Gln Phe Phe Gly Glu Gly Leu Phe 20 25 30 Glu Tyr Asp Leu Leu Pro Phe Leu Ser Ser Thr Ile Ser Pro Tyr Tyr 35 40 45 Arg Gln Ser Leu Phe Arg Thr Val Leu Asp Ser Gly Ile Ser Glu Val 50 55 60 Arg Ser Asp Arg Asp Lys Phe Val Ile Phe Leu Asp Val Lys His Phe 65 70 75 80 Ser Pro Glu Asp Leu Thr Val Lys Val Gln Asp Asp Phe Val Glu Ile 85 90 95 His Gly Lys His Asn Glu Arg Gln Asp Asp His Gly Tyr Ile Ser Arg 100 105 110 Glu Phe His Arg Arg Tyr Arg Leu Pro Ser Asn Val Asp Gln Ser Ala 115 120 125 Leu Ser Cys Ser Leu Ser Ala Asp Gly Met Leu Thr Phe Cys Gly Pro 130 135 140 Lys Ile Gln Thr Gly Leu Asp Ala Thr His Ala Glu Arg Ala Ile Pro 145 150 155 160 Val Ser Arg Glu Glu Lys Pro Thr Ser Ala Pro Ser Ser 165 170 <210> 30 <211> 175 <212> PRT <213> Artificial Sequence <220> <223> Alpha crystalline B <400> 30 Met Asp Ile Ala Ile His His Pro Trp Ile Arg Arg Pro Phe Phe Pro 1 5 10 15 Phe His Ser Pro Ser Arg Leu Phe Asp Gln Phe Phe Gly Glu His Leu 20 25 30 Leu Glu Ser Asp Leu Phe Pro Thr Ser Thr Ser Leu Ser Pro Phe Tyr 35 40 45 Leu Arg Pro Pro Ser Phe Leu Arg Ala Pro Ser Trp Phe Asp Thr Gly 50 55 60 Leu Ser Glu Met Arg Leu Glu Lys Asp Arg Phe Ser Val Asn Leu Asp 65 70 75 80 Val Lys His Phe Ser Pro Glu Glu Leu Lys Val Lys Val Leu Gly Asp 85 90 95 Val Ile Glu Val His Gly Lys His Glu Glu Arg Gln Asp Glu His Gly 100 105 110 Phe Ile Ser Arg Glu Phe His Arg Lys Tyr Arg Ile Pro Ala Asp Val 115 120 125 Asp Pro Leu Thr Ile Thr Ser Ser Leu Ser Ser Asp Gly Val Leu Thr 130 135 140 Val Asn Gly Pro Arg Lys Gln Val Ser Gly Pro Glu Arg Thr Ile Pro 145 150 155 160 Ile Thr Arg Glu Glu Lys Pro Ala Val Thr Ala Ala Pro Lys Lys 165 170 175

Claims

1. 1. A method for delivering a gene editing composition across the plasma membrane of a cell, comprising: Providing a cell population; and contacting the cell population with a volume of an aqueous solution comprising the gene editing composition and an alcohol at a concentration of at least about 2%, The volume is a function of (i) the exposed surface area of ​​the cell population or (ii) the number of cells in the cell population. The method.

2. The aqueous solution is (a) About 5% to about 50%; (b) at least about 5% and less than about 50, 40, 30, 20, 19, 18, 17, 16, or 15%; or (c) at least about 2.5, 3, 3.5, 4, 4.5, or 5 percent The method of claim 1, comprising:

3. 1. The gene editing composition, (a) a gene editing protein; (b) an RNA molecule; and / or (c) Ribonucleoproteins (RNPs) The method of claim 1, comprising one or more of:

4. The method of claim 3, wherein the gene editing composition comprises a gene editing protein, and the gene editing protein is a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a MegaTAL, a Cas protein, a Cre recombinase, a Hin recombinase, or an Flp recombinase.

5. 4. The method of claim 3, wherein the gene editing composition comprises an RNA molecule, the RNA molecule comprising an sgRNA, a crRNA, and / or a tracrRNA.

6. 4. The method of claim 3, wherein the gene editing composition comprises an RNP, the RNP comprising a Cas protein, and a gRNA or crRNA and a tracrRNA.

7. The gene editing composition is administered to the cell population or its progeny cells, (a) about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, 48, 60, 72, 0.5 to 2, 0.5 to 6, 6 to 12, or 0.5 to 72 hours after contacting the cell population with the aqueous solution; or (b) less than about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, 48, 60, 72, 0.5 to 2, 0.5 to 6, 6 to 12, or 0.5 to 72 hours after contacting the cell population with the aqueous solution; The method of claim 1, wherein the detection is possible.

8. The method of claim 4, wherein the genome of the cells in the cell population or progeny cells comprises at least one site-specific recombination site for Cre recombinase, Hin recombinase, or FLP recombinase.

9. 5. The method of claim 4, wherein the cells or progeny cells in the cell population comprise sgRNA, crRNA, and / or tracrRNA.

10. 10. The method of claim 9, wherein the cell population or its progeny expresses sgRNA, crRNA, and / or tracrRNA.

11. The method of claim 5, wherein the cells in the cell population or their progeny express a Cas protein.

12. 5. The method of claim 4, wherein the Cas protein is a Cas9 protein or a variant thereof.

13. 1. The gene editing composition, (a) a first sgRNA molecule and a second sgRNA molecule, wherein the nucleic acid sequence of the first sgRNA molecule differs from the nucleic acid sequence of the second sgRNA molecule; (b) a first RNP comprising a first sgRNA and a second RNP comprising a second sgRNA, wherein the nucleic acid sequence of the first sgRNA molecule differs from the nucleic acid sequence of the second sgRNA molecule; (c) a first crRNA molecule and a second crRNA molecule, wherein the nucleic acid sequence of the first crRNA molecule differs from the nucleic acid sequence of the second crRNA molecule; (d) a first crRNA molecule and a second crRNA molecule, wherein the nucleic acid sequence of the first crRNA molecule differs from the nucleic acid sequence of the second crRNA molecule, further comprising a tracrRNA molecule; or (e) a first RNP comprising a first crRNA and a tracrRNA and a second RNP comprising a second crRNA and a tracrRNA, wherein the nucleic acid sequence of the first crRNA molecule differs from the nucleic acid sequence of the second crRNA molecule; 4. The method of claim 3, comprising:

14. 14. The method of claim 13, wherein the first and second sgRNA or the first and second crRNA molecules together comprise nucleic acid sequences complementary to a target sequence adjacent to a gene, exon, intron, extrachromosomal sequence, or genomic nucleic acid sequence, and the gene, exon, intron, extrachromosomal sequence, or genomic nucleic acid sequence is about 1, 2, 3, 4, 5, 6, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1-100 kilobases in length, or is at least about 1, 2, 3, 4, 5, 6, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1-100 kilobases in length.

15. 15. The method of Claim 14, wherein the first and second sgRNA or the first or second crRNA molecule are complementary to sequences flanking an extrachromosomal sequence located within an expression vector.

16. The method of claim 1, wherein the gene editing composition comprises at least one gene editing protein and at least one nucleic acid, and the gene editing protein and the nucleic acid are not bound or complexed to each other.

17. 2. The method of claim 1, wherein at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99%, 1-99%, or more of the cell population or progeny cells become genetically modified after contact with the aqueous solution.

18. 2. The method of claim 1, wherein at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99%, 1-99%, or more of the cell population or progeny cells are viable after contact with the aqueous solution.

19. The method of claim 1, wherein the gene editing composition induces single-stranded or double-stranded breaks in DNA in the cell.

20. The method of claim 1, wherein the gene editing composition further comprises a repair template polynucleotide.

21. The repair template is (a) a first flanking region comprising nucleotides of a sequence complementary to about 40 to about 90 base pairs on one side of the single-stranded or double-stranded break, and a second flanking region comprising nucleotides of a sequence complementary to about 40 to about 90 base pairs on the other side of the single-stranded or double-stranded break; or (b) a first flanking region comprising nucleotides of a sequence complementary to at least about 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 90 base pairs on one side of the single-stranded or double-stranded break, and a second flanking region comprising nucleotides of a sequence complementary to at least about 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 90 base pairs on the other side of the single-stranded or double-stranded break.

21. The method of claim 20, comprising:

22. The method of claim 1 , wherein the volume of aqueous solution is delivered to the cell population in the form of a spray of aqueous particles.

23. (a) The volume of the aqueous particle spray is 6.0 × 10 -7 Microliters / cell ~ 7.4 x 10 -4 microliters per cell; and / or (b) the aqueous particles have discrete volume units in the size range of 10 nm to 100 μm, 30 to 100 μm, 50 to 80 μm, or 30 to 50 μm in diameter; 23. The method of claim 22.

24. 22. The method of claim 21, wherein the spray comprises colloidal particles or subparticles comprising diameters between 10 nm and 100 μm.

25. The volume is 2.6×10 -9 Microliters / square micrometer exposed surface area ~ 1.1 x 10 -6 13. The method of claim 1, in microliters per square micrometer exposed surface area.

26. 10. The method of claim 1, wherein the step of contacting the cell population with a volume of an aqueous solution is performed with a gas that propels the aqueous solution to form a spray.

27. 10. The method of claim 1, wherein the cell population is contacted with the aqueous solution for 0.1 to 10 minutes and then a second volume of buffer or culture medium is added to submerge or suspend the cell population.

28. 23. The method of claim 22, wherein the buffer or culture medium comprises phosphate buffered saline (PBS).

29. 10. The method of claim 1, wherein the aqueous solution comprises an ethanol concentration of about 2 to about 20%.

30. The aqueous solution is (a) about 75 to about 98% H 2 O; (b) about 2-20% ethanol; (c) about 6 to about 91 mM sucrose; (d) about 2 to about 35 mM potassium chloride; (e) about 2 to about 35 mM ammonium acetate; (f) about 1 to about 14 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES); and / or (g) about 0.1 to about 10 mM magnesium salt; and / or (h) about 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 mM magnesium salt The method of claim 1, comprising one or more of:

31. 31. The method of claim 30, wherein the aqueous solution comprises about 0.1 to about 10 mM magnesium chloride.

32. 31. The method of claim 30, wherein the aqueous solution comprises at least about 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 mM magnesium chloride.

33. The method of claim 1, wherein the cell population comprises primary cells or immortalized cells.

34. 34. The method of claim 33, wherein the cell population comprises at least one of primary or immortalized mesenchymal stem cells, lung cells, neuronal cells, fibroblasts, human umbilical vein (HUVEC) cells, and human embryonic kidney (HEK) cells, primary or immortalized hematopoietic stem cells (HSC), T cells, natural killer (NK) cells, cytokine-induced killer (CIK) cells, human umbilical cord blood CD34+ cells, B cells, A549 cells, Beas-2B cells, Jurkat cells, or peripheral blood mononuclear cells (PBMCs).

35. 1. A composition for delivering a payload across the plasma membrane of a cell, the composition comprising an aqueous solution comprising a payload and an alcohol at a concentration of at least 2%, the payload comprising a gene editing composition.

36. 36. The composition of claim 35, further comprising a magnesium salt.

37. 36. The composition of claim 35, comprising about 0.1 to about 10 mM of a magnesium salt.

38. 38. The composition of claim 37, comprising at least about 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 mM magnesium chloride.

39. 36. The composition of claim 35, wherein the alcohol comprises methanol, ethanol, isopropyl alcohol, butanol, or benzyl alcohol.

40. 40. The composition of claim 39, comprising about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50% ethanol.

41. 36. The composition of claim 35, having a pH of about 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.

0.

42. 1. A composition for delivering a payload across the plasma membrane of a cell, the composition comprising an aqueous solution comprising the payload, an alcohol at a concentration of at least 2%, less than about 46 mM salt lacking magnesium ions, less than about 121 mM sugar, less than 19 mM buffering agent, and about 0.1 to about 10 mM magnesium salt, wherein the payload comprises a gene editing composition.

43. 43. The composition of claim 42, wherein the buffering agent is a weak acid or a weak base effective to adjust or maintain the pH of the aqueous solution at about pH 6.5, 6.75, 7, 7.25, 7.5, 7.75, 8, 8.25, or 8.

5.

44. 43. The composition of claim 42, wherein the alcohol comprises methanol, ethanol, isopropyl alcohol, butanol, or benzyl alcohol.

45. 43. The composition of claim 42, wherein the salt lacking magnesium ions comprises Na ions or K ions.

46. Salts that lack magnesium ions are NaCl, KCl, and Na 2 HPO 4 , C 2 H 3 O 2 NH 4 , or K.H. 2 PO 4 43. The composition of claim 42, comprising:

47. 43. The composition of claim 42, wherein the sugar comprises sucrose.

48. 43. The composition of claim 42, wherein the buffer comprises 4-2-(hydroxyethyl)-1-piperazineethanesulfonic acid, 3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]-2-hydroxypropane-1-sulfonic acid, 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid, 3-(N-morpholino)propanesulfonic acid, or piperazine-N,N'-bis(2-ethanesulfonic acid).

49. About 32 mM sucrose, about 12 mM potassium chloride, about 12 mM ammonium acetate, about 5 mM HEPES, about 15% ethanol, and about 2.5 mM MgCl 2 43. The composition of claim 42, comprising:

50. 32 mM sucrose, 12 mM potassium chloride, 12 mM ammonium acetate, 5 mM HEPES, 2.5 mM MgCl 2 , 15% ethanol, and 85% H 2 50. The composition of claim 49, comprising O.

51. 50. The composition of claim 49, wherein the pH of the solution is adjusted to about pH 7.4 before adding ethanol.

52. 1. An apparatus for delivering a gene editing composition across the plasma membrane of a cell, comprising: An air pressure generating device for generating pressurized gas; a sprayer operatively connected to said air pressure generating device; a reservoir configured to contain a volume of an aqueous solution comprising the gene editing composition and a concentration of alcohol greater than 2 percent; a valve between the air pressure generator and the nebulizer switchable between a closed position to prevent actuation of the nebulizer by pressurized gas and an open position to allow actuation of the nebulizer by pressurized gas to generate colloidal droplets from the aqueous solution, the valve having a switching speed of less than 250 milliseconds. Including, the sprayer is oriented to contact the cell population with the aqueous solution, the volume being a function of (i) the exposed surface area of ​​the cell population or (ii) the number of cells in the cell population; The said instrument.

53. The method of claim 1 , wherein the aqueous solution further comprises an additive comprising a heat shock protein or a sugar alcohol.

54. 54. The method of claim 53, wherein the heat shock protein comprises alpha-crystallin.

55. 55. The method of claim 54, wherein the alpha-crystallin is present at a concentration of about 1 to about 500 μM.

56. 54. The method of claim 53, wherein the sugar alcohol comprises sorbitol at a concentration of about 1 to about 10% weight / volume.

57. 2. The method of claim 1, wherein the aqueous solution comprises an alcohol concentration of less than about 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5%.

58. The method of claim 1 , wherein the cells comprise stem cells.

59. 59. The method of claim 58, wherein the stem cells comprise mesenchymal stem cells (MSCs).

60. 60. The method of claim 59, wherein the MSCs comprise bone marrow-derived MSCs.

61. The aqueous solution may contain glycerol or MgCl 2 The method of claim 1, wherein the method does not include

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