Gene editing by microfluidic delivery

Abstract

This application relates to gene editing via microfluidic delivery. Gene editing can be performed by introducing gene-editing components into cells through mechanical cell disruption. Related devices, systems, techniques, and articles are also described. The methods and systems of this invention address the problem of intracellular delivery of gene-editing components and gene-editing complexes to target cells. The results described herein indicate that delivery of gene-editing components, such as proteins, ribonucleic acid (RNA), and deoxyribonucleic acid (DNA), via mechanical disruption of the cell membrane leads to successful gene editing. Because intracellular delivery of gene-editing materials is currently a challenge, these methods provide a robust mechanism for engineering target cells without the use of potentially harmful viral vectors or electric fields.

Description

[0001] This application is a divisional application of Chinese patent application No. 201680008278.0 entitled "Gene Editing by Microfluidic Delivery". The original application was PCT international application PCT / US2016 / 013113 filed on January 12, 2016, which entered the Chinese national phase on August 1, 2017.

[0002] Related applications

[0003] This application claims priority to U.S. Provisional Application No. 62 / 102,347, filed January 12, 2015, pursuant to Section 119(e) of 35 USC, the entire contents of which are incorporated herein by reference.

[0004] Statement on Federally Funded Research

[0005] This invention was carried out with government support under grant number R01GM101420-01A1 granted by the National Institutes of Health (NIH). The government has certain rights to this invention. Technical Field

[0006] The topic described in this article relates to gene editing achieved by introducing gene-editing components into cells through mechanical cell disruption.

[0007] References to sequence lists

[0008] This application incorporates nucleotide and / or amino acid sequences by reference to a file named "38172-510001WO_Sequence_Listing_ST25.txt", which is 517 kilobytes in size, created on January 12, 2016, in IBM-PC format with an MS-Windows compatible operating system, and is included in a text file filed on January 12, 2016, as part of this application. background

[0009] Genome editing technologies, such as clustered regularly distributed short palindromic repeats (CRISPR) – CRISPR-associated protein 9 (Cas9) and transcription activator-like effector nucleases (TALENs), have shown great potential in their ability to alter the cellular genetic code. Therefore, these technologies can enable new insights into drug discovery and lead to the development of next-generation gene therapies. Gene editing complexes, comprising protein and nucleic acid components (such as deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA)), cannot easily cross cell membranes. Therefore, the delivery of such complexes is a challenge. Summary of the Invention

[0010] The methods and systems of this invention address the problem of intracellular delivery of gene-editing components and gene-editing complexes to target cells. The results described herein indicate that delivery of gene-editing components, such as proteins, ribonucleic acid (RNA), and deoxyribonucleic acid (DNA), via mechanical disruption of the cell membrane leads to successful gene editing. Because intracellular delivery of gene-editing materials is currently challenging, these methods provide a robust mechanism for engineering target cells without the use of potentially harmful viral vectors or electric fields. Furthermore, the scalability and relative simplicity of the methods make them suitable for widespread adoption. This strategy and method are applicable to genome engineering applications in research and therapeutics.

[0011] Therefore, a method for delivering a protein-nucleic acid complex into cells is performed by: providing cells in a suspension; passing the solution through a microfluidic channel including a cell deformable contractile element; passing the cells through the contractile element such that pressure is applied to the cells, resulting in sufficient perturbation to allow the protein-nucleic acid complex to pass through; and incubating the cells in a solution containing the complex for a predetermined time before or after passing the cells through the contractile element. Exemplary protein-nucleic acid complexes contain gene-editing components. For example, a protein-nucleic acid complex contains a Cas protein (such as Cas9 protein) and guide RNA (gRNA) or donor DNA. In other examples, the protein-nucleic acid complex contains a TALEN protein, a zinc finger nuclease (ZFN), a broad range of nucleases, or a Cre recombinase.

[0012] The methods and systems are generally applicable to the cytoplasmic delivery of complexes, such as protein-protein complexes, small molecule + RNA complexes, etc.

[0013] This approach can be used to treat a variety of target cell types. For example, cells include mammalian cells such as immune cells (e.g., T cells) or stem cells such as hematopoietic stem cells.

[0014] Microfluidic systems may include multiple microfluidic channels. Each of the multiple microfluidic channels defines a cavity and is configured to allow cells suspended in a buffer solution to pass through the cavity. In some embodiments, the microfluidic channel includes one or more cell deformable contractile elements. In some embodiments, the diameter of the contractile element varies with the cell diameter. Therefore, a large number of microfluidic channels may be present within the microfluidic system of the present invention. For example, the microfluidic system may include multiple microfluidic channels arranged in parallel, such as 2, 5, 10, 20, 40, 45, 50, 75, 100, 500, 1000 or more.

[0015] Microfluidic systems with multiple parallel microfluidic channels allow for high-throughput delivery of payloads to cells. Many cells can pass through each parallel channel one by one. It should be understood that, depending on the context, the term "cell" in this document can refer to more than one cell.

[0016] The diameter of the shrinkage element is selected based on the size of the cell type to be processed. In some embodiments, the cells can be compressed primarily by a fluid flow. In some embodiments, the diameter is smaller than the cell diameter. For example, the diameter of the shrinkage element can be substantially or about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 20-99% of the cell diameter. For example, the shrinkage element is substantially 20-99% of the cell diameter, or for example, the diameter of the shrinkage element is substantially 60% of the cell diameter. Non-limiting examples of shrinkage element diameters include substantially or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 2-10 μm, or 10-20 μm. Different lengths of the shrinkage element are also possible. Non-limiting examples of shrinkage length include substantially or about 10, 15, 20, 24, 30, 40, 50, 60, 70, 80, 90, 100, 10-40, 10-50, 10-60 or 10-100 μm.

[0017] The subject matter described herein offers numerous technical advantages over methods that deliver components (or nucleic acids encoding the components) of complex fragments. Most gene editing systems require complex formation to occur intracellularly, which can be an inefficient process. The advantages of delivering RNA and Cas (such as Cas9) in complex form (or other protein / nucleic acid gene editing complex assemblies) include better efficiency and specificity compared to other methods. By avoiding the need for mRNA or DNA manipulation to express Cas proteins (such as Cas9), the time spent by the cell exposed to the protein can be reduced, thus reducing the chance of off-target effects. Delivering the editing component into the cell in a complex form also eliminates / minimizes the risk of Cas (such as Cas9) complexing with other RNA strands in the cell and cleaving at incorrect sites. For example, individual RNA can be detected by intracellular and extracellular Toll-like receptors (TLRs) and pattern recognition receptors, thereby promoting interferon responses or other antiviral pathways. The complex form does not interact with these pathways, thus avoiding undesirable side effects.

[0018] By complexing in vitro prior to delivery to cells, the complexation reaction of Cas (e.g., Cas9) and gRNA can be precisely controlled, ensuring optimal function, whereas complexes formed in the cytosol may be less effective. For example, cytosolic delivery of the complex ensures the simultaneous interaction of Cas (e.g., Cas9) and gRNA with the target DNA. As described herein, complexes formed in vitro and delivered to cells are fully functional and ready to act upon entering the cytoplasm of the target cell.

[0019] The method described in this article is relevant to any protein + RNA / DNA-based system that guides nucleases because the delivery process is independent of the precise size and composition of the complex, and because the formation of the complex of the edited material occurs in vitro and is controlled under its optimal conditions.

[0020] Embodiments of the present invention may also provide one or more of the following features: Cell deformation includes causing cell deformation to last substantially or about 1 μs to 10 ms, for example, 10 μs, 50 μs, 100 μs, 500 μs, and 750 μs. Incubation occurs from 0.0001 seconds to 20 minutes or longer, for example, substantially or about 1 second, 30 seconds, 90 seconds, 270 seconds, and 900 seconds.

[0021] The pressure and velocity at which cells pass through the microfluidic channel can also be varied. In some implementations, a pressure of approximately 10–35 psi is used to force the cell-containing solution through the microfluidic channel. The velocity can be adjusted for a variety of reasons, including improving the viability of the treated cells while maintaining high payload delivery. In some implementations, cells pass through the microfluidic channel at speeds of substantially or about 300 mm / s, 400 mm / s, 500 mm / s, 600 mm / s, 700 mm / s, 800 mm / s, 900 mm / s, 100-300 mm / s, 200-700 mm / s, 250-400 mm / s, 1-1000 mm / s, 1 m / s, 2 m / s, 3 m / s, 4 m / s, 5 m / s, 6 m / s, 7 m / s, 8 m / s, 9 m / s, 10 m / s, 0.01-5 m / s, 5-10 m / s, or 0.01-10 m / s. In cases where the cells are multiple cells, substantially or approximately 5, 10, 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 90-95, or 80-99% or more of the cells may be viable after passing through the shrinkage element. In some embodiments, after passing through the shrinkage element, the cells survive for at least approximately 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, 24 hours, or 48 hours.

[0022] In some instances, the apparatus of the present invention includes 2, 10, 20, 25, 45, 50, 75, 100, or more channels. In some embodiments, cells are moved (e.g., pushed) through channels or conduits by applying pressure. In some embodiments, cell actuators may apply pressure. Cell actuators may include, for example, pressure pumps, cylinders, compressors, vacuum pumps, syringes, syringe pumps, peristaltic pumps, manual syringes, pipettes, pistons, capillary actuators, and gravity. As an alternative to channels, cells may be passed through a constriction element in the form of a mesh. In either case, the width of the constriction element through which the cells pass is 20-99% of the width or diameter of the cells to be treated in their unconstricted (i.e., suspended) state. Temperature can affect the absorption of the composition and influence viability.

[0023] In some embodiments, a temperature of 0 to 45°C, such as 0-25°C, is used during cell treatment. In various embodiments, the method is performed at room temperature (e.g., 20°C), physiological temperature (e.g., 39°C), a temperature higher than physiological temperature or lower (e.g., 0.1°C), or a temperature between these exemplary temperatures (e.g., 0.1 to 40°C).

[0024] In some embodiments involving immune cells, treatment of unstimulated T cells, B cells, and / or monocytes is performed at a temperature of 4-8°C, for example, on ice. In another instance, the device is used to treat dendritic cells, activated T cells, and / or activated B cells at a temperature of 20-25°C, for example, at typical ambient room temperature.

[0025] In some embodiments, after controlled damage (e.g., perturbation) to cells via a contraction element, extension, and / or high-shear-rate pulses, the cells are incubated in a delivery solution containing a complex to be introduced into the cells. Controlled damage can be characterized as small (e.g., 200 nm in diameter) perturbations in the cell membrane. The cell recovery period is approximately several minutes to allow the damage induced by the contraction element to be reversed. Delivery periods include 1–10 minutes or longer, such as 15, 20, 30, 60 minutes or longer, with 2–5 minutes being optimal when operating at room temperature.

[0026] In some embodiments of the apparatus and methods described herein, stem cells or progenitor cells, such as induced pluripotent stem cells (iPSCs), are reliably induced to take up the composition into the cells via contractile channels without inducing differentiation. For example, gene-editing compounds are introduced into such cells without complications associated with the method of introducing factors into cells.

[0027] The magnitude and duration of temporary perturbations in the cell membrane can be altered by adjusting various factors, such as the diameter of the cell deforming contraction and the speed at which the cell passes through the contraction. The disclosure regarding the magnitude and duration of perturbations provided herein should not be construed as limiting. A non-limiting description of perturbations and recovery is provided in Sharei et al. (2014) Integr. Biol. 6, 470-475, the entire contents of which are incorporated herein by reference. In some embodiments, the perturbation of the cell membrane may be characterized by a maximum diameter substantially or about 1-20, 1-600, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 600 nm. In various implementations, perturbations of the cell membrane having a maximum diameter of substantially or about 1-20, 1-600, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 600 nm persist on the cell membrane for at least substantially or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 1-10 minutes or longer (11, 13, 15, 18, 20 minutes or longer).

[0028] In various embodiments, the diameter is smaller than the diameter of the cell. For example, the diameter of the shrinkage element can be substantially or about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 20-99% of the cell diameter. Non-limiting examples of shrinkage element diameters include substantially or about 4, 5, 6, 7, 8, 9, 10, 15, 20 (4-10 μm or 10-20 μm). Different lengths of the shrinkage element are also possible. Non-limiting examples of shrinkage element lengths include substantially or about 10, 15, 20, 24, 30, 40, 50, 60, 70, 80, 90, 100 (10-40, 10-50, 10-60, or 10-100 μm).

[0029] Many cells have a diameter between 5 and 20 μm; for example, unstimulated T cells have a diameter of 7-8 μm. For instance, retractor portions with diameters of 4.5, 5, 5.5, 6, or 6.5 μm are used for processing single cells. In another example, the size / diameter of the retractor portion used for processing human oocytes is between 60 μm and 80 μm, although larger and smaller retractors are possible (human oocytes are approximately 100 μm in diameter). In yet another example, embryos (e.g., clusters of 2-3 cells) are treated using retractor diameters between 12 μm and 17 μm. In non-limiting examples involving unstimulated T and B cells, the device includes a length of about 10, 15, 20, 25, 30, or 10-30 μm, a width of about 3, 3.5, 4, or 3-4 μm, a depth of about 15, 20, 25, or 15-25 μm, and / or a contractile element with an angle of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 5-15 degrees. Examples of microfluidic devices that can be used to deliver payloads to immune cells are described in PCT International Patent Application No. PCT / US2015 / 058489, Delivery of Biomolecules to Immune Cells, filed October 30, 2015, the entire contents of which are incorporated herein by reference.

[0030] In addition to single cells, even very large cells (e.g., eggs (approximately 200 μm in diameter)) and cell clusters (e.g., clusters of 2-5 cells, such as embryos containing 2-3 cells) can be processed to take up the target composition. The pore size is adjusted accordingly, i.e., the width of the constrictor is just below the size of the cluster. For example, the width of the channel is 20-99% of the width of the cell cluster.

[0031] Cells or cell clusters are purified / isolated or enriched for the desired cell type. Dendritic cells or other cells used in the purification or enrichment methods are, for example, immune cells such as macrophages, B cells, T cells, or stem cells such as embryonic stem cells or iPS cells. For example, cells are isolated or enriched because they express cell surface markers or other identifying characteristics. Dendritic cells are identified and isolated because they express β-integrin, CD11c, or other identifying cell surface markers. Regarding cells, the term "isolated" means that the cells are substantially free of other cell types or cellular material naturally present with them. For example, a cell sample of a particular tissue type or phenotype is "substantially pure" when it constitutes at least 60% of the cell population. Preferably, the preparation is at least 75% of the cell population, more preferably at least 90%, and most preferably at least 99% or 100%. Purity is measured by any suitable standard method, such as fluorescence-activated cell sorting (FACS).

[0032] Purification and / or isolation of payload compositions such as polynucleotides, peptides, or other agents (e.g., Cas9 and gRNA). Specifically, as used herein, “isolated” or “purified” nucleic acid molecules, polynucleotides, peptides, or proteins are substantially free of other cell material or culture medium when produced by recombinant technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. The purified compound is at least 60% by weight (dry weight) of the target compound. Preferably, the preparation is at least 75% by weight of the target compound, more preferably at least 90%, and most preferably at least 99%. For example, the purified compound is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w / w) of the desired compound by weight. Purity is measured by any suitable standard method, such as analysis by column chromatography, thin-layer chromatography, or high-performance liquid chromatography (HPLC). The purified or isolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) does not contain genes or sequences flanking it in its native 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 by either of the following two nucleic acid sequences, which are flanked by that part of the molecule in the genome of the organism in which the DNA is naturally occurring; (b) nucleic acids incorporated in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) individual molecules, such as cDNA, genomic fragments, fragments produced by polymerase chain reaction (PCR), or restriction fragments; and (d) recombinant nucleotide sequences as part of a heterozygous gene, i.e., a gene encoding a fusion protein. The isolated nucleic acid molecules according to the invention also include synthetically produced molecules and any nucleic acids that have been chemically altered and / or have a modified backbone.

[0033] Complexes are prepared from purified modules or components, such as purified proteins and purified nucleic acids (RNA and / or DNA). Therefore, the ratios of the components can be controlled and adjusted as needed to achieve the desired gene editing results. The method of this invention is particularly suitable for delivering sensitive payloads, such as protein-RNA / DNA complexes, for example, complexes of 40 kDa, 50 kDa, 75 kDa, 100 kDa, and up to 120, 130, 150, 200, 250, and 300 kDa.

[0034] A surfactant (e.g., 0.1-10% w / w) (e.g., poloxamer, animal serum, albumin protein) may be optionally used in the flow buffer. The delivery of molecules into cells is unaffected by the presence of the surfactant; however, a surfactant may be optionally used to reduce device clogging during operation.

[0035] In some aspects, the device is made of silicon, metals (e.g., stainless steel), plastics (e.g., polystyrene), ceramics, or any other material suitable for forming one or more channels or conduits of appropriate size. In other aspects, the device is formed of a material suitable for etching micron-level features and includes one or more channels or conduits through which cells pass. Silicon is particularly suitable because a fully micropatterning method is established with this material, making it easier to fabricate new devices, change designs, etc. Additionally, the stiffness of silicon can provide advantages over more flexible substrates such as polydimethylsiloxane (PDMS), such as higher delivery rates. For example, the device includes 2, 10, 20, 25, 45, 50, 75, 100, or more channels. The device is microfabricated by etching silicon. Cells are moved (e.g., pushed) through the channels or conduits by applying pressure. Cell actuators can apply pressure. Cell actuators can include, for example, pressure pumps, cylinders, compressors, vacuum pumps, syringes, syringe pumps, peristaltic pumps, manual syringes, pipettes, pistons, capillary actuators, and gravity. As an alternative to channels, cells can be passed through constrictive elements in the form of a mesh. In either case, the width of the contraction through which the cell passes is 20-99% of the width or diameter of the cell in its uncontracted (i.e., suspended) state.

[0036] Various embodiments of the present invention can also provide one or more of the following clinical and research capabilities: Quantitative delivery of gene-editing complexes or components thereof to cell models can be achieved to improve screening and dosing studies. This method can be deployed as a high-throughput method for screening protein activity in the cytosol to help identify protein therapeutics or understand disease mechanisms. The apparatus and techniques can be used to deliver gene-editing complexes intracellularly to specific subclasses of circulating blood cells (e.g., lymphocytes) or even whole blood; deliver complexes or components thereof in high-throughput to cells, particularly oocytes and zygotes; induce targeted cell differentiation to generate iPS cells by introducing gene editing (optionally along with genetic material such as donor DNA); deliver DNA and / or recombinases to embryonic stem cells for the development of transgenic or mutant stem cell lines; deliver DNA and / or recombinases to zygotes for the development of transgenic or mutant organisms; dendritic cell (DC) activation; iPS cell generation; generating mutations in normal or diseased cells (e.g., cancer cells) to study the contribution of one or more genes to cell function and / or disease; and stem cell differentiation. Skin cells used in conjunction with plastic surgery are also modified using the devices and methods described herein. The methods for delivering gene-edited proteins disclosed herein can also be used to generate CAR-T cells or genetically modified hematopoietic stem cells (HSCs) for the treatment of genetic and other diseases. In embodiments involving HSCs, a subject may receive autologous, syngeneically, or allogeneically edited HSCs. In various embodiments, the subject's cells may be ablated before the subject receives the gene-edited cells. For example, the subject's bone marrow cells may be ablated by radiation or chemical means before the subject receives the gene-edited HSCs. In some embodiments, genes associated with β-thalassemia or sickle cell anemia are edited using the methods or compositions disclosed herein. Cells treated ex vivo or in vitro (i.e., in vitro from the subject) according to the invention may be subsequently administered to subjects requiring treatment or diagnosis of pathology. In alternative embodiments, in vivo cell treatment is performed.

[0037] In various implementations, the SHP2 gene is edited / mutated to reduce its activity or knocked out or reduced SHP2 expression. In such implementations involving gene editing in T cells, T cells become less responsive to immunosuppressive signals and more active against tumors. In such implementations, T cells may be more responsive to tumor antigens and more effective in treating cancer.

[0038] This topic relates to the rapid and transient delivery of protein-protein and protein-nucleic acid complexes (e.g., gene editing complexes) into cells. The nucleic acid components of the complexes include deoxynucleic acid (DNA), ribonucleic acid (RNA, e.g., mRNA, gRNA), or other double-stranded or single-stranded nucleic acid compounds. For example, delivery of a gene editing complex (e.g., a ribonucleoprotein (RNP)) can achieve faster gene editing than when an expression vector encoding components of the gene editing complex (e.g., Cas protein and gRNA) is delivered into cells. For example, gene editing (e.g., mutation or substitution) in cells can be achieved 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, or 0.1–4 hours faster than in corresponding cells receiving microfluidic or electroporation-mediated delivery of an expression vector encoding components of the gene editing complex.

[0039] It is envisioned that each embodiment disclosed herein is applicable to every other disclosed embodiment. Therefore, all combinations of the various elements described herein are within the scope of this invention.

[0040] It also describes related devices, systems, technologies, and items.

[0041] Details of one or more variations of the subject matter described herein are set forth in the accompanying drawings and the description below. Other features and advantages of the subject matter described herein will become apparent from the description, the accompanying drawings, and the claims. Attached Figure Description

[0042] Figure 1A and 1B This is a schematic diagram of a microfluidic system in which cells are exposed to the delivery material (payload) after passing through a contraction element;

[0043] Figure 2A and 2B It is a schematic diagram illustrating an implementation scheme of a microfluidic system that depicts parameters such as channel depth, width, and length.

[0044] Figure 3A and 3B These are a series of flow cytometry images showing the recombination efficiency of K562 reporter cells delivered to the cytoplasm using the microfluidic device shown in Figure 1-2. Figure 3A ) and bar chart ( Figure 3BFor the reporter used, a frameshifted GFP gene is present in the cell line. To perform the editing, a Cas9 gRNA complex and a donor oligonucleotide are delivered. The complex cleaves near the GFP site, and the oligonucleotide inserts itself into the cleavage site. Successful insertion of the oligonucleotide corrects the gene and results in GFP expression, which is what is seen in this figure. Therefore, in this assay, something is turned on after gene editing, as opposed to being turned off. In the absence of a donor oligonucleotide, where the gene (or, depending on the context, its nucleotides or portions) is missing, the gene is expected to be turned off.

[0045] Figure 4 This is a series of FACS plots showing the expression of the β2-microglobulin component (B2M) of MHC class 1 in four different cell populations obtained using FACS relative to the delivered dextran. Compared to the endocytosis control, delivery of RNP at 90 psi using a 30-4 chip resulted in a 54.4% reduction in B2M expression, while delivery at 90 psi using a 10-4 chip resulted in a 25.2% reduction. B2M expression on the CAS9 control was not significantly different from the endocytosis control. Longer contraction chips resulted in greater delivery of the RNP complex and a larger reduction in B2M expression.

[0046] Figure 5 It is a series of FACS plots showing the dose-dependent reduction in B2M expression as a measure of functional editing under specified conditions, as determined by FACS.

[0047] Figure 6 Showing from Figure 6 First page to Figure 6 The last page contains the FoxP3 genome sequence (SEQ ID NO: 56). Exons belonging to FoxP3 are shown with underlined and highlighted letters. Other exons in this region that do not belong to FoxP3 are shown with un-underlined and unhighlighted letters.

[0048] Figure 7 The amino acid sequence translated from FoxP3 is shown (SEQ ID NO: 57). Alternating exons are shown as underlined and ununderlined. Bold and italicized text indicates residue overlap splicing sites.

[0049] Figure 8 Showing from Figure 8 First page to Figure 8 The last page contains the SHP1 genome sequence (SEQ ID NO: 58). Exons belonging to SHP1 are shown with underlined and highlighted letters. Other exons in this region that do not belong to SHP1 are shown with un-underlined and unhighlighted letters.

[0050] Figure 9The amino acid sequence translated from SHP1 is shown (SEQ ID NO: 59). Alternating exons are shown as underlined and ununderlined. Bold and italic text indicates residue overlap splicing sites.

[0051] Figure 10 Showing from Figure 10 First page to Figure 10 The SHP2 genome sequence (SEQ ID NO:60) is on the last page. Exons belonging to SHP2 are shown underlined and highlighted letters. SEQ ID NO: 60 is also shown below:

[0052]

[0053]

[0054] Figure 11 The amino acid sequence translated from SHP2 is shown (SEQ ID NO: 61). Alternating exons are shown as underlined and ununderlined. Bold and italic text indicates residue overlap splicing sites.

[0055] Similar reference symbols in the various figures indicate similar elements. Detailed Implementation Plan

[0056] Protein / nucleic acid complexes or assemblies are difficult to manipulate due to their fragility and requirements for structural integrity (e.g., three-dimensional conformation). Genome editing technologies, such as clustered regular short palindromic repeats (CRISPR)-Cas9 and transcription activator-like effector nucleases (TALENS), have shown great potential in their ability to alter the cellular genetic code. However, their activity is highly dependent on structural and conformational integrity.

[0057] Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are examples of a class of gene-editing tools. These chimeric nucleases consist of programmable, sequence-specific DNA-binding modules linked to nonspecific DNA cleavage domains. ZFNs and TALENs achieve a wide range of genetic modifications by inducing DNA double-strand breaks that stimulate error-prone non-homologous end joining or homology-guided repair at specific genomic locations (Gaj et al., 2013, Trends Biotechnol. 31(7):397-405; which is incorporated herein by reference).

[0058] Many publications describe the use of viruses, mRNA, and plasmids to encode Cas9 and / or gRNA and / or donor oligonucleotides (where applicable). In contrast to these methods, the methods and systems described herein utilize a different strategy: the delivery of the protein itself in conjunction with the guide RNA complex. This process is fundamentally different because it does not require cellular translation / transcription of anything to enable editing. For these reasons, and due to the short half-life of the protein / guide RNA complex, the methods of this invention result in faster and more efficient editing with fewer off-target effects.

[0059] Other methods include liposome-mediated protein delivery, microinjection, and cell-penetrating peptides (CPPs).

[0060] An example of the use of liposome-mediated protein delivery with GFP fused to Cas-9 has also been observed (Zuris et al., 2015, Nature Biotechnology 33:73-80). Due to charge interactions, GFP is able to complex with conventional liposome transfection agents (e.g., lipofectamine) and appears to mediate gene editing responses. The main advantages of this invention compared to the method are: 1) no fusion protein is required; 2) no liposome transfection agent is required, which can have problems / difficulties related to toxicity, endosome escape, and transfer to primary cells.

[0061] Microinjection-mediated complex delivery is characterized by extremely low throughput and is difficult to implement for most mammalian cell types. The fact that the study was conducted using embryos, cells much larger than fibroblasts or T cells (cells desired as target cells for gene editing), highlights this latter drawback. The method described herein offers significant advantages by achieving high throughput and convertibility with smaller primary cells such as fibroblasts, T cells, and stem cells.

[0062] CPP-based strategies do not involve complexes. An example of such a strategy is described in Ramakrishna et al., 2014, Genome Res. 24(6):1020-7. Individual component CPP-mediated delivery is also associated with drawbacks. Conjugating CPP to guide agents and Cas9 requires additional modifications that may inhibit function and limit scalability. CPP-mediated delivery is known to occur via endocytosis and is inefficient or ineffective in many primary cells, particularly immune cells.

[0063] Composition of target cells and payload

[0064] Any gene can be manipulated using the described gene-editing strategies. Some target genes / proteins are particularly relevant to clinical diseases, and therefore gene editing of these target genes / proteins is useful for therapy. Examples include CC chemokine receptor type 5 (CCR5): prevention of human immunodeficiency virus (HIV) infection; major histocompatibility complex class I (MHC-I): reduction of graft-versus-host disease; differentiation cluster 1 (CD1): reduction of graft-versus-host disease; programmed cell death protein 1 (PD-1), programmed death ligand 1 (PDL-1), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), interferon regulatory factor (IRF) protein family, TLR protein family, and pattern recognition receptor (PR). R): Modulating immunity to enhance or suppress effector / antiviral responses; Forkhead Box P3 (FoxP3): Eliminating Treg-mediated tolerance; Differentiation Cluster 80 (CD80), Differentiation Cluster 86 (CD86), and other co-stimulatory molecules: Promoting the knockout co-stimulatory capacity to enhance tolerance; T cell receptor (TCR), B cell receptor (BCR): Eliminating endogenous TCR or BCR to allow for the desired specific engineering of T cells and B cells; Oncogenes (e.g., Kras, Myc, Tp53): Cancer therapy. In another example, targeting transcription factors is used to alter cell fate, such as deleting FoxP3 to remove Treg-type function. Deletion of nuclear factor κB (NF-κB), t-bet, eomesodermin (Eomes), etc., alters T cell differentiation.

[0065] The protein coding sequence of Forkhead Box P3 (FoxP3) is as follows:

[0066]

[0067] Phosphatase-1 (SHP1), containing the Src homology region 2 domain, is also known as tyrosine protein phosphatase non-receptor type 6 (PTPN6). The protein coding sequence of SHP1 is as follows:

[0068]

[0069] Phosphatase-1 (SHP2), containing the Src homology region 2 domain, is also known as non-receptor tyrosine protein phosphatase type 11 (PTPN11). The protein coding sequence of SHP2 is as follows:

[0070]

[0071]

[0072] Other targets include regions of the genome into which plasmids or donor DNA can be inserted, enabling target cells to express new genes, such as recombinant TCRs, recombinant BCRs, chimeric antigen receptors, fluorescent proteins, and reprogramming factors.

[0073] In some implementations, the genome sequence is edited in the coding region. In other implementations, the genome sequence is edited in the non-coding region.

[0074] In various implementations related to FoxP3, gene regions upstream of FoxP3 can be edited. In such implementations, the region where the transcriptional repressor of FoxP3 can bind is edited. For example, sites approximately 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 0.5–2.5, or 0.5–5 kb upstream of the FoxP3 transcription start site can be edited.

[0075] Treatment subjects

[0076] Aspects of the present invention relate to editing the genome of cells from multiple subjects. In various embodiments, cells are removed from the subjects, a gene-editing complex is received using the methods of this subject, and then the cells are returned to the subjects. For example, mutant cells may be generated during the delivery of gene-editing complexes as described herein. For mutant alleles (genes involved in disease), the mutant cells may be heterozygous or homozygous. In some embodiments, the mutant cells are ineffective for genes involved in disease.

[0077] Cells, such as stem cells derived from bone marrow (e.g., hematopoietic stem cells) or circulating immune cells in whole blood, can be treated using the methods and apparatus described herein. Cells can be genetically modified to reduce the expression of receptors for pathogens (e.g., viral or bacterial pathogens) or toxins (e.g., microbial pathogen toxins). Since gene-edited protein complexes or components thereof can be directly introduced into target cells without expression, transgene delivery is unnecessary. This approach offers significant advantages over conventional gene therapy methods, which suffer from aberrant expression, insertion and silencing, and variable transgene copy number delivery.

[0078] In one implementation, a gene-editing complex targeting the CC chemokine receptor type 5 (CCR5) gene is introduced into the blood (e.g., CD4+ T cells) or bone marrow cells (e.g., hematopoietic stem cells) of a subject infected with human immunodeficiency virus (HIV). The gene-editing complex can be programmed to mutate the CCR5 gene, such that the cells receiving the gene-editing complex no longer express CCR5 or express CCR5 at a reduced level. In one instance, hematopoietic stem cells expressing the HIV-binding form of CCR5 (or producing progeny expressing CCR5) are taken from the subject, modified to no longer express the HIV-binding form of CCR5, and then transplanted into the subject. In another instance, the subject's CCR5-expressing CD4+ T cells receive the gene-editing complex using the methods and apparatus described herein, such that the CD4+ T cells no longer express the HIV-binding form of CCR5. The modified CD4+ T cells are then returned to the subject. This treatment of CD4+ T cells can be performed from whole blood from the subject. In these and other implementations, bone marrow cells or blood cells are modified to no longer express HIV-binding CXC chemokine receptor type 4 (CXCR4). Similarly, a subject's cells can be modified to have reduced CCR5 expression to treat or prevent infections associated with Yersinia pestis (bubonic plague) or Variolamajor (small pox).

[0079] It also provides subjects other than humans who contain cells modified by the methods and apparatus disclosed herein. Such subjects include non-human vertebrates, amphibians, mammals, and primates. Non-limiting examples include species of fish such as *Danio* sp., *Fugu* sp., *Xenopus* sp., *Mus* sp., and *Rattus* sp.

[0080] Introducing gene-editing proteins and complexes into cells

[0081] The delivery of pre-formed protein complexes allows for the study of cellular processes without genetically modifying the cells under study. This topic can be applied to the delivery of protein complexes and gene-editing complexes into cells, including CRISPR.

[0082] Advantages of using the methods and apparatus described herein to deliver protein complexes include controlled and transient introduction of assay reagents to study cellular and protein complex function. Since transgenic expression and cellular assembly of the complex components are not required, the timing and ratio of complex function (protein:RNA) can be controlled. Furthermore, the transient nature of delivery allows for observation of changes resulting from transient function, rather than long-term expression, which could lead to off-target or secondary effects. From the perspective of in vivo homing and gene expression patterns, microfluidic delivery has significantly fewer side effects on therapeutic cells than (10-fold) electroporation.

[0083] For example, compared to electroporation, microfluidic delivery results in fewer aberrant and nonspecific changes in gene expression. Furthermore, the structural and functional integrity of cells extruded via microfluidic delivery is preserved compared to electroporation-mediated delivery. For instance, an increased number of T cells exposed in vitro to microfluidic delivery (and then introduced into the subject in vivo) home to lymph nodes compared to T cells that have already undergone electroporation. Cells treated via electroporation and then administered to the subject (e.g., T cells) are more likely to be cleared from the subject compared to cells treated via microfluidic delivery. This clearing is associated with altered / abnormal gene expression in these cells that are marked for destruction or clearance by the body after electroporation.

[0084] Target cell and payload composition

[0085] Microfluidic devices can be used to process any eukaryotic cell, such as mammalian cells like human cells, to alter the cell membrane and introduce protein / nucleic acid complexes or assemblies into the cytosol of the target cell. Exemplary target cells include lymphocytes / immune cells: dendritic cells (DCs), B cells, T cells, natural killer cells (NK cells), neutrophils, basophils, eosinophils, innate lymphoid cells, monocytes, macrophages, hematopoietic stem cells, common lymphoid progenitor cells; stem cells: embryonic stem cells, mesenchymal stem cells, induced pluripotent stem cells; other primary cells: fibroblasts, hepatocytes, cardiomyocytes, neurons, epithelial cells, epidermal cells, endothelial cells, pancreatic islet cells; and cell lines, such as those used for disease research: T cell clones, Jurkat cells, HeLa cells, human embryonic kidney 293 (HEK293) cells, U2OS cells, and Chinese hamster ovary (CHO) cells. Prokaryotic cells can also be processed. The size of the device's shrinkage element is adjusted according to the type of cell to be processed.

[0086] In some embodiments, the cell is a prokaryotic cell. In other embodiments, the cell is a eukaryotic cell. Non-limiting examples of eukaryotic cells include protozoan, algae, fungi, yeast, plants, animals, vertebrates, invertebrates, arthropods, mammals, rodents, primates, and human cells. The cell can be, for example, a cell of a unicellular or multicellular organism. The cell can be, for example, a primary eukaryotic cell or an immortalized eukaryotic cell. In some embodiments, the cell is a cancer cell. In some embodiments, the cell is not a human cell. For example, compositions for treating cancer and / or methods for treating cancer, or methods for preparing compositions for treating cancer, include treating immune cells using gene-editing methods described herein to reduce the expression / production of immunosuppressive signals from tumor cells. One example includes reducing or knocking out SHP-2 to increase immune activity against tumors.

[0087] In various embodiments, the cells may be a mixture of two or more cell types, or multiple cell types may be a mixture of two or more cell types. The mixture of cell types may be a co-culture of multiple cell types (such as the two or more disclosed herein) or a mixture of cell types that are naturally present together, for example, in whole blood.

[0088] In some embodiments, the cells are peripheral blood mononuclear cells. In various embodiments, the cell suspension contains a purified cell population. In some embodiments, the cells are primary cells or cell lines.

[0089] In some embodiments, the cells are blood cells. In some embodiments, the blood cells are immune cells. In some embodiments, the immune cells are lymphocytes. In some embodiments, the immune cells are T cells, B cells, natural killer (NK) cells, dendritic cells (DCs), natural killer T (NKT) cells, mast cells, monocytes, macrophages, basophils, eosinophils, or neutrophils. In some embodiments, the immune cells are adaptive immune cells, such as T cells and B cells. In some embodiments, the immune cells are innate immune cells. Exemplary innate immune cells include innate lymphoid cells (ILCs; ILC1, ILC2, ILC3), basophils, eosinophils, mast cells, NK cells, neutrophils, and monocytes. In some embodiments, the immune cells are memory cells. In some embodiments, the immune cells are primary human T cells. In some embodiments, the cells are mouse, dog, cat, horse, rat, goat, monkey, or rabbit cells.

[0090] In some embodiments, the cells are human cells. In some embodiments, the cell suspension contains cells other than human cells or non-mammalian cells. In some embodiments, the cells are chicken, frog, insect, or nematode cells.

[0091] Any physiologically or cellularly compatible buffer system can be used as a solution for rinsing / incubating cells and processing them via a device. Examples include phosphate-buffered saline (PBS), Opti-MEM®, Roswell Park Memorial Institute (RPMI), and Dulbecco's modified Eagle medium (DMEM). Reduced serum or serum-free medium or buffer compositions are preferred. The buffer or medium is selected based on maintaining and preserving the health or viability of the target cells and / or its effect on gene expression. For example, in some cases, the presence of calcium in the buffer is desired to promote or support mRNA expression.

[0092] The payload composition comprises a protein-nucleic acid complex or assembly. Exemplary complexes include components or modules of gene editing systems as described above, such as nuclease / guide nucleic acid assemblies or assemblies. For example, the gRNA:Cas9 molar ratio ranges from 1:100,000 to 100,000:1, preferably from 1:10 to 10:1, such as 1:1, 1:2, or 2:1. The concentration (molar concentration) of the complex in the delivery-facilitating buffer typically ranges from 100 mM to 1 nM, such as 10 μM to 100 nM. The complex can be mixed with the cells before or after the cells pass through the contraction device.

[0093] Microfluidic delivery of gene editing complexes

[0094] To perform gene editing operations, microfluidic platforms (e.g., U.S. Patent Application Publication No. 20140287509, filed April 17, 2014; PCT International Application No. PCT / US2014 / 051343, filed August 15, 2014; PCT International Application No. PCT / US2015 / 060689, filed November 13, 2015; and PCT International Application No. PCT / US2015 / 058489, filed October 30, 2015, each of which is hereby incorporated herein by reference) can be used to deliver Cas proteins (such as Cas9 protein), guide RNA, and donor DNA into cells via mechanical deformation.

[0095] Figures 1-2 illustrate exemplary microfluidic systems that can be used to deliver genome editing proteins, RNA, and DNA. The microfluidic system 5 includes a channel 10 defining a lumen. The microfluidic channel 10 includes a constrictor 15, which is preferably configured such that only one target cell 20 can pass through the constrictor 15 at a time. Preferably, the cell 20, suspended in a solution buffer 25 that also includes delivery material 30, passes through the channel 10, although delivery material can be added to the solution buffer 25 after the cell 20 passes through the constrictor 15. As the cell 20 approaches and passes through the constrictor 15, the constrictor 15 applies pressure (e.g., mechanical compression) to the cell 20, squeezing the cell 20 (e.g., as shown in cell 201). The pressure applied to the cell by the constrictor 15 causes disturbances (e.g., cavitation) in the cell membrane (e.g., cell 202). Once the cell has passed through the constrictor 15, the cell 20 begins to absorb material from the solution buffer 25 through the cavitation, including delivery material 30 (e.g., cell 203). The cell membrane recovers over time, and at least a portion of the delivery material 30 is preferably retained and trapped within the cell.

[0096] In some embodiments, the device includes a shrinkage member length of about 5 μm to about 50 μm, or any length or range thereof. For example, the shrinkage member length ranges from about 5 μm to about 40 μm, about 5 μm to about 30 μm, about 5 μm to about 20 μm, or about 5 μm to about 10 μm. In some embodiments, the shrinkage member length ranges from about 10 μm to about 50 μm, about 20 μm to about 50 μm, about 30 μm to about 50 μm, or about 40 μm to about 50 μm. In some embodiments, the shrinkage member depth ranges from about 2 μm to about 200 μm, or any depth or range thereof. For example, the shrinkage member depth ranges from about 2 μm to about 150 μm, about 2 μm to about 100 μm, about 2 μm to about 50 μm, about 2 μm to about 25 μm, about 2 μm to about 15 μm, or about 2 μm to about 10 μm. In some embodiments, the shrinkage depth ranges from about 10 μm to about 200 μm, about 25 μm to about 200 μm, about 50 μm to about 200 μm, about 100 μm to about 200 μm, or about 150 μm to about 200 μm. In some embodiments, the angle of the inlet or outlet portion of the shrinkage element ranges from about 0 degrees to about 90 degrees, or any angle or angle range therebetween. For example, angles of about 5, about 10, about 15, about 20, about 30, about 40, about 50, about 60, about 70, about 80, or about 90 degrees or greater. In some embodiments, the pressure ranges from about 50 psi to about 200 psi, or any pressure or pressure range therebetween. For example, pressure ranges of about 50 psi to about 150 psi, about 50 psi to about 125 psi, about 50 psi to about 100 psi, or about 50 psi to about 75 psi. In some embodiments, the pressure range is approximately 75 psi to approximately 200 psi, approximately 100 psi to approximately 200 psi, approximately 125 psi to approximately 200 psi, approximately 150 psi to approximately 200 psi, or approximately 175 psi to approximately 200 psi. In some embodiments, the device includes a shrinkage element width between approximately 2 μm and approximately 10 μm, or any width or width range therebetween. For example, the shrinkage element width can be any of approximately 3 μm, approximately 4 μm, approximately 5 μm, approximately 6 μm, or approximately 7 μm.

[0097] The following data were generated using the following materials and methods. The complex was prepared as follows: 10 μl of 1 mg / ml nuclear localization signal (NLS)-labeled Cas9 protein was mixed with 5 μl of 1 mg / ml guide RNA. The mixture was incubated on ice for 20 minutes to allow the complex to form. For delivery, target cells were suspended in serum-free medium at 10 million cells / ml. The cells and Cas9-gRNA complex were mixed immediately before device treatment to a complex concentration of approximately 0.15 mg / ml. Cells were treated using pressure, temperature, chip design, and buffer conditions specific to the target cell type. For example, for primary human T cells, the pressure was approximately 100 psi, on ice, through a 30 μm long, 4 μm wide contraction element. After a 2-minute incubation following treatment, the cells were diluted in medium and washed to remove any undelivered complex. Cells are then cultured to allow gene editing to occur (e.g., 1, 2, 5, 12, 24 hours or longer; for non-clinical applications, the timeframe depends on the assay readings, e.g., 24 hours or longer). For clinical use, such as for patient therapy, cells can be immediately injected back into the patient after device treatment. Optionally, cells are incubated in vitro for a period of time (e.g., 1, 2, 5, 12, 24 hours or longer) before being injected into the patient recipient. The temperature, concentration, and iteration of the molecules vary depending on the target cell type.

[0098] Figure 3 shows K562 reporter cells (American Center for Type Culture Collection (ATCC)® CCL-243). TM A series of flow cytometry plots and bar charts of recombination efficiency (from bone marrow-derived cells (lymphoblast morphology) of K562 reporter cells) were generated using the microfluidic apparatus shown in Figures 1-2 to deliver gene-editing material into the cytoplasm. At the top is a flow cytometry plot of K562 reporter cells with delivered Cas9 protein, site-specific gRNA, and donor oligonucleotides. The CRISPR complex is approximately 150 kDa. These reporter cells possess the mCherry gene and therefore typically appear in Q1 of the plot. If site-specific DNA cleavage and insertion of the donor oligonucleotide is successful, it results in the expression of green fluorescent protein (GFP), which is indicated by cells appearing in Q2. The bottom shows a quantification of recombination efficiency based on various apparatus conditions, compared to endocytosis (entrapment) and untreated (NC) controls. Delivery of Cas9 protein, guide RNA, and donor DNA via cell squeezing resulted in successful changes to the reporter cell line genome. These data indicate that delivery of gene-editing components (in the form of complexes or assemblies) via mechanical cell disruption leads to efficient changes in the genome.

[0099] Delivery of TALEN proteins or mRNA, zinc finger nucleases, macronucleases, Cre recombinases, or any other enzymes capable of cleaving DNA can also be achieved through mechanical disruption of the cell membrane into the cytoplasm. An exemplary TALEN genome editing system comprising an exemplary TALEN protein is described in Ding et al. (2013) Cell Stem Cell, 12, 238-251, the entire contents of which are incorporated herein by reference. Ding et al. (2013) Cell Stem Cell, 12, 238-251 describes a non-limiting example of a general TALEN amino acid sequence that recognizes a 15-base-pair sequence. A non-limiting example of a general TALEN amino acid sequence is:

[0100]

[0101] In SEQ ID NO: 65 and 66, for identifying C: XX = HD; for identifying T: XX = NG; for identifying A: XX = NI; and for identifying G: XX = NN, except in the last position of XX = NK. Underlined terms indicate the N-terminus and C-terminus of the TAL effector.

[0102] While some variations have been described in detail above, other modifications or additions are possible. For example, gene-editing materials may include TALEN proteins, TALEN mRNA, zinc finger nucleases, macronucleases, Cre recombinases, or any other enzymes capable of mechanically breaking down and cleaving DNA delivered to the cytosol through the cell membrane.

[0103] RNA and Cas9 are delivered in a complex form.

[0104] Given the many factors that could potentially hinder successful gene editing via microfluidic delivery of gene complexes, the achieved results are surprising. For example, the Cas9-gRNA complex can elicit Toll-like receptor (TLR)-mediated or other PRR (pattern recognition receptor)-mediated responses, which would inhibit gene editing function and / or survival, but this potential problem was not observed. Since the complex is not guaranteed to be stable once it enters the cytoplasm, it can be degraded and become nonfunctional, but surprisingly, the delivered complex was still capable of editing.

[0105] The integrity of the gene-editing complex was preserved using microfluidic-based cell-squeezing delivery. This complex does not possess the same physical / chemical properties as individual gRNAs or proteins; therefore, it is uncertain whether the delivery process will behave the same when the complex is delivered relative to its individual components. The complex is larger and less stable than its components. The complex can separate due to, for example, shear forces. Furthermore, the complex may unfortunately be evaded membrane transport or cytosol transport because some other elements can break it down before they have the opportunity to function or act on the cellular target. The complex also has a different charge distribution, which can affect its ability to be delivered. The shape, and therefore transport properties, can also vary compared to the complex components. The delivery method successfully preserved the structural and functional integrity of the complex.

[0106] The shearing forces associated with the delivery process may have broken the protein / gRNA complex and rendered it nonfunctional, but surprisingly, the delivery system efficiently introduced the complex into the cell, and gene editing still functioned. Also notably, the complex retained appropriate nuclear localization behavior compared to standalone, uncomplexed Cas proteins (such as Cas9) with NLS; however, as demonstrated by the gene expression results above, behavior and function were preserved throughout the process.

[0107] The CRISPR-Cas system is known in the art. Non-limiting aspects of the system are described in U.S. Patent No. 8,697,359, published April 15, 2014, the entire contents of which are incorporated herein by reference.

[0108] 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, their homologs or modified forms thereof. These enzymes are known; for example, the amino acid sequence of the *Streptococcus pyogenes* Cas9 protein is available in the SwissProt database under accession number Q99ZW2. In some embodiments, the unmodified CRISPR enzyme has DNA cleaving activity, such as Cas9. In some embodiments, the CRISPR enzyme is Cas9, and may be Cas9 derived from *Streptococcus pyogenes* or *Streptococcus pneumoniae*. In some embodiments, the CRISPR enzyme directs the cleavage of one or both strands at a location within the target sequence (e.g., within the target sequence and / or within its complementary sequence). In some embodiments, the CRISPR enzyme directs the cleavage of one or both strands within approximately 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 mutated relative to the corresponding wild-type enzyme, such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence. For example, an aspartic-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from *Streptococcus pyogenes* transforms Cas9 from a two-strand cleaving nuclease into a nicking enzyme (cleaving a single strand). Other examples of mutations that make Cas9 a nicking enzyme include, but are not limited to, H840A, N854A, and N863A. In aspects of the present invention, the nicking enzyme can be used for genome editing via homologous recombination.

[0109] The following provides non-limiting examples of Cas9 amino acid and cDNA sequences.

[0110] The amino acid sequence of the *Streptococcus pyogenes* Cas9 protein can be found in the SwissProt database under accession number Q99ZW2. The amino acid sequence is:

[0111]

[0112]

[0113] SEQ ID NO: 1 can be encoded by the following nucleotide sequence found in the European Nucleotide Archive under accession number AAK33936.2:

[0114]

[0115]

[0116]

[0117]

[0118] The amino acid sequence of the *Streptococcus thermophilus* Cas9 protein can be found in the UniProt database under accession number Q03JI6.1. See also Sapranauskas et al., (2011) *Nucleic Acids Res. 39: 9275-9282*. The amino acid sequence is:

[0119]

[0120] SEQ ID NO: 3 can be encoded by the following nucleotide sequence found in the European Nucleotide Archive under accession number ABJ66636.1:

[0121]

[0122]

[0123] Examples of Cas9 proteins containing a nuclear localization signal (GGSGPPKKKRKV; SEQ ID NO: 5) at their C-terminus have the following amino acid sequence:

[0124]

[0125]

[0126] In some implementations, the Cas9 cleavage enzyme can be used in combination with guide sequences, such as two guide sequences that target the sense and antisense strands of the DNA target, respectively. This combination allows both strands to be cleaved and used to induce non-homologous end joining (NHEJ).

[0127] As another example, two or more catalytic domains (RuvC I, RuvC II, and RuvC III) of Cas9 can be mutated to produce a mutant Cas9 that substantially lacks all DNA cleavage activity. In some embodiments, the D10A mutation is combined with one or more of the H840A, N854A, or N863A mutations to produce a Cas9 enzyme that substantially lacks all DNA cleavage activity (where the amino acid numbers are as shown in SEQ ID NO: 1). In some embodiments, a CRISPR enzyme is considered substantially lacking in 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 lower relative to its non-mutated form. Other mutations can be useful; in cases where Cas9 or other CRISPR enzymes originate from species other than Streptococcus pyogenes, mutations in the corresponding amino acids can achieve similar effects.

[0128] In some implementations, the enzyme-coding sequence encoding a CRISPR enzyme is codon-optimized for expression in specific cells, such as eukaryotic cells. Eukaryotic cells can be cells of a specific organism or derived from a specific organism, such as mammals, including but not limited to humans, mice, rats, rabbits, dogs, or non-human primates. Generally, codon optimization refers to the process of modifying nucleic acid sequences to enhance expression in target host cells, which is performed by replacing at least one codon of the natural sequence with a codon from the gene that is more frequently or most frequently used in the host cell while maintaining the natural amino acid sequence (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons). Various species exhibit specific preferences for certain codons of specific amino acids. Codon preference (differences in codon use between organisms) is generally associated with the translation efficiency of messenger RNA (mRNA), which is considered to be particularly dependent on the characteristics of the codons translated and the utilization of specific transfer RNA (tRNA) molecules. The dominance of the chosen tRNA in the cell is generally a reflection of the most frequently used codons in peptide synthesis. Therefore, based on codon optimization, genes can be modified for optimal gene expression in a given organism. Codon usage tables are readily available, for example, in codon usage databases, and these tables can be adapted in various ways. See Nakamura, Y. et al., "Codon usage tabulated from the international DNAsequence databases: status for the year 2000," Nucl. Acids Res. 28:292 (2000). Computer algorithms for codon optimization of specific sequences for expression in specific host cells are also available, such as Gene Forge (Aptagen; Jacobus, Pa.). In some implementations, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more or all codons) in the sequence encoding the CRISPR enzyme correspond to the most frequently used codon for a particular amino acid.

[0129] Generally, a guide sequence is any polynucleotide sequence that is sufficiently complementary to the target polynucleotide sequence to hybridize with the target sequence and guide the CRISPR complex to bind sequence-specifically to the target sequence. In some embodiments, the complementarity between the guide sequence and its corresponding target sequence is about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more when optimal alignment is performed using a suitable alignment algorithm. Any suitable algorithm for sequence alignment can be used to determine optimal alignment; non-limiting examples of algorithms include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transformation (e.g., Burrows WheelerAligner), ClustalW, ClustalX, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Francisco)). Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some embodiments, the guide sequence is about or greater than 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 some embodiments, the guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides. The guide sequence's ability to guide CRIS can be evaluated by any suitable assay. The ability of the PR complex to bind sequence-specifically to the target sequence. For example, components of the CRISPR system sufficient to form a CRISPR complex (including the guide sequence to be tested) can be provided to host cells with the corresponding target sequence, such as by transfection with a vector encoding the CRISPR sequence, and then the preferential cleavage within the target sequence can be assessed, such as by a Surveyor assay as described herein. Similarly, the cleavage of the target polynucleotide sequence can be assessed in vitro by providing the target sequence, components of the CRISPR complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing the binding or cleavage rates at the target sequence between the reactions of the test and control guide sequences. Other assays are possible, and those skilled in the art will recognize them.

[0130] A guide sequence can be selected to target any target sequence. In some implementations, the target sequence is a sequence within the cell's genome. Exemplary target sequences include those unique within the target genome. For example, for *Streptococcus pyogenes* Cas9, a unique target sequence within the genome could include a Cas9 target site of the form MMMMMMMNNNNNNNNNNNNNXGGMMMMMMMMNNNNNNNNNNNXXAGAAW, where NNNNNNNNNNNNNXGG (N is A, G, T, or C; X can be a deoxynucleotide) has a single occurrence in the genome. A unique target sequence within the genome could also include a *Streptococcus pyogenes* Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXGG, where NNNNNNNNNNNNNXGG (N is A, G, T, or C; X can be a deoxynucleotide) has a single occurrence in the genome. For Streptococcus thermophilus CRISPR1 Cas9, unique target sequences in the genome may include Cas9 target sites of the form MMMMMMMMMNNNNNNNNNNNXXAAGAAW (SEQ ID NO: 7), wherein NNNNNNNNNNNNNXXAGAAW (SEQ ID NO: 8) (N is A, G, T, or C; X may be a deoxynucleotide; and W is A or T) appears once in the genome. Unique target sequences in the genome may include Streptococcus thermophilus CRISPR1 Cas9 target sites of the form MMMMMMMMMNNNNNNNNNNXXAGAAW (SEQ ID NO: 9), wherein NNNNNNNNNNNXXAAGAAW (SEQ ID NO: 10) (N is A, G, T, or C; X may be a deoxynucleotide; and W is A or T) appears once in the genome. For *Streptococcus pyogenes* Cas9, unique target sequences in the genome can include Cas9 target sites in the form MMMMMMMNNNNNNNNNNNXGGXG, where N (where N is A, G, T, or C; and X can be a deoxynucleotide) appears only once in the genome. In each of these sequences, "M" can be A, G, T, or C, and is not considered in identifying the sequence as unique.

[0131] In some implementations, a guide sequence is selected to reduce the degree of secondary structure within the guide sequence. Secondary structure can be determined by any suitable polynucleotide folding algorithm. Some procedures are based on calculating the minimum Gibbs free energy. An example of such an algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133–148). Another example folding algorithm is RNAfold, an online web server developed at the Institute for Theoretical Chemistry at the University of Vienna, which uses a centroid structure prediction algorithm (see, for example, AR Gruber et al., 2008, Cell 106(1): 23–24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12): 1151–62). Other algorithms can be found in U.S. Patent Application Serial No. 61 / 836,080; which is incorporated herein by reference.

[0132] This topic relates to CRISPR / CRISPR / CRISPR delivery of gene editing complexes from Prevotella and Francisella 1 (Cpf1) or their components (e.g., Cpf1 protein). Examples of human codon-optimized Cpf1-family proteins are provided below.

[0133] Human codon-optimized Cpf1 family proteins

[0134] Zetsche et al., Cell 163, 759-771, October 22, 2015, described non-restrictive instances of Cpf1-family protein sequences and aspects of CRISPR / Cpf1 gene editing; the entire contents of that reference are incorporated herein by reference.

[0135] Francisella tularensis Novicida subspecies U112 (FnCpf1; pY004), including NLS and HA tags:

[0136]

[0137] SEQ ID NO: 11 includes a nuclear localization signal (KRPAATKKAGQAKKKK) (SEQ ID NO: 12), followed by a glycine-serine linker (GS), followed by a human influenza lectin (HA) tag (YPYDVPDYAYPYDVPDYAYPYDVPDYA) (SEQ ID NO: 13).

[0138] SEQ ID NO: 11 can be encoded by the following nucleotide sequence:

[0139]

[0140]

[0141]

[0142] Lachnospiraceae bacterium MC2017 (Lb3Cpf1; pY005), including NLS and HA tags:

[0143]

[0144]

[0145] SEQ ID NO: 15 includes a nuclear localization signal (KRPAATKKAGQAKKKK) (SEQ ID NO: 12), followed by a glycine-serine linker (GS), followed by an HA tag (YPYDVPDYAYPYDVPDYAYPYDVPDYA) (SEQ ID NO: 13).

[0146] SEQ ID NO: 15 can be encoded by the following nucleotide sequence:

[0147]

[0148]

[0149]

[0150] Butyrivibrio proteoclasticus (BpCpf1; pY006), including NLS and HA tags:

[0151]

[0152] SEQ ID NO: 17 includes a nuclear localization signal (KRPAATKKAGQAKKKK) (SEQ ID NO: 12), followed by a glycine-serine linker (GS), followed by an HA tag (YPYDVPDYAYPYDVPDYAYPYDVPDYA) (SEQ ID NO: 13).

[0153] SEQ ID NO: 17 can be encoded by the following nucleotide sequence:

[0154]

[0155]

[0156]

[0157]

[0158] Peregrinibacteria bacterium GW2011_GWA_33_10 (PeCpf1; pY007), including NLS and HA tags:

[0159]

[0160] SEQ ID NO: 19 includes a nuclear localization signal (KRPAATKKAGQAKKKK) (SEQ ID NO: 12), followed by a glycine-serine linker (GS), followed by an HA tag (YPYDVPDYA YPYDVPDYA YPYDVPDYA) (SEQ ID NO: 13).

[0161] SEQ ID NO: 19 can be encoded by the following nucleotide sequence:

[0162]

[0163]

[0164]

[0165] Parcubacteria bacterium GWC2011_GWC2_44_17 (PbCpf1; pY008), including NLS and HA tags:

[0166]

[0167] SEQ ID NO: 21 includes a nuclear localization signal (KRPAATKKAGQAKKKK) (SEQ ID NO: 12), followed by a glycine-serine linker (GS), followed by an HA tag (YPYDVPDYAYPYDVPDYAYPYDVPDYA) (SEQ ID NO: 13).

[0168] SEQ ID NO: 21 can be encoded by the following nucleotide sequence:

[0169]

[0170]

[0171] Smithella sp. SC_K08D17 (SsCpf1; pY009), including NLS and HA tags:

[0172]

[0173]

[0174] SEQ ID NO: 23 includes a nuclear localization signal (KRPAATKKAGQAKKKK) (SEQ ID NO: 12), followed by a glycine-serine linker (GS), followed by an HA tag (YPYDVPDYAYPYDVPDYAYPYDVPDYA) (SEQ ID NO: 13).

[0175] SEQ ID NO: 23 can be encoded by the following nucleotide sequence:

[0176]

[0177]

[0178]

[0179] Acidaminococcus sp. BV3L6 (AsCpf1; pY010), including NLS and HA tags:

[0180]

[0181]

[0182] SEQ ID NO: 25 includes a nuclear localization signal (KRPAATKKAGQAKKKK) (SEQ ID NO: 12), followed by a glycine-serine linker (GS), followed by an HA tag (YPYDVPDYAYPYDVPDYAYPYDVPDYA) (SEQ ID NO: 13).

[0183] SEQ ID NO: 25 can be encoded by the following nucleotide sequence:

[0184]

[0185]

[0186] Bacteria of the family Trichophyceae, MA2020 (Lb2Cpf1; pY011), including NLS and HA tags:

[0187]

[0188]

[0189] SEQ ID NO: 27 includes a nuclear localization signal (KRPAATKKAGQAKKKK) (SEQ ID NO: 12), followed by a glycine-serine linker (GS), followed by an HA tag (YPYDVPDYAYPYDVPDYAYPYDVPDYA) (SEQ ID NO: 13).

[0190] SEQ ID NO: 27 can be encoded by the following nucleotide sequence:

[0191]

[0192]

[0193]

[0194] Candidatus Methanoplasma termitum (CMtCpf1; pY012), including NLS and HA tags:

[0195]

[0196]

[0197] SEQ ID NO: 29 includes a nuclear localization signal (KRPAATKKAGQAKKKK) (SEQ ID NO: 12), followed by a glycine-serine linker (GS), followed by an HA tag (YPYDVPDYAYPYDVPDYAYPYDVPDYA) (SEQ ID NO: 13).

[0198] SEQ ID NO: 29 can be encoded by the following nucleotide sequence:

[0199]

[0200]

[0201]

[0202] The picky eubacterium (Eubacterium eligens) (EeCpf1; pY013), including NLS and HA tags:

[0203]

[0204]

[0205] SEQ ID NO: 31 includes a nuclear localization signal (KRPAATKKAGQAKKKK) (SEQ ID NO: 12), followed by a glycine-serine linker (GS), followed by an HA tag (YPYDVPDYAYPYDVPDYAYPYDVPDYA) (SEQ ID NO: 13).

[0206] SEQ ID NO: 31 can be encoded by the following nucleotide sequence:

[0207]

[0208]

[0209]

[0210]

[0211] Moraxella bovoculi 237 (MbCpf1; pY014), including NLS and HA tags:

[0212]

[0213] SEQ ID NO: 33 includes a nuclear localization signal (KRPAATKKAGQAKKKK) (SEQ ID NO: 12), followed by a glycine-serine linker (GS), followed by an HA tag (YPYDVPDYAYPYDVPDYAYPYDVPDYA) (SEQ ID NO: 13).

[0214] SEQ ID NO: 33 can be encoded by the following nucleotide sequence:

[0215]

[0216]

[0217]

[0218] Leptospira inadai (LiCpf1; pY015), including NLS and HA tags:

[0219]

[0220]

[0221] SEQ ID NO: 35 includes a nuclear localization signal (KRPAATKKAGQAKKKK) (SEQ ID NO: 12), followed by a glycine-serine linker (GS), followed by an HA tag (YPYDVPDYAYPYDVPDYAYPYDVPDYA) (SEQ ID NO: 13).

[0222] SEQ ID NO: 35 can be encoded by the following nucleotide sequence:

[0223]

[0224]

[0225]

[0226] Bacteria of the family Trichophyceae, ND2006 (LbCpf1; pY016), including NLS and HA tags:

[0227]

[0228] SEQ ID NO: 37 includes a nuclear localization signal (KRPAATKKAGQAKKKK) (SEQ ID NO: 12), followed by a glycine-serine linker (GS), followed by an HA tag (YPYDVPDYAYPYDVPDYAYPYDVPDYA) (SEQ ID NO: 13).

[0229] SEQ ID NO: 37 can be encoded by the following nucleotide sequence:

[0230]

[0231]

[0232]

[0233] Porphyromonas crevioricanis (PcCpf1; pY017), including NLS and HA tags:

[0234]

[0235]

[0236] SEQ ID NO: 39 includes a nuclear localization signal (KRPAATKKAGQAKKKK) (SEQ ID NO: 12), followed by a glycine-serine linker (GS), followed by an HA tag (YPYDVPDYAYPYDVPDYAYPYDVPDYA) (SEQ ID NO: 13).

[0237] SEQ ID NO: 39 can be encoded by the following nucleotide sequence:

[0238]

[0239]

[0240]

[0241] Prevotella disiens (PdCpf1; pY018), containing NLS and HA tags:

[0242]

[0243] SEQ ID NO: 41 includes a nuclear localization signal (KRPAATKKAGQAKKKK) (SEQ ID NO: 12), followed by a glycine-serine linker (GS), followed by an HA tag (YPYDVPDYAYPYDVPDYAYPYDVPDYA) (SEQ ID NO: 13).

[0244] SEQ ID NO: 41 can be encoded by the following nucleotide sequence:

[0245]

[0246]

[0247]

[0248]

[0249] Porphyromonas macacae (PmCpf1; pY09), including NLS and HA tags:

[0250]

[0251] SEQ ID NO: 43 includes a nuclear localization signal (KRPAATKKAGQAKKKK) (SEQ ID NO: 12), followed by a glycine-serine linker (GS), followed by an HA tag (YPYDVPDYAYPYDVPDYAYPYDVPDYA) (SEQ ID NO: 13).

[0252] SEQ ID NO: 43 can be encoded by the following nucleotide sequence:

[0253]

[0254]

[0255]

[0256] Some of the non-limiting sequences shown above include sequences such as nuclear localization signals and / or tag sequences (e.g., HA tags). Different nuclear localization signals may be present in various embodiments. In some embodiments, no nuclear localization signal is used. In some embodiments, no tag is used (e.g., no HA tag is used).

[0257] In various embodiments involving proteins (e.g., proteins within gene-editing complexes), the proteins may include nuclear localization signals. For example, a protein (e.g., a Cas protein) may contain a nuclear localization signal (NLS). 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; and Dingwall et al. (1991) Trends in Biochemical Sciences 16(12): 478-81, the contents of which are hereby incorporated herein by reference. Specific, non-limiting examples of nuclear localization signals include GGSGPPKKKRKV (SEQ ID NO: 5), KRPAATKKAGQAKKKK (SEQ ID NO: 12), PKKKRKV (SEQ ID NO: 45), KR [PAATKKAGQA] KKKK (SEQ ID NO: 46), KR [XXXXXXXXXX] KKKK (SEQ ID NO: 47), KKXK (SEQ ID NO: 48), KRXK (SEQ ID NO: 49), KKXR (SEQ ID NO: 50), KRXR (SEQ ID NO: 51), AVKRPAATKKAGQAKKKKLD (SEQ ID NO: 52), MSRRRKANPTKLSENAKKLAKEVEN (SEQ ID NO: 53), PAAKRVKLD (SEQ ID NO: 54), and KLKIKRPVK (SEQ ID NO: 55).

[0258] General definitions and general techniques

[0259] Unless otherwise expressly defined, all technical and scientific terms used herein should be assumed to have the same meaning as commonly understood by a person skilled in the art (e.g., in cell culture, molecular genetics, and biochemistry).

[0260] As used herein, in the context of a number or range, the term “about” means ±10% of the stated or claimed number or range, unless the context requires a more limited range.

[0261] In the description above and in the claims, phrases such as “at least one” or “one or more” may appear, followed by a list of connected elements or features. The term “and / or” may also appear in a list of two or more elements or features. Unless implied or explicitly contradicted in the context of its use, such phrases are intended to refer to any one of the listed elements or features individually or in combination with any other stated 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 each intended to mean “A alone, B alone, or A and B together.” Similar interpretations are also intended for lists comprising three or more 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 each 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.” Furthermore, the use of the term “based on” in the above and claims is intended to mean “at least partially based on,” allowing for the inclusion of features or elements not explicitly stated.

[0262] The terms “plasma membrane” and “cell membrane” are used interchangeably in this document and refer to the semipermeable membrane that separates the inside of the cell from the outside environment.

[0263] As used herein, an "expression vector" is a DNA or RNA vector capable of expressing one or more polynucleotides. Preferably, the expression vector is also capable of replicating within a host cell. The expression vector can be prokaryotic or eukaryotic and is typically a plasmid. The expression vectors of the present invention include any vector that functions (i.e., directs gene expression) in the host cell of the present invention, including in one of the prokaryotic or eukaryotic cells described herein, such as protozoan, algae, fungi, yeast, plants, animals, vertebrates, invertebrates, arthropods, mammals, rodents, primates, or human cells.

[0264] The expression vectors of the present invention contain regulatory sequences, such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with host cells and control polynucleotide expression. In particular, the expression vectors of the present invention include transcription control sequences. Transcription control sequences are sequences that control transcription initiation, elongation, and termination. Particularly important transcription control sequences are those that control transcription initiation, such as promoter, enhancer, operon, and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one cell of the present invention. Many such transcription control sequences are known to those skilled in the art. In a preferred embodiment, the method does not involve using a viral vector (such as adenovirus) to deliver nucleic acid molecules or constructs.

[0265] It should be understood that, while providing a parameter range, the present invention also provides all integers within that range, and one-tenth of that range. For example, "0.2-5mg" is the disclosure of 0.2mg, 0.3mg, 0.4mg, 0.5mg, 0.6mg, etc., up to 5.0mg.

[0266] Unless implied or explicitly contradicted in the context of its use, references to cell “squeeze / squeezing,” “deformation,” etc., refer to processes used to deliver macromolecules directly into the cytosol of cells with minimal cytotoxicity. The underlying principle of this approach is the temporary membrane disruption caused by rapid mechanical deformation or squeezing of the target cell, which allows for uptake via diffusion of macromolecules in a fluid medium, followed by cell membrane repair (see, for example, U.S. Patent Application Publication No. 2014 / 0287509, published September 25, 2014; PCT International Patent Application No. PCT / US2015 / 058489, filed October 30, 2015; and PCT International Patent Application No. PCT / 2015 / 060689, filed November 13, 2015, the entire contents of which are incorporated herein by reference).

[0267] As used in this article, "gRNA" refers to the CRISPR-Cas system guide RNA.

[0268] As used herein, the term "protein complex" refers to a complex unit formed by the specific binding of a protein to a binding partner, wherein the binding partner can be one or more proteins, one or more nucleic acids, or a combination of one or more proteins and one or more nucleic acids, etc., to form the protein complex. Protein complexes can be protein-protein complexes, protein-nucleic acid complexes, etc. In some embodiments, the protein complex may include protein-protein interactions, such as interactions between dimers, trimers, tetramers, or higher oligomers of different proteins or the same protein. Interactions between subunits of a protein complex (e.g., in protein-protein complexes or protein-nucleic acid complexes containing more than one protein) or between proteins and nucleic acids (e.g., in protein-nucleic acid complexes) are typically non-binding interactions, such as those caused by hydrogen bridges, π-electron systems such as (arbitrarily conjugated) C-C double bonds, or aromatic rings such as phenyl and heteroaromatic rings such as pyrrole, imidazole, indole, pyrimidine, or purine rings, and interactions between metal atoms and oxygen, nitrogen, or sulfur atoms, but can also be weak, particularly reversible, covalently bonded interactions, such as sulfur-sulfur bridges.

[0269] A "protein-protein complex" refers to a complex unit composed of two or more proteins formed through interactions between them. Typically, but not always, two or more proteins are bound together by specific non-covalent affinity to form a protein complex. However, covalent bonds can also exist between interacting partners. For example, two interacting partners can be covalently cross-linked, making the protein complex more stable.

[0270] Similarly, a "protein-nucleic acid complex" refers to a complex unit consisting of at least one protein and at least one nucleic acid, formed through interactions involving interactions between proteins and nucleic acids. Typically, but not always, a "protein-nucleic acid complex" is formed by the non-covalent affinity binding of proteins and nucleic acids.

[0271] In various implementations, the gene-editing complex is a protein-nucleic acid complex, such as an RNP. A non-limiting example of an RNP is the CRISPR-Cas RNP, which comprises a Cas protein and gRNA.

[0272] The methods and apparatus described herein deliver intact and functional gene-editing complexes into cells. The components of the gene-editing complex do not separate during delivery and retain their function after delivery to cells.

[0273] Various assays can be used to determine whether intact and functional gene-editing complexes have been delivered to cells. For example, the detection of gene editing in a gene-editing complex can be used to indicate that intact and functional gene-editing complexes have been delivered to cells. Alternatively, cells that have been delivered with gene-editing complexes can be lysed using non-denaturing conditions (such as non-denaturing buffer or a French press), and the lysates can be analyzed using non-denaturing gels to determine whether the gene-editing complex is intact within the cells. Alternatively, cells can be lysed using non-denaturing conditions, and the gene-editing complex can then be isolated from the lysates using immunoprecipitation (i.e., verification that one component of the complex can be co-separated from another component using immunoprecipitation). The isolated gene-editing complex can be measured before or after delivery to cells using non-denaturing gels or denaturing assays (such as sodium dodecyl sulfate polyacrylamide gel electrophoresis) to determine the presence of the gene-editing complex in pre-delivery / pre-cell squeeze buffer and after microfluidic / squeezing treatment, and to identify intact and / or functional complexes in treated cells. In some implementations involving CRISPR-Cas9 RNPs, bands on a non-denaturing gel of approximately 145, 150, 155, or 145–160 kDa can indicate the delivery of the RNP as a complete and functional gene-editing complex into cells.

[0274] As used herein, device dimensions are represented by a series of numbers indicating length, width, and the number of optional shrinkable parts (e.g., 30μm-6mx5 indicates a device with a length of 30μm, a width of 6μm, and 5 shrinkable parts).

[0275] Exemplary Implementation

[0276] This subject matter provides a method for delivering proteins and nucleic acids into cells, the method comprising: providing cells in solution; passing the solution through a microfluidic channel including a cell deformable contractile element; passing the cells through the contractile element such that pressure is applied to the cells, causing a disturbance in the cells sufficiently large to allow the proteins and nucleic acids to pass through; and contacting the cells with the proteins and nucleic acids before, during, and / or after passing the cells through the contractile element.

[0277] In some embodiments, the solution contains the protein and the nucleic acid before, during, and / or after the cells are passed through the contractile element.

[0278] In some implementations, proteins and nucleic acids form protein-nucleic acid complexes.

[0279] In some implementations, proteins and nucleic acids are components of a protein-nucleic acid complex, but they do not recombine when delivered to a cell.

[0280] In some implementations, proteins and nucleic acids form protein-nucleic acid complexes after being delivered to cells.

[0281] In some implementations, proteins and nucleic acids form protein-nucleic acid complexes before being delivered into the cell.

[0282] In some implementations, proteins and nucleic acids contain gene-editing components.

[0283] In some embodiments, the protein-nucleic acid complex comprises a ribonucleoprotein (RNP).

[0284] In some implementations, (a) the protein is a Cas protein or a Cpf1 protein; and (b) the nucleic acid is a single guide RNA (sgRNA) or a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA).

[0285] In some embodiments, the complex is an RNP comprising a Cas protein or a Cpf1 protein and sgRNA, wherein the Cas protein or the Cpf1 protein is complexed with the sgRNA using an excess of about 0.5, 2.0, 2.5, or 3.0 moles of the Cas protein or the Cpf1 protein.

[0286] In some implementations, the Cas protein includes the Cas9 protein.

[0287] In some embodiments, the protein-nucleic acid complex includes a first RNP and a second RNP.

[0288] In some implementations, both the first RNP and the second RNP are nicking enzymes.

[0289] In some implementations, the first RNP cuts a target sequence that is different from the target sequence of the second RNP.

[0290] In some embodiments, the protein-nucleic acid complex comprises a TALEN protein, a zinc finger nuclease, a wide range of nucleases, or a Cre recombinase.

[0291] In some implementations, the nucleic acid includes mRNA encoding a TALEN protein, a zinc finger nuclease, a broad range of nucleases, or a Cre recombinase.

[0292] In some embodiments, the protein-nucleic acid complex comprises (a) a nucleic acid molecule that is complexed with the protein by electrostatic attraction; (b) a nucleic acid molecule that encapsulates the protein; (c) DNA and histones; (d) ribonucleoproteins (RNPs); (e) ribosomes, telomerase enzymes, fornix ribonucleoproteins, RNase P, hnRNPs, or small nucleus RNPs (snRNPs); or (f) a chromosome containing the protein.

[0293] In some implementations, the solution also contains donor DNA.

[0294] In some embodiments, the solution further contains donor DNA before, during, and / or after passing the cells through the contraction element.

[0295] In some implementations, the cells include mammalian cells.

[0296] In some implementations, the cells include human cells.

[0297] In some implementations, the diameter of the contractile element is selected such that it induces a temporary disturbance in the cell membrane large enough to allow the proteins and nucleic acids to pass through.

[0298] In some embodiments, the diameter of the contractile element is approximately 20-99% of the diameter of the cell.

[0299] In some embodiments, the diameter of the contractile element is approximately 60% of the diameter of the cell.

[0300] In some implementations, the microfluidic channel is one of a plurality of parallel microfluidic channels in the microfluidic system.

[0301] In some embodiments, the plurality of parallel microfluidic channels includes at least about 2, 5, 10, 20, 25, 30, 40, 45, 50, 75, 100, 500, 1,000 or 2-1,000 microfluidic channels.

[0302] In some embodiments, the cells are multiple cells, and each cell is passed through one of a plurality of parallel microfluidic channels, wherein each of the plurality of parallel microfluidic channels includes a cell deformable contraction element.

[0303] In some embodiments, (a) the diameter of the shrink member is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 2-10 μm or 10-20 μm; (b) the length of the shrink member is about 10, 15, 20, 24, 30, 40, 50, 60, 70, 80, 90, 100, 10-40, 10-50, 10-60 or 10-100 μm; (c) about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or 10-100 μm. (d) The cells are forced through the microfluidic channel at pressures of approximately 300, 400, 500, 600, 700, 800, 900, 100-300, 200-700, 250-400, 100-1000 mm / s, 1-1000 mm / s, 1 m / s, 2 m / s, 3 m / s, 4 m / s, 5 m / s, 6 m / s, 7 m / s, 8 m / s, 9 m / s, 10 m / s, 0.01-5 m / s, 5-10 m / s. (e) The microfluidic channel is oriented at a velocity of 0.01-10 m / s or s; (f) The microfluidic channel comprises multiple cell deformable contraction elements connected in series; (g) The microfluidic channel comprises a single-cell deformable contraction element; (h) The perturbation of the cell membrane comprises approximately 1-20, 1-600, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 600. The disturbance of the cell membrane having a maximum diameter of about 1-20, 1-600, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500 or 600 nm on the cell membrane lasts for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 1-10 minutes.

[0304] In some embodiments, after delivering the protein and the nucleic acid into the cell, (a) the expression of the target gene in the cell is reduced by at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 80, 85, 90, 95, or 99% or more; or (b) the cell is a plurality of cells, and the expression of the target gene in the plurality of cells is reduced by at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 80, 85, 90, 95, or 99% or more.

[0305] In some embodiments, after about 1, 2, 5, 12, 24, 1–12, 6–12, 6–18, 12–24, or 1–24 hours following the delivery of the protein and the nucleic acid into the cells, (a) the expression of the target gene in the cells is reduced by at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 80, 85, 90, 95, or 99% or more; or (b) the cells are multiple cells, and the expression of the target gene in the multiple cells is reduced by at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 80, 85, 90, 95, or 99% or more.

[0306] In some embodiments, after the protein and the nucleic acid are delivered into the cell, (a) the expression of the target gene in the cell is increased by at least about 5, 10, 25, 50, 75, 100, 250, 500% or more; or (b) the cell is a plurality of cells, and the expression of the target gene in the plurality of cells is increased by at least about 5, 10, 25, 50, 75, 100, 250, 500% or more.

[0307] In some embodiments, about 1, 2, 5, 12, 24, 1–12, 6–12, 6–18, 12–24, or 1–24 hours after the protein and nucleic acid are delivered into the cells, (a) the expression of the target gene in the cells is increased by at least about 5, 10, 25, 50, 75, 100, 250, 500% or more; or (b) the cells are multiple cells, and the expression of the target gene in the multiple cells is increased by at least about 5, 10, 25, 50, 75, 100, 250, 500% or more.

[0308] An aspect of this subject provides an apparatus for delivering a protein-nucleic acid complex into a cell, the apparatus comprising at least one microfluidic channel, wherein the channel comprises a contraction length of about 30 μm and a contraction width of about 4 μm.

[0309] The following examples are provided to provide a more complete understanding of the invention. These examples illustrate exemplary modes of generating and practicing the invention. However, the scope of the invention is not limited to the specific embodiments disclosed in these examples, which are merely illustrative, as similar results can be obtained using alternative methods.

[0310] Example 1: Using CellSqueeze (SQZ) to deliver CRISPR / Cas9 gene editing complexes to edit primary human genes. B2M locus in T cells

[0311] A series of experiments were conducted in unstimulated human T cells to demonstrate the SQZ platform’s ability to deliver Cas9 ribonucleoprotein (RNP; recombinant Cas9 protein complexed with a single guide RNA) and achieve efficient genome editing at a model locus (the β2 microglobulin component (B2M) of MHC class 1).

[0312] Cas9 RNP delivered to unstimulated human T cells

[0313] Fresh PBMCs were isolated from human blood using a standard Ficoll gradient. Next, T cells were negatively selected (using a human T cell enrichment kit (StemCell Technologies)), counted, washed, and cultured in OptiMEM at a gradient of 10-20x10⁻¹⁰. 6 Cells / mL were resuspended for delivery. 10 μg of recombinant CAS9 (PNA Bio) was pre-complexed with 2.5 moles of unmodified gRNA (PNA Bio) designed to specifically target the B2M locus. Recombinant CAS9 was reconstituted into a solution with a final concentration of 20 mM Hepes, 150 mM KCl, and 1% sucrose. gRNA was added directly to the CAS9 solution and incubated on ice for 20 min to form the complex. The complex was added directly to the resuspended cells. The RNP complex was incubated on ice for 20 min prior to SQZ-mediated delivery. RNP (2.2 μM) was co-delivered with 3kD-Cascade Blue Dextran (0.15 mg / mL), which was used as a representative of delivery efficiency. The complex was delivered using two different chips, 10⁻⁴ and 30⁻⁴, at pressures of 60 and 90 psi. The chip has a shrinkage piece of the same width (4 micrometers), but with two different shrinkage piece lengths (30 micrometers versus 10 micrometers).

[0314] B2M protein levels were determined using FACS-based readings 48 hours post-delivery. Reduced B2M expression was used as a measure of functional editing. Two controls were used: 1) T cells incubated with the RNP complex at room temperature for the same duration as the delivery process using the cell squeeze method (endocytosis control; "endo control"), and 2) T cells with Cas9 protein but without gRNA squeeze. A graph showing B2M expression relative to delivered dextran for four different cell populations is presented. Figure 4 B2M expression in the Cas9 control was not significantly different from that in the endocytosis control. Compared with the endocytosis control, RNP delivery using a 30-4 chip at 90 psi resulted in a 54.4% reduction in B2M expression, while a 10-4 chip at 90 psi resulted in a 25.2% reduction in B2M expression. Longer shrinkage chips resulted in greater delivery of the RNP complex and a larger reduction in B2M expression.

[0315] Dextran delivery was used to define low, intermediate, and high delivery populations. The differences in B2M knockdown efficiency among these specific populations were then determined using the mean fluorescence intensity (MFI) of B2M staining. For a 10⁻⁴ microarray, the MFI was 18,637 for the high-delivery population, 71,173 for the intermediate-delivery population, and 83,676 for the low- or non-delivery population. This almost 5-fold decrease in B2M staining intensity for the high-delivery population demonstrates the extent to which delivery affects RNP activity. Similarly, for a 30⁻⁴ microarray, the MFI was 16,460 for the high-delivery population, 44,207 for the intermediate-delivery population, and 54,159 for the low-delivery population. These data demonstrate the importance of cellular extrusion delivery systems of gene-editing complexes into the cytosol of cells for editing efficiency, even within a single population.

[0316] To confirm the FACS reads, a second sequence-based analysis was employed, in which DNA was extracted and amplified using primers flanking the target region to generate amplicons for the edited region used in next-generation sequencing (NGS). The sequencing results were analyzed using a simple algorithm designed to detect CRISPR variants from NGS reads. As expected, the sequence-based reads showed higher editing efficiency. In fact, some insertions and deletions (indels) identified in the sequencing still resulted in functional full-length proteins (i.e., without altering single-base substitutions of the resulting amino acids).

[0317] Table: Comparison of FACS-based and sequence-based readings from the 10-4 edit experiment.

[0318]

[0319] These data demonstrate the successful editing capability of the RNP complex when delivered via a cell extrusion platform.

[0320] The effect of RNP complex amount on editing efficiency

[0321] RNP complexes were delivered to unstimulated human T cells using a 30-4 chip and at two different amounts: 1) standard 1X RNP complex (10 μg Cas9, 2.5 mol excess gRNA) and 2) 0.1X standard RNP complex. B2M protein levels were determined using FACS-based readings at 48 h post-delivery. Reduced B2M expression was used as a measure of functional editing. The following plot shows B2M expression relative to delivered dextran for four different cell populations. Two controls were used: 1) T cells incubated at room temperature in the 1X RNP complex for the same duration as the delivery process using a cell squeezing method (endocytosis control), and 2) T cells with Cas9 protein but without gRNA squeezing (…). Figure 5 ).

[0322] B2M expression on the Cas9 control (Cas9 protein without gRNA) was not significantly different from that on the endocytosis control. Compared to a 55.4% reduction in B2M-positive cells with a higher amount of RNP complex (1X RNP complex (10 μg CAS9, 2.5 mol excess gRNA), a lower amount of RNP complex (0.1X RNP) resulted in a 20.7% reduction in B2M-positive cells. This experiment demonstrates a dose-dependent response directly related to RNP delivery.

[0323] Other implementation plans

[0324] The references cited are incorporated herein by reference. In the event of any inconsistency between the incorporated material and this disclosure, this disclosure shall prevail. Furthermore, where necessary, material incorporated herein by reference should be disregarded to maintain the validity of the claims.

[0325] Furthermore, while the above description relates to the present invention, the description may include more than one invention.

[0326] Depending on the desired configuration, the subject matter described herein can be embodied in systems, apparatus, methods, and / or articles. The embodiments set forth in the foregoing description do not represent all embodiments consistent with the subject matter described herein. Rather, they are merely some examples consistent with aspects related to the stated subject matter. While some variations have been described in detail above, other modifications or additions are possible. In particular, further features and / or variations may be provided in addition to those set forth herein. For example, the above embodiments may be applicable to various combinations or sub-combinations of the features disclosed above and / or combinations and sub-combinations of several further features disclosed above. Furthermore, the logical flows described in the drawings and / or herein do not necessarily require the specific order or sequence shown to achieve the desired results. Other embodiments are within the scope of the appended claims.

[0327] The technical solutions corresponding to the original claims of the parent application are hereby incorporated in this specification:

[0328] 1. A method for delivering proteins and nucleic acids into cells, the method comprising:

[0329] Provide cells in solution;

[0330] The solution is passed through a microfluidic channel that includes cell deformation and contraction elements;

[0331] The cell is forced through the contractile member, thereby applying pressure to the cell and causing sufficient disturbance to allow the proteins and nucleic acids to pass through; and

[0332] The cells are brought into contact with the proteins and nucleic acids before, during, and / or after passing the cells through the contractile element.

[0333] 2. The method according to Project 1, wherein the solution contains the protein and the nucleic acid before, during and / or after passing the cells through the contractile element.

[0334] 3. The method according to item 1 or 2, wherein the protein and the nucleic acid form a protein-nucleic acid complex.

[0335] 4. The method according to any one of items 1-3, wherein the protein and the nucleic acid are components of the protein-nucleic acid complex, but are not complexed when delivered to the cell.

[0336] 5. The method according to Project 4, wherein the protein and the nucleic acid form a protein-nucleic acid complex after delivery into the cell.

[0337] 6. The method according to any one of items 1-4, wherein the protein and the nucleic acid form a protein-nucleic acid complex prior to delivery to the cell.

[0338] 7. The method according to any one of items 1-6, wherein the protein and the nucleic acid comprise gene editing components.

[0339] 8. The method according to any one of items 3-7, wherein the protein-nucleic acid complex comprises a ribonucleoprotein (RNP).

[0340] 9. According to the method described in Project 7, wherein

[0341] (a) The protein is a Cas protein or a Cpf1 protein; and

[0342] (b) The nucleic acid is a single guide RNA (sgRNA) or CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA).

[0343] 10. The method according to item 9, wherein the complex is an RNP comprising a Cas protein or a Cpf1 protein and sgRNA, wherein the Cas protein or the Cpf1 protein is complexed with the sgRNA using an excess of about 0.5, 2.0, 2.5, or 3.0 moles of the Cas protein or the Cpf1 protein.

[0344] 11. The method according to Project 9, wherein the Cas protein includes the Cas9 protein.

[0345] 12. The method according to any one of items 3-11, wherein the protein-nucleic acid complex comprises a first RNP and a second RNP.

[0346] 13. The method according to Item 11, wherein both the first RNP and the second RNP are nicking enzymes.

[0347] 14. The method according to item 13, wherein the first RNP cuts a target sequence that is different from the target sequence of the second RNP.

[0348] 15. The method according to any one of items 3-8, wherein the protein-nucleic acid complex comprises a TALEN protein, a zinc finger nuclease, a broad range of nucleases, or a Cre recombinase.

[0349] 16. The method according to Project 1, wherein the nucleic acid comprises mRNA encoding a TALEN protein, a zinc finger nuclease, a broad range of nucleases, or a Cre recombinase.

[0350] 17. The method according to any one of items 3-8, wherein the protein-nucleic acid complex comprises

[0351] (a) Nucleic acid molecules that combine with proteins through electrostatic attraction;

[0352] (b) Nucleic acid molecules that encapsulate proteins;

[0353] (c) DNA and histones;

[0354] (d) Ribonuclear protein (RNP);

[0355] (e) Ribosomes, telomerase, fornix ribonucleoproteins, RNase P, hnRNP, or small nucleus RNP (snRNP); or

[0356] (f) Chromosomes containing proteins.

[0357] 18. The method according to any one of items 1-17, wherein the solution further comprises donor DNA.

[0358] 19. The method according to item 18, wherein the solution further comprises donor DNA before, during and / or after passing the cells through the contraction element.

[0359] 20. The method according to any one of items 1-19, wherein the cell comprises a mammalian cell.

[0360] 21. The method according to any one of items 1-20, wherein the cells comprise human cells.

[0361] 22. The method according to any one of items 1-21, wherein the diameter of the contractile element is selected to induce a temporary perturbation of the cell membrane sufficiently large to allow the protein and the nucleic acid to pass through.

[0362] 23. The method according to any one of items 1-22, wherein the diameter of the contractile member is about 20-99% of the diameter of the cell.

[0363] 24. The method according to any one of items 1-23, wherein the diameter of the contractile member is about 60% of the diameter of the cell.

[0364] 25. The method according to any one of items 1-24, wherein the microfluidic channel is one of a plurality of parallel microfluidic channels in the microfluidic system.

[0365] 26. The method according to item 25, wherein the plurality of parallel microfluidic channels comprises at least about 2, 5, 10, 20, 25, 30, 40, 45, 50, 75, 100, 500, 1,000 or 2-1,000 microfluidic channels.

[0366] 27. The method according to any one of items 1-26, wherein the cell is a plurality of cells, and each cell is passed through one of a plurality of parallel microfluidic channels, and each of the plurality of parallel microfluidic channels includes a cell deformable contraction element.

[0367] 28. The method according to any one of items 1-27, wherein

[0368] (a) The diameter of the shrinkage member is approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 2-10 μm or 10-20 μm;

[0369] (b) The length of the shrinkage member is approximately 10, 15, 20, 24, 30, 40, 50, 60, 70, 80, 90, 100, 10-40, 10-50, 10-60 or 10-100 μm;

[0370] (c) The solution is passed through the microfluidic channel using a pressure of approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or 10-100 psi;

[0371] (d) The cells pass through the microfluidic channel at speeds of approximately 300, 400, 500, 600, 700, 800, 900, 100-300, 200-700, 250-400, 100-1000 mm / s, 1-1000 mm / s, 1 m / s, 2 m / s, 3 m / s, 4 m / s, 5 m / s, 6 m / s, 7 m / s, 8 m / s, 9 m / s, 10 m / s, 0.01-5 m / s, 5-10 m / s, or 0.01-10 m / s;

[0372] (e) The microfluidic channel comprises multiple cell deformable contraction elements connected in series;

[0373] (f) The microfluidic channel includes a single-cell deformable contractile element;

[0374] (g) The perturbation of the cell membrane includes a maximum diameter of approximately 1-20, 1-600, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 600 nm; and / or

[0375] (h) The perturbation of the cell membrane having a maximum diameter of about 1-20, 1-600, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500 or 600 nm on the cell membrane lasts for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 1-10 minutes.

[0376] 29. The method according to any one of items 7-11, wherein after delivering the protein and the nucleic acid into the cell,

[0377] (a) Reducing the expression of the target gene in the cells by at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 80, 85, 90, 95, or 99% or more; or

[0378] (b) The cells are multiple cells, and the expression of the target gene in the multiple cells is reduced by at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 80, 85, 90, 95 or 99% or more.

[0379] 30. The method according to item 29, wherein approximately 1, 2, 5, 12, 24, 1–12, 6–12, 6–18, 12–24, or 1–24 hours after delivering the protein and the nucleic acid into the cells,

[0380] (a) Reducing the expression of the target gene in the cells by at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 80, 85, 90, 95, or 99% or more; or

[0381] (b) The cells are multiple cells, and the expression of the target gene in the multiple cells is reduced by at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 80, 85, 90, 95 or 99% or more.

[0382] 31. The method according to any one of items 7-11, wherein after delivering the protein and the nucleic acid into the cell,

[0383] (a) Increasing the expression of the target gene in the cells by at least about 5, 10, 25, 50, 75, 100, 250, 500% or more; or

[0384] (b) The cells are multiple cells, and the expression of the target gene in the multiple cells is increased by at least about 5, 10, 25, 50, 75, 100, 250, 500% or more.

[0385] 32. The method according to item 31, wherein approximately 1, 2, 5, 12, 24, 1–12, 6–12, 6–18, 12–24, or 1–24 hours after delivering the protein and the nucleic acid into the cells,

[0386] (a) Increasing the expression of the target gene in the cells by at least about 5, 10, 25, 50, 75, 100, 250, 500% or more; or

[0387] (b) The cells are multiple cells, and the expression of the target gene in the multiple cells is increased by at least about 5, 10, 25, 50, 75, 100, 250, 500% or more.

[0388] 33. An apparatus for delivering a protein-nucleic acid complex into a cell, comprising at least one microfluidic channel, wherein the channel comprises a contraction length of about 30 μm and a contraction width of about 4 μm.

Claims

1. A method for delivering proteins and nucleic acids into cells, the method comprising: Provide cells in solution; The solution is passed through a microfluidic channel that includes cell deformation and contraction elements; The cell is passed through the contractile member, thereby applying pressure to the cell and causing sufficient disturbance to allow the proteins and nucleic acids to pass through; and The cells are brought into contact with the proteins and nucleic acids before, during, and / or after passing the cells through the contractile element.

2. The method of claim 1, wherein the solution contains the protein and the nucleic acid before, during and / or after passing the cell through the contractile member.

3. The method according to claim 1 or 2, wherein the protein and the nucleic acid form a protein-nucleic acid complex.

4. The method according to any one of claims 1-3, wherein the protein and the nucleic acid are components of the protein-nucleic acid complex, but are not complexed when delivered to the cell.

5. The method of claim 4, wherein the protein and the nucleic acid form a protein-nucleic acid complex after delivery into the cell.

6. The method according to any one of claims 1-4, wherein the protein and the nucleic acid form a protein-nucleic acid complex prior to delivery to the cell.

7. The method according to any one of claims 1-6, wherein the protein and the nucleic acid comprise gene editing components.

8. The method according to any one of claims 3-7, wherein the protein-nucleic acid complex comprises a ribonucleoprotein (RNP).

9. The method of claim 7, wherein (a) The protein is a Cas protein or a Cpf1 protein; and (b) The nucleic acid is a single guide RNA (sgRNA) or CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA).

10. The method of claim 9, wherein the complex is an RNP comprising a Cas protein or a Cpf1 protein and sgRNA, wherein the Cas protein or the Cpf1 protein is complexed with the sgRNA using about 0.5, 2.0, 2.5, or 3.0 moles of the Cas protein or the Cpf1 protein in excess.

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