Compositions and methods for RNA synthesis

Abstract

To provide a method for RNA synthesis.SOLUTION: Provided is a method comprising: providing an RNA polymerase immobilized on a surface; and synthesizing a plurality of RNAs at a rate of extension of at least 50 nucleotides per hour, wherein each of the plurality of RNAs has a preselected sequence, and wherein the synthesizing comprises extending by a single base in an extension reaction.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 104,735, filed October 23, 2020, which is incorporated herein by reference in its entirety. [Background technology]

[0002] RNA-based therapies are an attractive class of drugs for treating various diseases due to the important and diverse roles that RNA molecules play in cells. RNA therapies regulate gene expression by delivering messenger RNA (mRNA) of a target gene or non-coding RNAs such as microRNA (miRNA), small interfering RNA (siRNA), and antisense oligonucleotides (ASO). Significant progress has been made in RNA therapy. Additionally, guide RNAs (gRNAs) for the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) protein system play an essential role in guiding the CRISPR-Cas system to the target site for genome editing. The use of synthetic RNA instead of plasmid DNA provides a reliable approach with minimal off-target activity due to the relatively short half-life of RNA molecules.

[0003] Currently available RNA synthesis methods include chemical synthesis for short RNA molecules and enzymatic synthesis for long RNA molecules, such as in vitro transcription (IVT). While chemical synthesis provides pure, high-quality RNA molecules and offers a wide variety of site-specific custom modifications, it suffers from high costs and slow speed of scale-up. While IVT offers a less expensive solution, it tends to have a high error rate, is labor-intensive, and limits sequence editing. There is a need to develop a fast, accurate, automated, scalable, and cost-effective RNA synthesis platform. Summary of the Invention

[0004] In one aspect, provided herein is a method for synthesizing RNA, the method comprising: providing an RNA polymerase immobilized on a surface; and synthesizing a plurality of gRNAs at an extension rate of at least 50 nucleotides per hour, where each gRNA has a preselected sequence and the synthesizing comprises extending each gRNA by one base in a single extension reaction. In another aspect, provided herein is a method for synthesizing RNA, the method comprising: providing a fusion RNA polymerase, or a functional fragment or variant thereof; and synthesizing a plurality of gRNAs at an extension rate of at least 50 nucleotides per hour, where each gRNA has a preselected sequence and the synthesizing comprises extending each gRNA by one base in a single extension reaction. In some embodiments, the extension rate is at least 50 nucleotides per minute. In some embodiments, the extension rate is at least 50 nucleotides per second.

[0005] In another aspect, provided herein is a nucleic acid library comprising a plurality of purified RNAs and at least one single-stranded DNA (ssDNA) encoding a truncated RNA polymerase promoter region. Further provided herein is a nucleic acid library comprising a plurality of purified guide RNAs (gRNAs) at least 80 nucleotides in length and at least one oligonucleotide 2-10 nucleotides in length. In some aspects, provided herein is a method for generating a nucleic acid library comprising at least 50 guide RNAs (gRNAs), the method comprising: synthesizing at least 50 gRNAs using an RNA polymerase, wherein at least one of the at least 50 gRNAs comprises a spacer sequence complementary to a target sequence in a target gene, and the 5'-terminal nucleotide of the spacer sequence is complementary to the 3'-terminal nucleotide of the target sequence. In some aspects, provided herein is a nucleic acid library comprising at least 50 purified guide RNAs (gRNAs) comprising a gRNA sequence comprising a 5'-terminal guanine (G) analog.

[0006] In some aspects, provided herein are nucleic acid libraries, wherein the nucleic acid library comprises at least 50 RNAs, each of the at least 50 RNAs encoding a different guide RNA (gRNA) sequence, and at least about 90% of the at least 50 RNAs are each present in the nucleic acid library at an amount within 1.5-fold of the average frequency for the at least 50 RNAs in the library. In some embodiments, at least about 95% of the at least 50 RNAs are each present in the nucleic acid library at an amount within 1.5-fold of the average frequency for the at least 50 RNAs in the library. In some embodiments, at least about 99% of the at least 50 RNAs are each present in the nucleic acid library at an amount within 1.5-fold of the average frequency for the at least 50 RNAs in the library.

[0007] In one aspect, provided herein is a modified polypeptide composition comprising a purified RNA polymerase, or a functional fragment or variant thereof, and a purified nucleic acid binding protein, optionally a zinc finger containing protein, or a functional fragment or variant thereof, wherein the purified RNA polymerase and the nucleic acid binding protein are heterologous, and the purified RNA polymerase and the purified nucleic acid binding protein are linked. In another aspect, provided herein is a composition comprising a fusion RNA polymerase, or a functional fragment or variant thereof, and a DNA polynucleotide, wherein the fusion RNA polymerase comprises (i) an RNA polymerase, or a fragment thereof, and (ii) a DNA binding protein, and the RNA polymerase and the DNA binding protein are heterologous.

[0008] In one aspect, provided herein is a modified polypeptide, comprising a variant T7 RNA polymerase or a functional fragment thereof, wherein the variant T7 RNA polymerase comprises at least four variations selected from the group consisting of K172L, P266L, H772R, N748X, R756M, Q758X, and E775V, the positions of which are determined by alignment with SEQ ID NO: 1. In another aspect, provided herein is a modified polypeptide, comprising a variant T7 RNA polymerase or a functional fragment thereof, wherein the variant T7 RNA polymerase comprises at least one variation selected from the group consisting of K172L, P266L, H772R, N748X, R756M, Q758X, and E775V, the positions of which are determined by alignment with SEQ ID NO: 1, and wherein the modified polypeptide is immobilized on a surface.

[0009] In one aspect, provided herein is a device comprising: a surface; a nucleic acid binding protein, or a functional fragment or variant thereof, linked to the surface; T7 RNA polymerase, or a variant thereof, linked to a DNA binding protein; and a DNA template comprising a truncated T7 promoter sequence.

[0010] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief explanation of the drawings]

[0011] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings.

[0012] [Figure 1]FIG. 1 depicts an exemplary workflow for microfluidic polynucleotide synthesis. [Figure 2] FIG. 1 depicts an exemplary process for surface functionalization for microfluidic polynucleotide synthesis. [Figure 3A] 1 depicts a diagram of an exemplary microfluidic polynucleotide synthesis. [Figure 3B] 1 depicts a diagram of an exemplary microfluidic polynucleotide synthesis. [Figure 4] A schematic representation of the DNA template for RNA transcription (top) and its predicted second hairpin structure (bottom) is shown. V represents the template for RNA transcription, W represents the DNA melting site, X represents the sequence that is reverse complementary to and hybridizes with the polymerase promoter sequence Z, Y represents the loop region connecting X and Z, and Z represents the polymerase promoter sequence that hybridizes with X. [Figure 5] An exemplary guide RNA (gRNA) is depicted that contains an additional 5' guanine (G) nucleotide that does not match the target DNA sequence. [Figure 6] An exemplary guide RNA (gRNA) that perfectly matches the target DNA sequence is depicted. [Figure 7] A computer system is described. [Figure 8] FIG. 1 is a block diagram illustrating the structure of a computer system. [Figure 9] 1 illustrates a network configured to incorporate multiple computer systems, multiple mobile phones and personal digital assistants, and network attached storage (NAS). [Figure 10] 1 is a block diagram of a multiprocessor computer system using a shared virtual address memory space. [Figure 11] Plots comparing guide RNA (gRNA) purity are shown. Each graph represents gRNA produced by the microfluidic polynucleotide synthesis method (orange), gRNA produced by chemical synthesis (blue), or gRNA produced by in vitro transcription (green). The x-axis is elution time, and the y-axis is normalized fluorescence units. [Figure 12] Plots comparing the purity of gRNAs are shown. Each graph represents gRNAs produced by the microfluidic polynucleotide synthesis method (blue) or three different in vitro transcription (IVT) kits (green, red, and purple). The x-axis is run time (seconds), and the y-axis is normalized fluorescence units. [Figure 13] Figure 1 depicts plots comparing gRNA purity. Each graph represents eight different gRNA samples produced by the microfluidic polynucleotide synthesis method. The X-axis is run time (seconds), and the Y-axis is normalized fluorescence units. [Figure 14] Depicts DNA agarose gel electrophoresis analysis of an in vitro cleavage assay for CRISPR-Cas9 target DNA cleavage at various time points. Lane 1: ladder; Lane 2: blank; Lane 3: gRNA produced by in vitro transcription using a hairpin DNA template at 0 minutes; Lane 4: commercially available in vitro transcribed gRNA1 at 5 minutes; Lane 5: commercially available chemically synthesized gRNA2 at 5 minutes; Lane 6: gRNA produced by in vitro transcription using a hairpin single-stranded DNA template at 5 minutes. [Figure 15A] Figure 1 depicts a Tracking Indels by Degradation of Single-Guide RNA (sgRNA) (TIDE) analysis showing the spectrum of insertions and / or deletions (indels) and their frequency. The X-axis shows the spectrum of indels, and the Y-axis shows the percentage of sequences (%). The overall editing efficiency (%) is shown in the upper left corner, and the coefficient of determination (r²) is shown in the upper right corner. [Figure 15B] Figure 1 depicts the Tracking Indels by Degradation of sgRNA (TIDE) analysis showing the spectrum of indels and their frequency. The X-axis shows the spectrum of indels, and the Y-axis shows the percentage of sequences (%). The overall editing efficiency (%) is shown in the upper left corner, and the coefficient of determination (r2) is shown in the upper right corner. [Figure 16A]Figure 1 depicts a plot of real-time measurements of ribonucleotide transcription comparing the enzymatic activity of commercially available enzymes and fusion enzymes containing RNA polymerase and DNA binding domains. The X-axis shows time, and the Y-axis shows the fluorescence reading in relative fluorescence units (RFU). [Figure 16B] A plot of real-time measurement of ribonucleotide transcription is depicted, with the X-axis showing time and the Y-axis showing fluorescence readings in relative fluorescence units (RFU). [Figure 17A] A plot of real-time measurement of ribonucleotide transcription is depicted, with the X-axis showing time and the Y-axis showing fluorescence readings in relative fluorescence units (RFU). [Figure 17B] Figure 1 depicts a plot of RNA yield improvement comparing truncated promoters with conventional promoters, with the X-axis showing the length of the truncated promoter and the Y-axis showing the fold improvement. DETAILED DESCRIPTION OF THE INVENTION

[0013] Provided herein are methods, compositions, and devices for generating polynucleotides using a microfluidic workflow that allows for rapid, highly efficient, accurate, and pure RNA production. The methods, compositions, and devices described herein provide a means for synthesizing polynucleotides using efficient RNA polymerase enzymes, microfluidic dynamics, enhanced kinetics, and reduced off-target effects. In some embodiments, the methods, compositions, and devices described herein provide a single-unit, cartridge-based, portable system for polynucleotide synthesis.

[0014] definition

[0015] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless expressly stated otherwise. It should also be noted that the term "or" is generally used in its sense to include "and / or" unless the content clearly dictates otherwise. As used herein, the terms "and / or," "any combination thereof," and their grammatical equivalents can be used interchangeably. These terms can convey that any combination is specifically contemplated. For illustrative purposes only, the phrase "A, B, and / or C" or "A, B, C, or any combination thereof" can mean "A alone, B alone, C alone, A and B, B and C, A and C, and A, B, and C." The term "or" can be used conjunctively or disjunctively unless the context specifically dictates disjunctive use.

[0016] The terms "about" or "approximately" can mean within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean 1 or more than 1 standard deviation per practice in the art. Alternatively, "about" can mean within up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5-fold, or more preferably within 2-fold of a value. When particular values ​​are described in this application and claims, unless otherwise specified, the term "about" meaning within an acceptable error range of the particular value should be assumed.

[0017] Throughout this disclosure, numerical characteristics are presented in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of any embodiment. Accordingly, the description of a range should be considered to specifically disclose all possible subranges and individual numerical values ​​within that range, to two decimal places of the lower limit, unless the context clearly dictates otherwise. For example, the description of a range such as 1 to 6 should be considered to specifically disclose subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual numerical values ​​within that range, for example, 1.1, 2, 2.3, 5, and 5.9. This applies regardless of the broadness of the range. The upper and lower limits of these intervening ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the invention, unless the context clearly dictates otherwise.

[0018] As used in this specification and claims, the words "comprising" (and "comprising" forms such as "comprise" and "comprises"), "having" (and "having" forms such as "have" and "has"), "including" (and "including" forms such as "includes" and "include"), or "containing" (and "containing" forms such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. Any embodiment discussed herein can be implemented with respect to the methods or compositions of the present disclosure, and vice versa. Furthermore, the compositions of the present disclosure can be used to achieve the methods of the present disclosure.

[0019] References herein to "some embodiments," "embodiments," "one embodiment," or "other embodiments" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least some embodiments of the present disclosure, but not necessarily in all embodiments of the present disclosure. To facilitate understanding of this disclosure, a number of terms and phrases are defined below.

[0020] As used herein, the terms "preselected sequence," "predefined sequence," or "predetermined sequence" are used interchangeably. The terms mean that the polynucleic acid sequence is known and is selected prior to the synthesis or assembly of the polynucleic acid. In particular, the various aspects of the invention described herein are primarily directed to the preparation of nucleic acid molecules, where the polynucleic acid sequence is known and is selected prior to the synthesis or assembly of the nucleic acid molecule.

[0021] The nomenclature used to describe a polypeptide or protein follows conventional practice, with the amino group of each amino acid residue at the left (amino- or N-terminus) and the carboxyl group at the right (carboxy- or C-terminus). When amino acid residue positions are referenced in a polypeptide or protein, they are numbered from amino to carboxyl, with the first position being the residue at the amino terminus of the polypeptide or protein of which it may be a part. The amino acid sequences of peptides described herein are typically designated using standard single-letter symbols (A is alanine, C is cysteine, D is aspartic acid, E is glutamic acid, F is phenylalanine, G is glycine, H is histidine, I is isoleucine, K is lysine, L is leucine, M is methionine, N is asparagine, P is proline, Q is glutamine, R is arginine, S is serine, T is threonine, V is valine, W is tryptophan, and Y is tyrosine).

[0022] Certain details in this description are set forth to provide a thorough understanding of various embodiments. However, it will be understood by those skilled in the art that the present disclosure may be practiced without these details. In other instances, well-known structures have not been depicted or shown in detail to avoid unnecessarily obscuring the description of the embodiments. Unless the context otherwise requires, throughout the following specification and claims, the word "comprise" and variations thereof (e.g., "comprises" or "comprising") are intended to be interpreted in an open and inclusive sense, i.e., "including, but not limited to." Additionally, headings provided herein are for convenience only and do not interpret the scope or meaning of the present disclosure.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below.

[0024] Microfluidic polynucleotide synthesis, devices, and systems

[0025] Provided herein are methods, compositions, devices, and systems for producing polynucleotides in a fast, pure, and accurate manner. Additionally, provided herein are devices and systems for producing polynucleotides using a single-unit cartridge-based system that enables highly accurate and efficient polynucleotide synthesis using microfluidics, enhanced kinetics, and reduced off-target effects. The terms oligonucleotide, oligo, and polynucleotide are defined synonymously throughout. The nucleic acid (e.g., DNA or RNA) libraries described herein may contain multiple polynucleotides that collectively encode coding or non-coding RNA sequences. In some examples, the coding sequence may include messenger RNA (mRNA). In some examples, the non-coding sequence may include guide RNA (gRNA), microRNA (miRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), piwi-interacting RNA (piRNA), RNA aptamer, transfer RNA (tRNA), or antisense oligonucleotide (ASO). In some examples, the nucleic acid library contains multiple polynucleotides, each encoding a different sequence.

[0026] Provided herein are methods, compositions, devices, and systems for the production of polynucleotides using microfluidic workflows. Microfluidics utilizes the science of controlling and manipulating fluids in the μL to pL range within a network of geometrically constrained channels on a small scale (10-100 μm), where capillary permeation directs mass transport. Microfluidics offers many advantages, including reduced sample and reagent consumption, shorter experiment times, and reduced overall application costs. Additionally, microfluidics enables the automation and miniaturization of polynucleotide synthesis, improving precision and accuracy of synthesis and enabling extremely rapid synthesis by simultaneously operating multiple chambers in a single microfluidic chip or cartridge.

[0027] In an exemplary workflow, the microfluidic polynucleotide synthesis in the cartridge described herein can include three stages: surface functionalization, RNA synthesis, and RNA purification and quality control (Figure 1). In Stage 1, the surface is functionalized to set up a transcription system in the microfluidic cartridge. The surface can be functionalized by surface activation with functional groups, including, but not limited to, N-hydroxysuccinimide ester (NHS) functional groups, trifluoroacetic anhydride (TFAA) functional groups, or glutaraldehyde (GA) functional groups. For example, to support polynucleotide attachment and synthesis, surfaces such as magnetic or agarose beads activated with standard N-hydroxysuccinimide (NHS) functional groups are provided in each reaction chamber of the microfluidic cartridge (Figures 1 and 2). The NHS functional groups (201) can react with primary amines on target proteins, e.g., DNA-binding proteins such as streptavidin (strep) (202) or functional fragments thereof, to form stable amide bonds (Step 1 in Figure 2). The NHS reaction may then be quenched before the addition of other necessary components for microfluidic polynucleotide synthesis (e.g., transcription proteins). This two-step reaction protects other transcription complex proteins, including polymerases, from the NHS reaction chemistry. The resulting immobilized proteins are covalently conjugated to beads (203) and may be highly resistant to leaching from the bead surface. Each bead represents one chamber in which one synthesis reaction can occur in Figure 2, although more than one bead may be provided in each chamber. The transcription complex (204) is then assembled (step 2 in Figure 2). As an example of a polymerase, T7 RNA polymerase (205) is added to and binds to a DNA binding protein or functional fragment thereof (e.g., strep, or zinc finger array protein) through fusion partner interactions (206) (blue and purple triangles in step 2 of Figure 2) to form a stable transcription complex (204), optionally with linkers linking the DNA binding protein to fusion partner 1 and the polymerase to fusion partner 2.The DNA template is then added to and binds to a DNA-binding protein or functional fragment thereof (step 3 in Figure 2). For example, the DNA template can be biotinylated (207) for interaction with strep as shown in Figure 2. In another example, the DNA template can contain a DNA-binding target sequence (e.g., a zinc finger array (ZFA) binding sequence) for interaction with a polymerase through a DNA-binding protein (e.g., a ZFA) fused to the polymerase. A DNA template as described herein can contain double-stranded DNA (dsDNA), partially double-stranded DNA, or single-stranded DNA (ssDNA). The DNA template can be ssDNA containing a secondary structure, such as a hairpin (Figure 4). The DNA templates described herein can contain a promoter region to which RNA polymerase binds and initiates RNA synthesis. In some embodiments, the promoter regions described herein can include essential and / or non-essential promoter regions. The RNA polymerases described herein may include bacteriophage RNA polymerases, bacterial RNA polymerases, or eukaryotic RNA polymerases. Non-limiting examples of phage RNA polymerases include T3 RNA polymerase, T7 RNA polymerase, KP34 RNA polymerase, N4 RNA polymerase, and SP6 RNA polymerase. For example, the RNA polymerase may be T7 RNA polymerase as shown in FIG. 2. The RNA polymerases described herein may include wild-type or variant RNA polymerases containing one or more amino acid substitutions that may affect DNA recognition, DNA binding affinity, polymerase activity, or polymerase stability. As described herein, the RNA polymerase may be provided as a fusion protein comprising an RNA polymerase (e.g., T7 RNA polymerase or a variant thereof) and a heterologous DNA-binding protein or domain (e.g., strep or ZFA) capable of recognizing and binding to a DNA template for microfluidic RNA synthesis, as shown in FIG. 2. In some instances, the polymerase may be dried into the biomatrix in the cartridge.In this example, the polymerase can be rehydrated before using the cartridge for microfluidic polynucleotide synthesis. In some examples, cartridges containing polymerase can be shipped on dry ice, ice, or at room temperature to preserve the polymerase in the cartridge. In some examples, the DNA template can be directly linked to a surface such as a bead. In some examples, the surface can include magnetic beads, agarose beads, fused silica, sol-gel, silica polymer, silica monolith, cellulose, agar, acrylamide, gold beads, or a gel matrix. In some examples, the solid surface can include a gel matrix for encapsulation or entrapment of RNA polymerase, DNA template, or nucleic acid binding protein. In some examples, RNA polymerase can bind to a DNA template linked to a surface.

[0028] In stage 2, transcription reaction reagents (301) are injected into the microfluidic cartridge by a syringe pump and flowed over transcription complexes (302) containing immobilized RNA polymerase (303) and immobilized DNA templates (304) in each reaction chamber for microfluidic RNA synthesis (Figure 3A). The transcription reaction reagents include, but are not limited to, a mixed group, a 5' initiator oligo, and a 3' terminator oligo. In some instances, modified nucleobases may be used to synthesize RNA containing one or more modifications. For example, mixed groups may include natural nucleosides, nucleoside analogs, chemically modified bases, biologically modified bases, intercalated bases, modified sugars, and / or modified phosphate groups. For microfluidic RNA synthesis, a thermoelectric Peltier element heater (305) may be utilized to maintain the temperature of the reaction chamber at 37 °C (Figure 3B). As transcription reaction reagents flow unidirectionally through each reaction chamber, RNA is produced and removed from the reaction chamber in approximately seconds as controlled by the flow rate (Figure 3B).

[0029] In stage 3, newly synthesized polynucleotides (e.g., RNA) are extracted and purified using a micropillar array and / or isotachophoresis (ITP) purification process, or any known purification method known to those skilled in the art, followed by spectroscopic quantification of UV using a UV LED and UV detector (Figure 1). ITP is a robust electrophoretic separation and preconcentration technique that generates a strong electric field gradient, allowing selective focusing and separation of charged species based on their electrophoretic mobility. The chemistry of the ITP electrolyte can be controlled to purify RNA within a target size range. Alternatively, capillary electrophoresis (CE), a separation technique in which charged species are separated based on their charge and size by their different migration rates in an electric field, can be used for purification. The completed polynucleotide products can be stored, and nucleic acid libraries can be synthesized. Synthesized polynucleotides can also be purified using a micropillar array. The array of micropillars can capture or confine the synthesized polynucleotides while other components of microfluidic polynucleotide synthesis continuously flow through the micropillars in wash buffer. Polynucleotides captured or entrapped in the micropillars can be extracted using an elution buffer.

[0030] Provided herein are methods for storing polynucleotides synthesized using the microfluidic polynucleotide synthesis methods, compositions, and / or devices described herein. The synthesized polynucleotides can be used immediately after synthesis or stored for later use. In some embodiments, the synthesized polynucleotides can be stored in a refrigerator or freezer. For example, the synthesized polynucleotides can be stored at any temperature known to be suitable for polynucleotide storage. In some embodiments, the synthesized polynucleotides can be stored at 4°C, -20°C, or -80°C. In some embodiments, the synthesized polynucleotides can be dried in a biomatrix using a vacuum pump. In this embodiment, the dried polynucleotide product can be rehydrated before use.

[0031] In some embodiments, the device for microfluidic polynucleotide synthesis further includes a piezoelectric vibrator, a rotary valve, a Peltier heater, a voltage controller, a syringe pump, a UV LED and a sensor, or a vacuum pump. In some embodiments, the piezoelectric vibrator may be used to refine the mixing of components for microfluidic polynucleotide synthesis, such as transcription reaction reagents. In some embodiments, one or more rotary valves may be used to select initiator oligos. In some embodiments, a Peltier heater may be used to maintain the optimal temperature for microfluidic polynucleotide synthesis. In some embodiments, a voltage controller may be used to apply voltage for ITP purification. In some embodiments, a UV LED and a sensor may be used to detect and quantify synthesized polynucleotides. In some embodiments, a vacuum pump may be used to dry the synthesized polynucleotides.

[0032] Provided herein are compositions, methods, devices, and systems for synthesizing RNA, which enable rapid synthesis of RNA. Provided herein are methods for synthesizing multiple RNAs at an extension rate of at least 50 nucleotides per hour. Also provided herein are compositions, methods, devices, and systems for synthesizing RNAs with preselected sequences. Also provided herein are compositions, methods, devices, and systems for synthesizing RNAs, where the synthesis comprises extending one base at a time in a single extension reaction. As described herein, RNAs may include guide RNAs (gRNAs), messenger RNAs (mRNAs), small interfering RNAs (siRNAs), microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), RNA aptamers, transfer RNAs (tRNAs), or antisense oligonucleotides (ASOs).

[0033] Provided herein are compositions, methods, devices, and systems for synthesizing multiple RNAs at an extension rate of at least 50 nucleotides per hour. For example, the extension rate may be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 11000, 12000, 13000, 1400, 2600, 2700, 2800, 3900, 4000, 4500, 5000, 5500, 6000, 6500, 7000 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000 nucleotides. In some embodiments, the extension rate is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 4100, 4200, 4300, 4400, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 31 In some embodiments, the extension rate is at least 400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000 nucleotides per minute. In some embodiments, the extension rate is at least 50 nucleotides per minute. In some embodiments, the extension rate is at least 3000 nucleotides per minute. In some embodiments, the extension rate is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides per second. In some embodiments, the extension rate is at least 50 nucleotides per second.

[0034] Provided herein are devices with multi-feature surfaces for microfluidic polynucleotide synthesis, offering technical advantages for polynucleotide synthesis, such as improved enzymatic reaction kinetics and accuracy. The devices provided herein may include pneumatic and peristaltic pumping systems for programmable fluid manipulation. Peristaltic pumping systems may operate continuously or deliver small volumes of fluid. In some instances, minimizing circulating flow rates is desirable. In some instances, peristaltic pumping systems may incorporate micropump structures into microfluidic circuits. Provided herein are devices that include a microfluidic cartridge, an inlet, an outlet, a membrane, a pneumatic microvalve, a pneumatic micropump, or one or more holes for a pneumatic microvalve and a pneumatic micropump. The channels and holes in the devices described herein may be created by laser ablation techniques.

[0035] Further provided herein are devices including microfluidic cartridges containing flow cells or chambers. The microfluidic cartridges described herein may include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 flow cells or chambers. In some examples, the microfluidic cartridges may include at least three flow cells or chambers. In some examples, the microfluidic cartridges may include a reaction chamber, a collection chamber, or an ITP chamber. In some embodiments, the microfluidic cartridge includes a reaction chamber, a collection chamber, and an ITP chamber. The reaction chambers and collection chambers described herein may utilize glass wafers or silicon wafers. In some examples, the reaction chambers and collection chambers may utilize glass wafers and silicon wafers. The ITP chambers described herein may utilize glass wafers. In some examples, the ITP chambers may utilize glass wafers only with an applied voltage for ITP.

[0036] As described herein, the silicon wafer can have a diameter of 25 to 450 mm. For example, the silicon wafer can have a diameter of 25, 30, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, or 450 mm. In some embodiments, the silicon wafer can have a diameter of 200 mm. As described herein, the silicon wafer can have a thickness or height of 100 μm to 15 mm. For example, the silicon wafer can have a thickness or height of 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1,000 μm. In some embodiments, the silicon wafer can have a thickness or height of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mm.

[0037] As described herein, the glass wafer can have a diameter of 25 to 450 mm. For example, the glass wafer can have a diameter of 25, 30, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, or 450 mm. In some embodiments, the glass wafer can have a diameter of 200 mm. As described herein, the glass wafer may have a thickness or height of 0.1 to 1.3 mm, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, or 1.3 mm.

[0038] As described herein, a silicon wafer or glass wafer can include one or more dies. In some examples, each of the one or more dies has a width of 0.5 to 70 mm. For example, each of the one or more dies can have a width of 0.5, 0.6, 0.7, 0.8, 0.9, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 mm. In some embodiments, each of the one or more dies has a width of 25 mm. In some examples, each of the one or more dies has a height of 0.5 to 70 mm. For example, each of the one or more dies can have a height of 0.5, 0.6, 0.7, 0.8, 0.9, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 mm. In some embodiments, each of the one or more dies has a height of 37 mm. In some embodiments, each of the one or more dies has a height of 75 mm. In some examples, the silicon wafer or glass wafer can have a die per wafer (DPW) count of 1 to 30. For example, the number of DPWs can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000. In some embodiments, the number of DPWs is 8. In some embodiments, the number of DPWs is 20.

[0039] Provided herein are devices comprising multiple microchambers, wherein the height or depth of the chambers is 100 nm to 100 μm. For example, the height or depth of the chambers can be 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1,000 nm. For example, the height or depth of the chambers can be 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 μm. Further provided herein are devices comprising multiple microchambers, wherein the width of the chambers is 100 nm to 100 μm. For example, the width of the chamber can be 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1,000 nm. For example, the width of the chamber can be 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 μm.

[0040] Provided herein are devices including one or more reaction chambers, each containing a DNA template. The DNA template can be added to the reaction chamber after assembly of the microfluidic cartridge. In some examples, the microfluidic cartridge can include two or more reaction chambers, each containing the same DNA template, thereby producing multiple RNAs (e.g., gRNA, miRNA, siRNA, mRNA, etc.) having the same RNA sequence. In some examples, the microfluidic cartridge can include two or more reaction chambers, each containing a different DNA template, thereby producing multiple groups of two or more RNAs, each containing a different RNA sequence. For example, the microfluidic cartridge can include three reaction chambers, each containing a different DNA template, thereby producing multiple RNAs with three different sequences.

[0041] Devices for microfluidic polynucleotide synthesis are provided herein, and fluids can flow from an inlet to an outlet of the devices described herein for microfluidic polynucleotide synthesis. As fluids flow from the inlet to the outlet, various flow rates are used herein for transcription reaction reagents for microfluidic polynucleotide synthesis or purification of synthesized polynucleotides. In some examples, the flow rate is about 0.001 to 1000 nL / second (nL / s). In some examples, the flow rate is about 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 5, 10, 15, 20, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 500, 800, or about 1000 nL / second (nL / s). In some examples, the flow rate is at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 5, 10, 15, 20, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 500, or at least 800 nL / second (nL / s). In some examples, the flow rate is less than or equal to 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 5, 10, 15, 20, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 500, 800, or 1000 nL / second (nL / s). In some examples, the flow rate is about 1, 5, 10, 15, 20, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, or about 500 μL / second (μL / s). In some examples, the flow rate is at least 1, 5, 10, 15, 20, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, or at least 500 μL / s. In some examples, the flow rate is no more than 5, 10, 15, 20, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, or no more than 500 μL / s.In some examples, the flow rate is about 1-500 μL / s, about 5-500 μL / s, about 10-400 μL / s, about 20-300 μL / s, about 50-500 μL / s, about 50-400 μL / s, about 50-300 μL / s, about 75-300 μL / s, about 100-400 μL / s, about 200-500 μL / s, or about 40-350 μL / s. In some examples, the flow rate is about 40-350 μL / s. In some examples, the flow rate is about 75-250 μL / s. In some examples, the flow rate is about 50-400 μL / s.

[0042] Devices for microfluidic polynucleotide synthesis are provided herein, and the devices can include channels having widths, heights, or diameters of 1 μm to 1 cm. For example, channels on a microfluidic polynucleotide synthesis device can have widths, heights, or diameters of at least 1, 5, 10, 15, 20, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, or at least 950 μm. For example, the channels on a microfluidic polynucleotide synthesis device may be: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 , 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 3 00, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 73 In some embodiments, the channels on the microfluidic polynucleotide synthesis device can have a total volume of about 1 μL to 100 mL. For example, channels on a microfluidic polynucleotide synthesis device can have a total volume of at least about 1, 5, 10, 15, 20, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, or at least about 950 μL.In some embodiments, the channels on the microfluidic polynucleotide synthesis device can have a total volume of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mL.

[0043] A device for microfluidic polynucleotide synthesis is provided herein, and the synthesized polynucleotides can be purified using an ITP purification process. The voltage used for the ITP purification process can depend on the length of the column or channel used. For example, the voltage can be 10 to 500 V. For example, the voltage can be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 130 , 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500V.

[0044] A device for microfluidic polynucleotide synthesis is provided herein, and synthesized polynucleotides can be purified using an array of micropillars. The micropillars can have a diameter of 0.1 to 10.0 μm. For example, the micropillars can have a diameter of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0, 8.0, 9.0, 9.5, or 10.0 μm. In some embodiments, the height of the micropillars can be 10 to 50 μm. For example, the height of the micropillars can be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 μm. In some embodiments, the array of micropillars can include micropillars spaced 100 to 1500 nm apart. For example, the micropillars can be spaced 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, or 1500 nm apart.

[0045] Provided herein are devices for microfluidic polynucleotide synthesis, where the surface chemistry and fluid dynamics of microfluidic polynucleotide synthesis can increase enzymatic reaction kinetics, enhance purity, and enable RNA modifications, including 5'-end, 3'-end, and internal chemical modifications. Provided herein are devices for microfluidic polynucleotide synthesis that allow integrated RNA purification and quality control, thereby reducing production costs.

[0046] DNA template

[0047] Methods, compositions, devices, and systems for producing polynucleotides using DNA templates are provided herein. The DNA templates described herein can be double-stranded DNA (dsDNA), partially double-stranded DNA, or single-stranded DNA (ssDNA). DNA templates can include, but are not limited to, linearized plasmid constructs engineered by cloning, such as plasmid vectors, PCR products, oligonucleotides, such as two complementary oligonucleotides annealed together, or complementary DNA (cDNA) templates generated from RNA precursors by first-strand and second-strand synthesis. The DNA template can include a promoter region through which RNA polymerase binds and initiates RNA synthesis. For example, T7 RNA polymerase requires a dsDNA promoter to initiate transcription. Because the unwinding process of the double-stranded template is not required, ssDNA templates are preferable to dsDNA templates for transcription because ssDNA requires less energy and is less likely to separate from the template, allowing for faster initiation and elongation without requiring a dsDNA template. The ssDNA templates described herein may contain secondary structures to create a partially double-stranded promoter region for recognition by RNA polymerase. Examples of secondary structures include, but are not limited to, stems, pseudoknots, hairpin loops, internal loops, multi-branched loops, and bulge loops. In some embodiments, the promoter regions described herein may contain essential and / or non-essential promoter regions. An exemplary ssDNA template is shown in FIG. 4 and includes, from 5' to 3', a template sequence for RNA transcription (401), a DNA melting region (402), a promoter sequence (403), a loop (404) connecting (403) and (405), and a reverse-complementary counterpart of the promoter sequence (405). When (405) hybridizes to (403), the ssDNA template forms a secondary structure, e.g., a hairpin, resulting in a partially double-stranded region that can be recognized by RNA polymerase. In some embodiments, the secondary structure includes a hairpin. In some embodiments, an exemplary sequence for the hairpin structure may include a sequence comprising SEQ ID NO:7.

[0048] Provided herein are DNA templates for microfluidic polynucleotide synthesis, comprising a promoter sequence as described herein. The DNA templates described herein can comprise a promoter sequence corresponding to the RNA polymerase selected for RNA synthesis. For example, the DNA template can comprise a phage promoter sequence, such as a T3 promoter sequence, a T7 promoter sequence, a KP34 promoter sequence, an N4 promoter sequence, or an SP6 promoter sequence, depending on the RNA polymerase selected for RNA synthesis. Provided herein are DNA templates comprising a T7 promoter sequence. The DNA templates provided herein can comprise an essential T7 promoter and / or a non-essential T7 promoter. In some embodiments, the T7 promoter sequence comprises an essential T7 promoter sequence. In some embodiments, the T7 promoter sequence comprises a non-essential T7 promoter sequence. In some embodiments, the T7 promoter sequence comprises an essential T7 promoter sequence and a non-essential T7 promoter sequence. In some embodiments, the essential T7 promoter sequence comprises a sequence comprising SEQ ID NO: 15. In some embodiments, the non-essential T7 promoter sequence comprises a sequence comprising SEQ ID NO: 16. Further provided herein are DNA templates comprising truncated promoter sequences. In some examples, the DNA templates described herein may comprise a truncated T7 promoter sequence. In one example, the truncation may be on the 5' end of the T7 promoter sequence. In another example, the truncation may be on the 3' end of the T7 promoter sequence. In some embodiments, the T7 promoter sequence comprises a sequence comprising SEQ ID NO: 3. In some embodiments, the T7 promoter sequence comprises a sequence comprising SEQ ID NO: 4. In some embodiments, the T7 promoter sequence comprises a sequence comprising SEQ ID NO: 5. In some embodiments, the T7 promoter sequence comprises a sequence comprising SEQ ID NO: 6. In some embodiments, the truncation may be in a non-essential T7 promoter sequence.

[0049] Provided herein are DNA templates for microfluidic polynucleotide synthesis, further comprising a DNA-binding target sequence recognized by a DNA-binding protein or DNA-binding domain. In some examples, the DNA-binding target sequence recognized by the DNA-binding protein or DNA-binding domain can comprise a zinc finger array (ZFA)-binding sequence. In some embodiments, the DNA template can comprise one or more copies of the ZFA-binding sequence. For example, the DNA template can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies of the ZFA-binding sequence. As described herein, the DNA template can comprise one or more copies of the ZFA-binding sequence, and the one or more copies of the ZFA-binding sequence can be arranged in tandem with a nucleotide linker or insert between each of the one or more copies of the ZFA-binding sequence (e.g., 5'-ZFA-nucleotide linker-ZFA-nucleotide linker-...-3'). In some examples, the nucleotide linker or insert can comprise a length of 2 to 30 nucleotides. For example, the nucleotide linker or insert can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the nucleotide linker or insert comprises 8, 9, or 10 nucleotides in length. Provided herein are DNA templates comprising copies of one or more ZFA-binding sequences upstream or 5' to the promoter sequence (e.g., 5'-(ZFA-nucleotide linker) n -promoter sequence-3', where n is an integer from 1 to 10. In some embodiments, the promoter sequence is a truncated promoter sequence.

[0050] The present invention provides a DNA template for programmable RNA synthesis, which comprises one or more DNA binding target sequences (e.g., one or more copies of ZFA binding sequences) and a truncated promoter sequence.The DNA template described herein can be used in a programmable RNA synthesis system that utilizes a fusion protein comprising a DNA binding domain (e.g., ZFA) and an RNA polymerase (e.g., T7 RNA polymerase).In this system, binding affinity is an important factor for RNA transcription, and RNA synthesis can be programmed by manipulating copies of DNA binding target sequences, such as ZFA binding sequences, to match the binding affinity of the fusion protein to the DNA template.

[0051] As described herein, the DNA template can be of various lengths. For example, the DNA template can be at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, or at least 300 nucleotides in length. In some examples, the DNA template is at most 70, at most 80, at most 90, at most 100, at most 120, at most 140, at most 160, at most 180, or at most 200 nucleotides in length. In some examples, the DNA template is about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 120, about 140, about 160, about 180, about 200, about 250, or about 300 nucleotides in length. In some examples, the DNA template is at least 60 nucleotides in length. In some embodiments, the DNA template is at least 120 nucleotides in length. In some embodiments, the DNA template is at most 80 nucleotides in length.

[0052] The DNA template as described herein may comprise a sequence for synthesizing coding RNA or non-coding RNA. Non-limiting examples of coding RNA include mRNA, and non-limiting examples of non-coding RNA include gRNA, miRNA, siRNA, shRNA, and ASO. For example, the DNA template may comprise a sequence for synthesizing gRNA, which can be used to target specific genes involved in diseases or disorders for CRISPR-Cas9-mediated gene editing. In some examples, the target gene is a gene involved in cancer, such as acute lymphocytic carcinoma, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer, breast cancer, anal, anal canal, rectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck, gallbladder, or pleural cancer, nose, nasal cavity, or middle ear cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid carcinoma, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, The gene may include genes found in Hodgkin's lymphoma, hypopharyngeal cancer, renal cancer, laryngeal cancer, leukemia, liquid tumors, liver cancer, lung cancer, lymphoma, malignant mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal carcinoma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, peritoneal, omental, and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer, and / or bladder cancer.In some examples, the target genes include MAGE-1, MAGE-2, MAGE-3, CEA, tyrosinase, midkine, BAGE, CASP-8, β-catenin, CA-125, CDK-1, ESO-1, gp75, gplOO, MART-1, MUC-1, MUM-1, p53, PAP, PSA, PSMA, ras, trp-1, HER-2, TRP-1, TRP-2, IL13Ralpha, IL13Ralpha2, AIM-2, AIM-3, NY-ESO-1, C9orfll2, SART1, SART2, SART3, BRAP, RTN4, GLEA2, T KS2, KIAA0376, ING4, HSPH1, C13orf24, REIC, RBPSUH, C6orfl53, KTR, NSEP1, U2AF1L, CY L2, TPR, SOX2, GOLGA, BMI1, COX-2, EGFRvIII, EZH2, LICAM, Livin, MRP-3, Nestin, OLIG2, ART1, ART4, B-cyclin, Glil, Cav-1, Cathepsin B, CD74, e-cadherin, EphA2 / Eck, Fra -l / Fosll, GAGE-1, ganglioside / GD2, GnT-V, β1,6-N, Ki67, Ku70 / 80, PROX1, PSCA, SOX10, SOX11, survivin, UPAR, mesothelin, WT-1, RET, ERBB2 or HER2, PDGF-Rβ, SRC, PRAD1 / These may include Icrin D1, C-Myc, BCL2, ABL, MDM2, p110α, B-RAF, IDH1, IDH2, JAK2, KIT, MET, FLT-3, VEGF, VEGFR, SKY, p53, RB, BCL2, SWI / SNF, RAP1A, DCC, K-REV, WT1, TBR-11, INK4A / ARF, SMAD2, SMAD3, SMAD4 / DPC4, e-cadherin, APC, VHL, PTEN / MMAC1, NF1, NF2, BRCA1, BRCA2, MSH2, MLH1, PMS1, PMS2, REIC, SDHB, SDHD, and / or GP43 / Merlin.In some examples, target genes may include genes involved in immune disorders, such as PTPN22, TRAF1-C5, PADI4, STAT4, TNF, IL-1, IL-6, IL-4, IL-5, OPN, PRF1, IFIH1, TRAF3IP2, IL12A, IL12RB2, AIRE, Fas, FasL, caspase 10, caspase 8, PRKCD, NRAS, CTLA-4, FOXP3, LRBA, HLA-DQ8, INS, IL2RA, SH2B3, ERBB3, PTPN2, CLEC16A, IL18RAP, CTSH, CD226, IL2RA, PRKCQ, IL2, BACH2, UBASH3A, RGS1, IL17RA, CIQTNF6, TNFAIP3, TYK2, and / or TAGAP. In some examples, the target gene may include a gene that needs to be knocked down, knocked out, modified, or edited. In some examples, the target gene may include a gene that needs to be transcriptionally regulated, e.g., up-regulated or down-regulated.

[0053] In some embodiments, the amount of RNA transcribed using a DNA template described herein may be increased compared to the amount of RNA transcribed using a conventional DNA template, i.e., an uncharacterized DNA template described herein. For example, the amount of RNA transcribed using a DNA template described herein may be increased by at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3.0-fold, 3.1-fold, 3.2-fold, 3.3-fold, 3.4-fold, 3.5-fold, 4.0-fold, 5.0-fold, 6.0-fold, 7.0-fold, 8.0-fold, 9.0-fold, 10.0-fold, 11.0-fold, 12.0-fold, 13.0-fold, 14.0-fold, 15.0-fold, 16.0-fold, 17.0-fold, 18.0-fold, 19.0-fold, 20.0-fold, 21.0-fold, 22.0-fold, 23.0-fold, 24.0-fold, 25.0-fold, 26.0-fold, 27.0-fold, 28.0-fold, 29.0-fold, 30.0-fold, 31.0-fold, 32.0-fold, 33.0-fold, 34.0-fold, 35.0-fold, 36.0-fold, 37.0-fold x, 4.5x, 5.0x, 5.5x, 6.0x, 7.0x, 8.0x, 9.0x, 9.5x, 10.0x, 10.5x, 11.0x, 11.5x, 12.0x, 12.5x, 13.0x, 14.5x, 15.0x, 15.5x, 16.0x, 16. 5x, 17.0x, 17.5x, 18.0x, 18.5x, 19.0x, 19.5x, 20x, 21x, 22x, 23x, 24x, 25x, 26x, 27x, 28x, 29x, 30x, 31x, 32x, 33x, 34x, 35x, 36x, 37x, 3 8x, 39x, 40x, 41x, 42x, 43x, 44x, 45x, 46x, 47x, 48x, 49x, 50x, 60x, 70x, 80x, 90x, 100x, 110x, 120x, 130x, 140x, 150x, 160x, 170x, 180x , 190x, 200x, 210x, 220x, 230x, 240x, 250x, 260x, 270x, 280x, 290x, 300x, 310x, 320x, 330x, 340x, 350x, 360x, 370x, 380x, 390x, 400x, 4 10x, 420x, 430x, 440x, 450x, 460x, 470x, 480x, 490x, 500x, 610x, 620x, 630x, 640x, 650x, 660x, 670x, 680x, 690x, 700x, 710x, 720x, 730x x, 740x, 750x, 760x, 770x, 780x, 790x, 800x, 810x, 820x, 830x, 840x, 850x, 860x, 870x, 880x, 890x, 900x, 910x, 920x, 930x, 940x, 950x,960x, 970x, 980x, 990x, 1000x, 1100x, 1200x, 1300x, 1400x, 1500x, 1600x, 1700x, 1800x x, 1900x, 2000x, 2100x, 2200x, 2300x, 2400x, 2500x, 2600x, 2700x, 2800x, 2900x, 3000x , 3100x, 3200x, 3300x, 3400x, 3500x, 3600x, 3700x, 3800x, 3900x, 4000x, 4100x, 4200x , 4300x, 4400x, 4500x, 4600x, 4700x, 4800x, 4900x, 5000x, 5100x, 5200x, 5300x, 5400x, 5500x, 5600x, 5700x, 5800x, 5900x, 6000x, 6100x, 6200x, 6300x, 6400x, 6500x, 6600x, 6700x, 6800x, 6900x, 7000x, 7100x, 7200x, 7300x, 7400x, 7500x, 7600x, 7700x, 7800x, 7 The increase may be 900-fold, 8000-fold, 8100-fold, 8200-fold, 8300-fold, 8400-fold, 8500-fold, 8600-fold, 8700-fold, 8800-fold, 8900-fold, 9000-fold, 9100-fold, 9200-fold, 9300-fold, 9400-fold, 9500-fold, 9600-fold, 9700-fold, 9800-fold, 9900-fold, or at least 10,000-fold.

[0054] In some embodiments, the amount of DNA template required for RNA synthesis using the DNA templates described herein may be less than the amount of DNA template required for RNA synthesis using conventional DNA templates, i.e., the uncharacterized DNA templates described herein. For example, the amount of DNA template required for RNA synthesis using a DNA template described herein may be at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3.0-fold, 3.1-fold, 3.2-fold, 3.3-fold, 3.4-fold, 3.5-fold, 4.0-fold, 4.5-fold, 5.0-fold, 5.5-fold, 6.0-fold, 6.5-fold, 7.0-fold, 8.0-fold, 9.0-fold, 9.5-fold, or at least 10-fold less than the amount of DNA template required for RNA synthesis using a conventional DNA template, i.e., a DNA template without the features described herein.

[0055] In some embodiments, the amount of nucleoside triphosphate (NTP) required for RNA synthesis using the DNA templates described herein may be less than the amount of NTP required for RNA synthesis using conventional DNA templates, i.e., the uncharacterized DNA templates described herein. For example, the amount of nucleoside triphosphate (NTP) required for RNA synthesis using a DNA template described herein may be at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3.0-fold, 3.1-fold, 3.2-fold, 3.3-fold, 3.4-fold, 3.5-fold, 4.0-fold, 4.5-fold, 5.0-fold, 5.5-fold, 6.0-fold, 6.5-fold, 7.0-fold, 8.0-fold, 9.0-fold, 9.5-fold, or at least 10-fold less than the amount of NTP required for RNA synthesis using a conventional DNA template, i.e., a DNA template without the features described herein.

[0056] RNA polymerase

[0057] RNA polymerases for microfluidic RNA synthesis are provided herein. For example, the RNA polymerase may include a phage RNA polymerase, a bacterial RNA polymerase, or a eukaryotic RNA polymerase. In some examples, the bacterial RNA polymerase may include an RNA polymerase from any bacterial species, including, but not limited to, E. coli. In some examples, the eukaryotic RNA polymerase may include any eukaryotic RNA polymerase I, RNA polymerase II, RNA polymerase III, RNA polymerase IV, or RNA polymerase V. Non-limiting examples of phage RNA polymerases include T3 RNA polymerase, T7 RNA polymerase, KP34 RNA polymerase, N4 RNA polymerase, and SP6 RNA polymerase. In some embodiments, the RNA polymerase used in the microfluidic RNA synthesis described herein is T7 RNA polymerase. In some embodiments, the T7 RNA polymerase comprises a sequence comprising SEQ ID NO: 1.

[0058] Further provided herein are RNA polymerases containing one or more variations or substitutions in the amino acid sequence, for example, variations related to enzymatic activity such as stability, DNA recognition, DNA binding affinity, and / or RNA polymerase activity. In some examples, the one or more variations may enhance the stability of the variant RNA polymerase during the transcription initiation and / or elongation process compared to an RNA polymerase lacking the one or more variations. In some examples, the one or more variations may enhance the DNA recognition ability of the RNA polymerase compared to an RNA polymerase lacking the one or more variations. In some examples, the one or more variations may enhance the DNA binding affinity of the RNA polymerase compared to an RNA polymerase lacking the one or more variations. In some examples, the one or more variations may reduce the DNA binding affinity of the RNA polymerase compared to an RNA polymerase lacking the one or more variations. In some examples, the one or more variations may enhance the RNA polymerase activity compared to an RNA polymerase lacking the one or more variations.

[0059] T7 RNA polymerase variants for microfluidic RNA synthesis are provided herein. Non-limiting examples of variations or amino acid substitutions in T7 RNA polymerase include I4M, I119V, N165S, K172L, G175R, E222K, G225S, Q239K, Q239R, Q239L, A255T, P266L, K333N, D366N, F400L, V426L, V426I, V426F, S430P, N433T, G542V, V574A, E593G, V625L, S633V, S633M, S633P, Y639F, Y6 39L, Y639V, E643K, V650L, T654L, S661G, G675R, V685A, A702V, R756C, Q758K, Q758R, V783I, V795I, H772R, N748X, R756M, Q758X, E775K, E775V, H784A, H784G, H784S, F849I, and F880Y, the positions of which are determined by alignment with SEQ ID NO: 1, and where X is any amino acid different from the wild-type amino acid. Provided herein are T7 RNA polymerase variants for microfluidic RNA synthesis, wherein the T7 RNA polymerase variants comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 40, or at least 41 amino acid variations or substitutions. For example, a T7 RNA polymerase variant described herein can include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 40, or at least 41 variations or amino acid substitutions in the DNA recognition or binding domain (e.g., the AT-recognition loop).For example, a T7 RNA polymerase variant described herein can include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 40, or at least 41 variations or amino acid substitutions in an RNA polymerase domain (e.g., the thumb subdomain, palm domain, or fingers subdomain). For example, a T7 RNA polymerase variant described herein can contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 40, or at least 41 variations or amino acid substitutions in the specificity loop.

[0060] Provided herein are T7 RNA polymerase variants that include at least one, at least two, at least three, or at least four or more of the variations described herein, e.g., K172L, P266L, H772R, N748X, R756M, Q758X, and E775V, the positions of which are determined by alignment with SEQ ID NO: 1, where X is any amino acid different from the wild-type amino acid. Also provided herein are T7 RNA polymerase variants that include at least one variation selected from the group consisting of K172L, P266L, H772R, N748X, R756M, Q758X, and E775V, the positions of which are determined by alignment with SEQ ID NO: 1, where X is any amino acid different from the wild-type amino acid. Further provided herein is a T7 RNA polymerase variant comprising at least four variations selected from the group consisting of K172L, P266L, H772R, N748X, R756M, Q758X, and E775V, the positions of which are determined by alignment with SEQ ID NO: 1, where X is any amino acid different from the wild-type amino acid. In some examples, the variant T7 RNA polymerase or functional fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 1. In some embodiments, a variant T7 RNA polymerase comprising at least one or at least four of the variations described herein may exhibit increased stability during the transcription initiation and / or elongation process compared to a T7 RNA polymerase without at least one variation, e.g., a T7 RNA polymerase comprising the sequence set forth in SEQ ID NO: 1. In some embodiments, a variant T7 RNA polymerase comprising at least one or at least four of the variations described herein may exhibit reduced DNA binding affinity, e.g., binding affinity to a T7 promoter sequence, during the transcription initiation and / or elongation process, compared to a T7 RNA polymerase without at least one variation, e.g., a T7 RNA polymerase comprising the sequence set forth in SEQ ID NO:1.

[0061] In some embodiments, the amount of RNA transcribed using an RNA polymerase described herein (e.g., a modified RNA polymerase) may be increased compared to the amount of RNA transcribed using a conventional RNA polymerase, i.e., an RNA polymerase without the features or modifications described herein. In some embodiments, the amount of RNA transcribed using an RNA polymerase described herein (e.g., a modified RNA polymerase) may be increased by at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, or more compared to the amount of RNA transcribed using a conventional RNA polymerase, i.e., an RNA polymerase without the features or modifications described herein. , 2.6x, 2.7x, 2.8x, 2.9x, 3.0x, 3.1x, 3.2x, 3.3x, 3.4x, 3.5x, 4.0x, 4.5x, 5.0x, 5.5x, 6.0x, 7.0x, 8.0x, 9.0x, 9.5x, 10.0x, 10.5x , 11.0x, 11.5x, 12.0x, 12.5x, 13.0x, 14.5x, 15.0x, 15.5x, 16.0x, 16.5x, 17.0x, 17.5x, 18.0x, 18.5x, 19.0x, 19.5x, 20x, 21x, 22x , 23x, 24x, 25x, 26x, 27x, 28x, 29x, 30x, 31x, 32x, 33x, 34x, 35x, 36x, 37x, 38x, 39x, 40x, 41x, 42x, 43x, 44x, 45x, 46x, 47x, 48x, 49 x, 50x, 60x, 70x, 80x, 90x, 100x, 110x, 120x, 130x, 140x, 150x, 160x, 170x, 180x, 190x, 200x, 210x, 220x, 230x, 240x, 250x, 260x, 270x, 280x, 290x, 300x, 310x, 320x, 330x, 340x, 350x, 360x, 370x, 380x, 390x, 400x, 410x, 420x, 430x, 440x, 450x, 460x, 470x, 48 0x, 490x, 500x, 610x, 620x, 630x, 640x, 650x, 660x, 670x, 680x, 690x, 700x, 710x, 720x, 730x, 740x, 750x, 760x, 770x, 780x, 790x,800x, 810x, 820x, 830x, 840x, 850x, 860x, 870x, 880x, 890x, 900x, 910x, 920x, 930x, 940x, 950x, 96 0x, 970x, 980x, 990x, 1000x, 1100x, 1200x, 1300x, 1400x, 1500x, 1600x, 1700x, 1800x, 1900x, 2000x , 2100x, 2200x, 2300x, 2400x, 2500x, 2600x, 2700x, 2800x, 2900x, 3000x, 3100x, 3200x, 3300x, 340 0x, 3500x, 3600x, 3700x, 3800x, 3900x, 4000x, 4100x, 4200x, 4300x, 4400x, 4500x, 4600x, 4700x, 48 00x, 4900x, 5000x, 5100x, 5200x, 5300x, 5400x, 5500x, 5600x, 5700x, 5800x, 5900x, 6000x, 6100x, 6200x, 6300x, 6400x, 6500x, 6600x, 6700x, 6800x, 6900x, 7000x, 7100x, 7200x, 7300x, 7400x, 7500x , 7600-fold, 7700-fold, 7800-fold, 7900-fold, 8000-fold, 8100-fold, 8200-fold, 8300-fold, 8400-fold, 8500-fold, 8600-fold, 8700-fold, 8800-fold, 8900-fold, 9000-fold, 9100-fold, 9200-fold, 9300-fold, 9400-fold, 9500-fold, 9600-fold, 9700-fold, 9800-fold, 9900-fold, or at least 10,000-fold increase.

[0062] In some embodiments, the amount of DNA template required for RNA synthesis using an RNA polymerase described herein (e.g., a modified RNA polymerase) may be less than the amount of DNA template required for RNA synthesis using a conventional RNA polymerase, i.e., an RNA polymerase without the characteristics or modifications described herein. In some embodiments, the amount of DNA template required for RNA synthesis using an RNA polymerase described herein (e.g., a modified RNA polymerase) may be at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3.0-fold, 3.1-fold, 3.2-fold, 3.3-fold, 3.4-fold, 3.5-fold, 4.0-fold, 4.5-fold, 5.0-fold, 5.5-fold, 6.0-fold, 7.0-fold, 8.0-fold, 9.0-fold, 9.5-fold, or at least 10.0-fold less than the amount of DNA template required for RNA synthesis using a conventional RNA polymerase, i.e., an RNA polymerase without the features or modifications described herein.

[0063] In some embodiments, the amount of nucleoside triphosphates (NTPs) required for RNA synthesis using an RNA polymerase described herein (e.g., a modified RNA polymerase) may be less than the amount of NTPs required for RNA synthesis using a conventional RNA polymerase, i.e., an RNA polymerase without the characteristics or modifications described herein. For example, the amount of nucleoside triphosphates (NTPs) required for RNA synthesis using an RNA polymerase described herein (e.g., a modified RNA polymerase) may be at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 1.10-fold, 1.20-fold, 1.30-fold, 1.40-fold, 1.50-fold, 1.60-fold, 1.70-fold, 1.80-fold, 1.90-fold, 1.90-fold, 1.90-fold, 1.10-fold, 1.11-fold, 1.12-fold, 1.13-fold, 1.14-fold, 1.15-fold, 1.16-fold, 1.17-fold, 1.18-fold, 1.19-fold, 1.20-fold, 1.21-fold, 1.22-fold, 1.23-fold, 1.24-fold, 1.25-fold, 1.26-fold, 1.27-fold, 1.28-fold, 1.29-fold, 1.30-fold, 1.31-fold, 1.32-fold, 1.33-fold, 1.34-fold, 1.35-fold, 1.36-fold, 1.37-fold, 1.38-fold, 1. It may be 0.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2.0x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3.0x, 3.1x, 3.2x, 3.3x, 3.4x, 3.5x, 4.0x, 4.5x, 5.0x, 5.5x, 6.0x, 7.0x, 8.0x, 9.0x, 9.5x, or at least 10.0x less.

[0064] Fusion proteins

[0065] The present invention provides a fusion protein, comprising the RNA polymerase or its functional fragment and DNA binding domain for use in RNA synthesis as described herein, wherein the RNA polymerase or its functional fragment and DNA binding domain are heterologous.As used herein, fusion protein or fusion polypeptide can comprise the RNA polymerase as described herein, for example, T7 RNA polymerase or its variant, and heterologous DNA binding domain that can recognize and bind to the DNA template for RNA synthesis.

[0066] Exemplary DNA-binding domains include, but are not limited to, zinc finger domains, leucine zippers, helix-turn-helix (HTH) motifs, helix-loop-helix (HLH) motifs, winged helix (WH), winged HTH (WHTH) motifs, high-mobility group (HMG) boxes, white-opaque regulator 3 (Wor3) domains, oligonucleotide / oligosaccharide-binding (OB) fold domains, immunoglobulin folds, B3 domains, transcription activator-like effectors (TALEs), TALE-like proteins, and clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) proteins. In some examples, the DNA-binding domain comprises avidin. Non-limiting examples of avidin include streptavidin, rhizavidin, and neutravidins such as Extravidin, NeutrAvidin, and NeutraLite. In some examples, a DNA-binding domain as used herein may not comprise a mutation or variation. In some examples, a DNA-binding domain may comprise a mutation or variation. In some examples, a DNA-binding domain comprises a DNA methyltransferase. In some examples, the DNA methyltransferase is Haemophilus pungens HaeIII methyltransferase (HaeIIIM). In some embodiments, the DNA methyltransferase can covalently bind to DNA, e.g., a DNA template, in the presence of modified bases, including, but not limited to, 5-bromo-cytidine, 5-iodo-cytidine, 5-fluoro-cytidine, 5-bromo-deoxyuracil, 5-iodo-deoxyuracil, or 5-fluoro-deoxyuracil. In some examples, a DNA-binding domain is a zinc finger domain. In some embodiments, the zinc finger domain comprises a zinc finger array (ZFA). In some embodiments, a DNA-binding domain comprises a sequence comprising SEQ ID NO: 8.In some examples, the DNA-binding domain is a leucine zipper. In some embodiments, the DNA-binding domain is streptavidin. In some embodiments, the DNA-binding domain is monomeric streptavidin. In some embodiments, the DNA-binding domain is lysavidin. In some embodiments, the DNA-binding domain comprises a sequence comprising SEQ ID NO:9.

[0067] Provided herein is an RNA polymerase or functional fragment thereof linked to a DNA-binding domain. The general structure of an exemplary RNA polymerase fusion protein with a DNA-binding domain may include the following structure: NH2-[DNA-binding domain]-[linker]-[RNA polymerase]-COOH, where NH2 is the N-terminus of the fusion protein and COOH is the C-terminus of the fusion protein. In some examples, the RNA polymerase or functional fragment thereof is covalently linked to the DNA-binding domain. For example, the RNA polymerase or functional fragment thereof may be linked to the DNA-binding domain via a linker. Non-limiting examples of linkers include peptide linkers, non-peptide linkers, nucleotide linkers, chemical linkers, and flexible linkers. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is a non-peptide linker. In some embodiments, the linker comprises at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 35, or at least 40 amino acid residues in length. In some embodiments, the linker comprises the amino acid sequence SGGS. In some embodiments, the linker is (SGGS) n , (GGGS) n , (GGGGS) n , (G) n , (EAAAK) n , (GGS) n , GS(GGGS) n , GS(GGGGS) n , or (XP) nmotif, or any combination thereof, wherein n is independently an integer from 1 to 30, and X is any amino acid. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The peptide linker can be a flexible linker or a rigid linker. In some embodiments, the linker contains multiple proline residues and is 5 to 21, 5 to 14, 5 to 9, or 5 to 7 amino acids in length, e.g., PAPAP, PAPAPA, PAPAPAP, PAPAPAPA, P(AP) n where n is an integer between 1 and 10. Such proline-rich linkers are also termed "rigid" linkers. In some embodiments, the linker is GS(GGGGS) n wherein n is an integer from 1 to 10. In some embodiments, the linker comprises a sequence comprising SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In some embodiments, the linker comprises an XTEN linker. In some embodiments, the linker comprises a sequence comprising SEQ ID NO: 13.

[0068] Provided herein is a programmable RNA synthesis system that includes a customizable DNA-binding protein (e.g., ZFA) fused or crosslinked to a wild-type or variant RNA polymerase (e.g., T7 RNA polymerase) along with a DNA template containing a truncated RNA promoter (e.g., T7 promoter) that exhibits recruitment-dependent activity when placed adjacent to a DNA-binding target sequence recognized by the DNA-binding protein. The RNA transcription activity of the fusion protein can be programmed by directing the binding affinity of the fusion protein to the DNA template or the stability of the fusion protein on the DNA template during initiation and / or elongation. For example, the binding affinity between the fusion protein and the DNA template can be adjusted by modifying any element in the system, such as the DNA-binding domain, the RNA polymerase, the DNA-binding target sequence in the DNA template recognized by the DNA-binding protein, and / or the truncated promoter sequence. For example, a variant T7 RNA polymerase with reduced binding affinity for a truncated T7 promoter bound to a ZFA can be used in a system with a DNA template containing one or more copies of the ZFA binding sequence and the truncated T7 promoter, and the binding affinity of the fusion protein can depend on the number of copies of the ZFA binding sequence in the DNA template.

[0069] In some embodiments, the amount of RNA transcribed using a fusion protein described herein (e.g., a fusion protein comprising an RNA polymerase or a functional fragment thereof and a DNA-binding domain) may be increased compared to the amount of RNA transcribed using a conventional RNA polymerase, i.e., an RNA polymerase without the characteristics or modifications described herein. In some embodiments, the amount of RNA transcribed using a fusion protein described herein (e.g., a fusion protein comprising an RNA polymerase or a functional fragment thereof and a DNA-binding domain) may be increased by at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, or 1.8-fold compared to the amount of RNA transcribed using a conventional RNA polymerase, i.e., an RNA polymerase without the characteristics or modifications described herein. x, 2.0x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3.0x, 3.1x, 3.2x, 3.3x, 3.4x, 3.5x, 4.0x, 4.5x, 5.0x, 5.5x, 6. 0x, 7.0x, 8.0x, 9.0x, 9.5x, 10.0x, 10.5x, 11.0x, 11.5x, 12.0x, 12.5x, 13.0x, 14.5x, 15.0x, 15.5x, 16.0x, 16.5x, 17.0x, 17. 5x, 18.0x, 18.5x, 19.0x, 19.5x, 20x, 21x, 22x, 23x, 24x, 25x, 26x, 27x, 28x, 29x, 30x, 31x, 32x, 33x, 34x, 35x, 36x, 37x, 38x, 3 9x, 40x, 41x, 42x, 43x, 44x, 45x, 46x, 47x, 48x, 49x, 50x, 60x, 70x, 80x, 90x, 100x, 110x, 120x, 130x, 140x, 150x, 160x, 170x, 1 80x, 190x, 200x, 210x, 220x, 230x, 240x, 250x, 260x, 270x, 280x, 290x, 300x, 310x, 320x, 330x, 340x, 350x, 360x, 370x, 380x, 390x, 400x, 410x, 420x, 430x, 440x, 450x, 460x, 470x, 480x, 490x, 500x, 610x, 620x, 630x, 640x, 650x, 660x, 670x, 680x, 690x,700x, 710x, 720x, 730x, 740x, 750x, 760x, 770x, 780x, 790x, 800x, 810x, 820x, 830x, 840x, 850x, 860x, 870 x, 880x, 890x, 900x, 910x, 920x, 930x, 940x, 950x, 960x, 970x, 980x, 990x, 1000x, 1100x, 1200x, 1300x, 140x 0x, 1500x, 1600x, 1700x, 1800x, 1900x, 2000x, 2100x, 2200x, 2300x, 2400x, 2500x, 2600x, 2700x, 2800x, 29 00x, 3000x, 3100x, 3200x, 3300x, 3400x, 3500x, 3600x, 3700x, 3800x, 3900x, 4000x, 4100x, 4200x, 4300x, 4 400x, 4500x, 4600x, 4700x, 4800x, 4900x, 5000x, 5100x, 5200x, 5300x, 5400x, 5500x, 5600x, 5700x, 5800x , 5900x, 6000x, 6100x, 6200x, 6300x, 6400x, 6500x, 6600x, 6700x, 6800x, 6900x, 7000x, 7100x, 7200x, 7300x fold, 7400-fold, 7500-fold, 7600-fold, 7700-fold, 7800-fold, 7900-fold, 8000-fold, 8100-fold, 8200-fold, 8300-fold, 8400-fold, 8500-fold, 8600-fold, 8700-fold, 8800-fold, 8900-fold, 9000-fold, 9100-fold, 9200-fold, 9300-fold, 9400-fold, 9500-fold, 9600-fold, 9700-fold, 9800-fold, 9900-fold, or at least 10,000-fold increase.

[0070] In some embodiments, the amount of DNA template required for RNA synthesis using a fusion protein described herein (e.g., a fusion protein comprising an RNA polymerase, or a functional fragment thereof, and a DNA-binding domain) may be less than the amount of DNA template required for RNA synthesis using a conventional RNA polymerase, i.e., an RNA polymerase without the characteristics or modifications described herein. In some embodiments, the amount of DNA template required for RNA synthesis using a fusion protein described herein (e.g., a fusion protein comprising an RNA polymerase, or a functional fragment thereof, and a DNA-binding domain) may be at least 1.1-fold, 1.2-fold, or more than the amount of DNA template required for RNA synthesis using a conventional RNA polymerase, i.e., an RNA polymerase without the characteristics or modifications described herein. , 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2.0x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3.0x, 3.1x, 3.2x, 3.3x, 3.4x, 3.5x, 4.0x, 4.5x, 5.0x, 5.5x, 6.0x, 7.0x, 8.0x, 9.0x, 9.5x, or at least 10.0x less.

[0071] In some embodiments, the amount of nucleoside triphosphates (NTPs) required for RNA synthesis using a fusion protein described herein (e.g., a fusion protein comprising an RNA polymerase or a functional fragment thereof and a DNA-binding domain) may be less than the amount of NTPs required for RNA synthesis using a conventional RNA polymerase, i.e., an RNA polymerase without the characteristics or modifications described herein. For example, the amount of nucleoside triphosphates (NTPs) required for RNA synthesis using a fusion protein described herein (e.g., a fusion protein comprising an RNA polymerase or a functional fragment thereof and a DNA-binding domain) may be at least 1.1-fold, 1.2-fold, or more than the amount of NTPs required for RNA synthesis using a conventional RNA polymerase, i.e., an RNA polymerase without the characteristics or modifications described herein. , 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2.0x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3.0x, 3.1x, 3.2x, 3.3x, 3.4x, 3.5x, 4.0x, 4.5x, 5.0x, 5.5x, 6.0x, 7.0x, 8.0x, 9.0x, 9.5x, or at least 10.0x less.

[0072] In some embodiments, binding of a fusion protein described herein (e.g., a fusion protein comprising an RNA polymerase, or a functional fragment thereof, and a DNA-binding domain) to a promoter described herein (e.g., a promoter in a DNA template having a DNA-binding target sequence described herein) may be stronger than binding of a fusion protein described herein (e.g., a fusion protein comprising an RNA polymerase, or a functional fragment thereof, and a DNA-binding domain) to a conventional promoter or DNA template without the characteristics or modifications described herein (e.g., a promoter or DNA template without a DNA-binding domain).For example, binding of a fusion protein described herein (e.g., a fusion protein comprising an RNA polymerase, or a functional fragment thereof, and a DNA-binding domain) to a promoter described herein (e.g., a promoter in a DNA template having a DNA-binding target sequence described herein) is comparable to binding of a fusion protein described herein to a conventional promoter or DNA template lacking the features or modifications described herein (e.g., a promoter or DNA template without a DNA-binding domain). and / or a fusion protein (e.g., a fusion protein comprising an RNA polymerase or a functional fragment thereof and a DNA binding domain) that is at least 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 1100-fold, 1200-fold, 1300-fold, 1400-fold, 1500-fold, 1600-fold, 1700-fold, 1800-fold, 1900-fold, 2000-fold, 2100-fold, 2200-fold, 2300-fold, 2400-fold, 2500-fold, 2600-fold, 2700-fold, 2800-fold, 2900-fold, 3000-fold, 3100-fold, 3200-fold, 3300-fold, 3400-fold, 3500-fold, 3600-fold, 3700-fold, 3800-fold, 3900-fold, 4000-fold, 4100-fold, 4200-fold, 4300-fold, 4400-fold, 4500-fold, 4600-fold, 4700-fold, 4800-fold, 4900-fold, 5000-fold, 5100-fold, 5200-fold, 5300-fold, 5400-fold, 5500-fold, 5600-fold, 5700-fold, 5800-fold, 5900-fold, 6000-fold, 61 x, 3000x, 3100x, 3200x, 3300x, 3400x, 3500x, 3600x, 3700x, 3800x, 3900x, 4000x, 4100x, 4200x, 4300x, 4400x, 4500x, 4600x, 4700x, 4 800x, 4900x, 5000x, 5100x, 5200x, 5300x, 5400x, 5500x, 5600x, 5700x, 5800x, 5900x, 6000x, 6100x, 6200x, 6300x, 6400x, 6500x, 6600x , 6700x, 6800x, 6900x, 7000x, 7100x, 7200x, 7300x, 7400x, 7500x, 7600x, 7700x, 7800x, 7900x, 8000x, 8100x, 8200x, 8300x, 8400x, 8500x, 8600x, 8700x, 8800x, 8900x, 9000x, 9100x, 9200x, 9300x, 9400x, 9500x, 9600x, 9700x, 9800x, 9900x, or at least 10,000x more powerful.

[0073] Synthesized RNA

[0074] Provided herein are RNAs synthesized by the compositions, methods, devices, and systems for microfluidic polynucleotide synthesis described herein. The synthesized RNAs provided herein include coding RNAs or non-coding RNAs. In one example, coding RNAs can include messenger RNAs (mRNAs). In another example, non-coding RNAs can include guide RNAs (gRNAs), small interfering RNAs (siRNAs), microRNAs (miRNAs), short hairpin RNAs (shRNAs), piwi-interacting RNAs (piRNAs), RNA aptamers, transfer RNAs (tRNAs), or antisense oligonucleotides (ASOs).

[0075] Provided herein is a gRNA synthesized by the microfluidic polynucleotide synthesis method described herein. The gRNA can be used to guide the CRISPR-Cas system to the target sequence for targeted genome editing, including genetic screening, targeted transcriptional regulation, targeted knock-in, and the introduction of double-strand breaks (DSBs) for base editing, epigenetic editing, and nucleotide sequence insertion via homologous recombination. Genome editing can refer to the targeted modification of DNA sequences, including, but not limited to, adding, removing, replacing, or modifying existing DNA sequences to change gene expression, and inducing chromosomal rearrangements or modifying transcriptional regulators (e.g., methylation / demethylation of gene promoter sequences). The CRISPR-Cas system requires a guide system that can position the Cas protein to the target DNA site in the genome. In some examples, the guide system includes a crispr RNA (crRNA) having a 17-20 nucleotide sequence complementary to a target DNA site and a trans-activating crRNA (tracrRNA) scaffold recognized by a Cas protein (e.g., Cas9). The 17-20 nucleotide sequence complementary to the target DNA site is called a spacer, while the 17-20 nucleotide target DNA sequence is called a protospacer. While crRNA and tracrRNA naturally exist as two separate RNA molecules, a single guide RNA (sgRNA or gRNA) can be engineered to combine and fuse elements of crRNA and tracrRNA into one single RNA molecule. Thus, in one embodiment, the gRNA includes two or more RNAs, e.g., a crRNA and a tracrRNA. In another embodiment, the gRNA includes an sgRNA that includes a spacer sequence for genome targeting and a scaffold sequence for Cas protein binding. In some examples, the guide system naturally includes an sgRNA. For example, Cas12a / Cpf1 utilizes a guide system that lacks tracrRNA and includes only crRNA containing spacer sequences and a scaffold for Cas12a / Cpf1 binding.The spacer sequence can vary depending on the target site in the genome, but the scaffold sequence for Cas protein binding can be identical for all gRNAs.

[0076] The CRISPR-Cas system described herein can comprise various CRISPR enzymes.For example, the CRISPR-Cas system can comprise Cas9, Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, or Cas12i. Non-limiting examples of Cas enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csn1 or Csx12), Cas10, Cas10d, Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, and Cas12d / Cas Y, Cas12e / CasX, Cas12f / Cas14 / C2c10, Cas12g, Cas12h, Cas12i, Cas12k / C2c5, Cas13a / C2c2, Cas13b, Cas13c, Cas 13d, C2c4, C2c8, C2c9, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, C sx3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, These include, but are not limited to, GSU0054, Type II Cas effector proteins, Type V Cas effector proteins, Type VI Cas effector proteins, CARF, DinG, their homologs, or modified or engineered versions thereof, such as dCas9 (endonuclease-inactive Cas9) and nCas9 (Cas9 nickase with an inactive DNA cleavage domain). In some cases, the compositions, methods, devices, and systems described herein may use the Cas9 nuclease from Streptococcus pyogenes, whose amino acid sequence and structure are well known to those of skill in the art.

[0077] Binding of a Cas protein to its target DNA sequence (i.e., target dsDNA) requires the presence of a protospacer adjacent motif (PAM), a short sequence adjacent to the protospacer, on the non-target DNA strand, which limits the region in the genome that can be targeted by the Cas protein. Cas proteins from various bacterial species recognize various PAM sequences and cleave 3–4 nucleotides upstream (e.g., Cas9) or 18–23 nucleotides downstream (e.g., Cas12a / Cpf1). For example, Streptococcus pyogenes Cas9 recognizes the PAM sequence 5'-NGG-3' (where "N" can be any nucleotide) and cleaves three nucleotides upstream of the PAM sequence. In another example, Staphylococcus aureus Cas9 recognizes the PAM sequence 5'-NNGRRN-3' (where "N" can be any nucleotide) and cleaves four nucleotides upstream of the PAM sequence. Although the PAM sequence is essential for Cas-mediated cleavage, in some cases, the gRNA sequence does not contain a PAM sequence.

[0078] Provided herein is a gRNA comprising a target sequence that includes a spacer complementary to the sequence of a target site in a genome. As described herein, the spacer can be about 10 to about 25 nucleotides in length. For example, the spacer sequence complementary to the target site sequence in a genome can be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 27, 28, 30, or more nucleotides in length. As described herein, the target site can include a sequence of about 20 nucleotides immediately upstream or 5' of the first nucleotide of the PAM.

[0079] Commercially available gRNAs currently produced by in vitro transcription (IVT) typically contain one to three additional guanines (Gs) at the 5' end of the gRNA sequence (Figure 5). T7 RNA polymerase with a minimized T7 promoter is commonly used for IVT of gRNAs. While the minimum specification for transcription initiation is one G at the +1 position, naturally occurring transcription initiation sequences can have up to three Gs at positions +1, +2, and +3 from the 5' end of the gRNA. Such additional Gs in IVT-synthesized gRNAs can result in an extra G at the 5' end of the spacer sequence, which, for example, in Cas9 gRNAs, can cause an unpaired overhang or mismatch between the gRNA and its target sequence. This mismatch can reduce the efficiency of targeted genome editing (Figure 5). To improve 5'-end pairing with the target site DNA sequence, gRNAs without mismatched nucleotides at the 5' end of the spacer are provided herein. A gRNA lacking one or more additional Gs at the 5' end of the spacer can support highly accurate genome editing due to reduced off-target effects (Figure 6). Provided herein are gRNAs in which at least one, at least two, or at least three consecutive nucleotides at the 5' end of the gRNA sequence are 100% identical to the 3' end of the target sequence in the genome. The gRNAs synthesized by the compositions, methods, devices, and systems described herein exhibit improved or enhanced pairing with the target sequence in the target gene through complementary base pairing compared to gRNAs containing additional 5'-terminal G nucleotides that are not present in the protospacer sequence or are not complementary to the 3' end of the target sequence.

[0080] The present specification provides gRNAs that exhibit enhanced editing efficiency of target sequences when the gRNA contacts a target sequence in a complex with a CRISPR-Cas system (e.g., CRISPR-Cas9, CRISPR-Cas12a / Cpf1, etc.). For example, the gRNAs provided herein (e.g., gRNAs that do not contain one or more additional G nucleotides at the 5'-end, or gRNAs that do not contain a G nucleotide in the 5'-end codon) can exhibit enhanced editing efficiency of target sequences when in a complex with a CRISPR-Cas system, compared to gRNAs that contain one or more additional 5'-end G nucleotides, where the one or more additional 5'-end G nucleotides are not present in the protospacer sequence or are not complementary to one or more nucleotides at the 3'-end of the target sequence in the genome. As used herein, the term "codon" generally refers to three consecutive nucleotides, and may or may not code for an amino acid. As used herein, "editing efficiency" or "editing efficiency" may refer to the ability of a gRNA-directed effector protein (e.g., a CRISPR-Cas protein) to modify a target DNA sequence. Non-limiting examples of target sequence modifications may include introducing double-strand breaks, modifying nucleic acid bases, inducing chromosomal rearrangements, and modifying the methylation / demethylation reaction of a gene's promoter sequence. The target sequence may be located in a gene or promoter region in the genome. The effector protein may be a gRNA-directed nuclease, such as a Cas protein, such as Cas9 or any other Cas protein described herein. Editing efficiency can be measured by any method known to those skilled in the art.For example, genome editing efficiency or editing efficiency can be measured using tracking indels by degradation (TIDE) analysis, surveyor nuclease assay, junction PCR, droplet digital PCR (ddPCR), denaturing high-performance liquid chromatography (DHPLC), PCR single-strand conformation polymorphism (SSCP), high-resolution melting curve (HRM), suppressed restriction enzyme digestion PCR (RE-PCR), engineered nuclease-induced translocation (ENIT), restriction enzyme digestion, deep sequencing such as Sanger DNA sequencing, next-generation sequencing (NGS), or any combination thereof. As used herein, the term "indel" refers to the insertion or deletion of a nucleotide base within a nucleic acid. In some embodiments, the efficiency of genome editing, e.g., the generation of double-strand breaks, can be measured by TIDE analysis, a three-step method whereby the region targeted by a nuclease (e.g., Cas9) is PCR-amplified from DNA isolated from cells transfected with a CRISPR-Cas system and gRNA. The 500-1500 bp amplicons generated around the target site are subjected to conventional Sanger DNA sequencing, followed by analysis using the web-based TIDE software. Any sequence modifications made by the nuclease are visualized in the sequence graph as aberrant base signals. The software also provides precise localization of the cut site and an estimated statistical significance of each indel. In some embodiments, the editing efficiency, e.g., the efficiency of generating DNA breaks within the intended target site, is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0081] Further provided herein are gRNAs that exhibit reduced off-target editing (e.g., editing of non-target sequences) when contacted with a target sequence in a complex with a CRISPR-Cas system (e.g., CRISPR-Cas9, CRISPR-Cas12a / Cpf1, etc.). For example, the gRNAs provided herein (e.g., gRNAs that do not contain one or more additional G nucleotides at the 5'-end or gRNAs that do not contain a G nucleotide in the 5'-terminal codon) can exhibit reduced off-target editing of the target sequence when in a complex with a CRISPR-Cas system, compared to gRNAs that contain one or more additional 5'-terminal G nucleotides, where the one or more additional 5'-terminal G nucleotides are not present in the protospacer sequence or are not complementary to one or more nucleotides at the 3'-end of the target sequence in the genome. In some embodiments, off-target editing may be reduced by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0082] The present specification provides gRNAs that contain secondary structures. For example, the scaffold portion of a gRNA recognized by a Cas protein can form a secondary structure such as a stem, a hairpin, and / or a loop. The stem or hairpin described herein can be approximately 3 to 10 nucleotides long. The loop can be approximately 6 to 20 nucleotides long. The stem can include one or more bulges each 1 to 10 nucleotides long.

[0083] Further provided herein are gRNAs comprising target sequences containing one or more mismatched nucleotides, i.e., the spacer sequence can contain one or more nucleotides that are not complementary to the target site sequence in the genome. The spacers described herein can have various numbers of mismatches, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mismatches. In some embodiments, the spacer contains up to 1, 2, 3, 4, or 5 mismatches. In some embodiments, the spacer does not contain any mismatches compared to the protospacer sequence of the target site, i.e., the spacer hybridizes 100% with the target sequence. The spacers described herein can contain at least one to at least five mismatched nucleotides. For example, the spacer can contain at least one, at least two, at least three, at least four, or at least five mismatched nucleotides. In some embodiments, the spacer may contain up to three, up to four, up to five, up to six, or up to seven mismatched nucleotides. In some embodiments, one or more mismatched nucleotides may be located at the 5' end of the spacer sequence. In some embodiments, one or more mismatched nucleotides may be located at the 3' end of the spacer sequence. In some embodiments, one or more mismatched nucleotides may be located within the spacer sequence.

[0084] Provided herein are gRNAs containing sequence extensions. The sequence extensions can be on the 5' or 3' end, or can be added internally. For example, the 5' end of a gRNA Cas12a / Cpf1 (e.g., crRNA) can be extended by 2 to 59 nucleotides. The 5' end extension of a Cas12a / Cpf1 gRNA includes a scaffold sequence for Cas12a / Cpf1 binding at the 5' end and a target sequence at the 3' end, which can increase the editing efficiency and delivery of Cas12a / Cpf1 in vitro and in vivo. Furthermore, the gRNA can be made more resistant to chemical modifications, resulting in enhanced gRNA stability. In another example, a Cas9 gRNA containing an internal extension of 2 to 10 nucleotides to extend the stem region of the stem-loop structure can increase gene knockout efficiency in CRISPR-Cas9-mediated genome editing. In some instances, a gRNA may contain two or more crRNA and tracrRNA sequences and may bind two or more Cas proteins and target DNA sites at two or more distinct regions in the genome. In some embodiments, a gRNA described herein may contain a 5' sequence extension. In some embodiments, a gRNA described herein may contain a 3' sequence extension. In some embodiments, a gRNA described herein may contain an internal sequence extension. In some embodiments, a sequence extension may comprise at least about 1 to at least 70 nucleotides. In some embodiments, a sequence extension may comprise at least 1, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, or at least 70 nucleotides.

[0085] Provided herein are gRNAs that contain nucleotide analogs, such as guanine (G) analogs. Nucleotide analogs include modifications of the phosphate backbone, sugar, and / or nucleobase. Non-limiting examples of nucleotide analogs include 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, hexose, phosphorothioate linkage, 5'-N-phosphoramidite linkage, intercalating bases, and / or chemically modified bases. Provided herein are gRNAs comprising a 5'-terminal G analog that can exhibit enhanced editing efficiency of a target sequence when in a complex with a CRISPR-Cas system compared to gRNAs lacking the 5'-terminal G analog.

[0086] Further provided herein are RNAs comprising 10 to 100 nucleotides in length. In some embodiments, the synthesized RNA comprises 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides in length. In some embodiments, the synthesized RNA comprises 10 to 20 nucleotides, 10 to 30 nucleotides, 10 to 40 nucleotides, 10 to 50 nucleotides, 10 to 60 nucleotides, 10 to 70 nucleotides, 10 to 80 nucleotides, 10 to 90 nucleotides, 10 to 100 nucleotides, 20 to 30 nucleotides, 20 to 40 nucleotides, 20 to 50 nucleotides, 20 to 60 nucleotides, 20 to 70 nucleotides, 20 to 80 nucleotides, 20 to 90 nucleotides, 20 to 100 nucleotides, 30 to 40 nucleotides, 30 to 50 nucleotides, 30 to 60 nucleotides, 30 to 70 nucleotides, 30 to 80 nucleotides, 30 to 80 nucleotides, 30 to 90 nucleotides, 30 to 100 nucleotides, 30 to 40 nucleotides, 30 to 50 nucleotides, 30 to 60 nucleotides, 30 to 7 ... The length may be 0-90 nucleotides, 30-100 nucleotides, 40-50 nucleotides, 40-60 nucleotides, 40-70 nucleotides, 40-80 nucleotides, 40-90 nucleotides, 40-100 nucleotides, 50-60 nucleotides, 50-70 nucleotides, 50-80 nucleotides, 50-90 nucleotides, 50-100 nucleotides, 60-70 nucleotides, 60-80 nucleotides, 60-90 nucleotides, 60-100 nucleotides, 70-80 nucleotides, 70-90 nucleotides, 70-100 nucleotides, 80-90 nucleotides, 80-100 nucleotides, or 90-100 nucleotides. In some embodiments, the synthesized RNA comprises at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, or at least 90 nucleotides in length. In some embodiments, the synthesized RNA comprises up to 20, up to 30, up to 40, up to 50, up to 60, up to 70, up to 80, up to 90, or up to 100 nucleotides in length. In some embodiments, the synthesized RNA comprises at least 20 nucleotides in length. In some embodiments, the synthesized RNA comprises at least 80 nucleotides in length. In some embodiments, the synthesized RNA comprises up to 30 nucleotides in length.

[0087] The synthesized RNA may contain one or more modifications as described herein. For example, the synthesized RNA may contain synthetic nucleotides, synthetic nucleotide analogs, nucleotide derivatives, and / or modified nucleotides. In some embodiments, the one or more modifications may increase the stability of the synthesized RNA. In some embodiments, the one or more modifications may enhance the biological activity of the synthesized RNA. In some embodiments, modification of internucleotide linkages using phosphorothioate (PS) linkage surrogates may be introduced to inhibit exonuclease-mediated degradation of the RNA. In some embodiments, the one or more modifications may occur at any position in the synthesized RNA. The synthesized RNA may be synthesized using natural nucleosides (e.g., adenosine, guanosine, cytidine, and uridine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deoxy-2-methyl ... The bases may include bases such as 7-azaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose), and / or modified phosphate groups (e.g., phosphorothioate linkages and 5'-N-phosphoramidite linkages).

[0088] RNAs containing chemical modifications are provided herein. Chemical modifications as described herein may include one or more 5' modifications selected from the group consisting of 5' triphosphate, 5' diphosphate, 5' monophosphate, and 5' hydroxyl. In another embodiment, the chemical modifications include one or more ribose modifications selected from the group consisting of 2'-O methylation (2'OMe), 2'-O-methoxy-ethyl (2'-MOE), 2'-fluoro (2'F), 2-deoxy-2'-thio, and 2'-azido. In some embodiments, the chemical modifications include one or more internucleotide bond modifications selected from the group consisting of phosphorothioate, methylphosphonate, phosphonocarboxylate, phosphonothiocarboxylate, boranophosphonate, alkylphosphonate, and alkylphosphonate.

[0089] Chemical modifications further include modified nucleotides containing one or more heterocyclic modifications selected from the group consisting of 2,6-diaminopurine, 2-aminopurine, inosine, 2-aminoadenosine, N6-methyladenosine, N6,2'-O-dimethyladenosine, N1-methyladenosine, 2-amino-6-chloropurine riboside, 5-methylcytidine, 5-hydroxymethylcytidine, 8-oxo-7,8-dihydroguanosine, pseudouridine, N4-acetylcytidine, 5-bromouridine, 5-methyluridine, and 5-nitroindole. In some embodiments, chemical modifications include, but are not limited to, modified nucleotides containing one or more 5' cap modifications selected from the group consisting of GpppG, 7-methylguanylate (m7GpppG), m2,2,7GpppG, and m7-3'-OGpppG (ARCA).

[0090] In some examples, the chemical modification comprises a modified nucleotide comprising one or more 5' cap modifications selected from the group consisting of attachment chemistry (e.g., biotin), a dye, a cell targeting moiety, an activation chemistry, and an amino modifier. In some embodiments, the attachment chemistry may comprise biotin. In some embodiments, the dye comprises fluorescein. In some embodiments, the cell targeting moiety comprises digoxigenin. In some embodiments, the activation chemistry comprises an azide, an acrydite, a thiol, or an alkyne. In some embodiments, the amino modifier comprises an aminoallyl.

[0091] Nucleic Acid Library

[0092] Provided herein is a nucleic acid library containing purified RNA synthesized using the microfluidic polynucleotide synthesis described herein. The nucleic acid library provided herein contains highly pure and homogeneous RNA molecules. The purified RNA may include guide RNA (gRNA), messenger RNA (mRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), piwi-interacting RNA (piRNA), RNA aptamer, transfer RNA (tRNA), or antisense oligonucleotide (ASO), as described herein. As described herein, RNA purity can be measured and analyzed using methods including, but not limited to, isotachophoresis (ITP), capillary electrophoresis (CE), and / or ITP coupled with microchip-based CE. Further provided herein is a nucleic acid library containing RNA with highly accurate sequences compared to a predetermined RNA sequence encoded by a DNA template. In some examples, for a nucleic acid library in which each RNA contains at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, the percentage of full-length sequences is at least 40%, 45%, 50%, 52%, 55%, 58%, 60%, 62%, 65%, 68%, 70%, 72%, 75%, 78%, 80%, 82%, 85%, 88%, 90%, or 95%. In some examples, at least about 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more of the RNAs in the nucleic acid library do not have errors in the sequence, such as nucleotide changes. In some examples, at least about 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more of the RNAs in the nucleic acid library have insertions or deletions (indels) in the sequence. In some examples, the nucleic acid libraries described herein have an error rate of less than 1:100, 1:500, 1:1000, 1:1500, 1:2000, 1:3000, 1:5000, 1:10,000, or less compared to a predetermined RNA sequence encoded by a DNA template.

[0093] The nucleic acid libraries described herein can be measured in terms of uniformity, which is a measure of RNA species representation. Uniformity can be measured both per cluster and per device. In some examples, 99% of RNAs have abundances within about 0.05, 0.1, 0.2, 0.5, 0.7, 1, 1.2, 1.5, 1.7-fold, or within about 2-fold of the average abundance. In some examples, 97% of polynucleotides have abundances within about 0.05, 0.1, 0.2, 0.5, 0.7, 1, 1.2, 1.5, 1.7-fold, or within about 2-fold of the average abundance. In some examples, 60%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% of sequences have abundances within 2-fold of the average. In some examples, 60%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% of the sequences have abundances within 1.5-fold of the average, hi some examples, 60%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% of the sequences have abundances within 1-fold of the average.

[0094] Provided herein is a nucleic acid library containing RNA molecules with high uniformity after microfluidic polynucleotide synthesis.For example, at least about 60%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% of the RNAs in the nucleic acid library described herein can be represented within 1.5 times the average frequency for the entire library.In some embodiments, provided herein is a nucleic acid library, wherein the nucleic acid library comprises at least 50 RNAs, each of which encodes a different guide RNA (gRNA) sequence, and at least about 90% of the at least 50 RNAs are present in the nucleic acid library at an amount within 1.5 times the average frequency for the at least 50 RNAs in the library.In some embodiments, the nucleic acid library comprises at least 50 RNAs, each of which encodes a different guide RNA (gRNA) sequence, and at least about 95% of the at least 50 RNAs are present in the nucleic acid library at an amount within 1.5 times the average frequency for the at least 50 RNAs in the library. In some embodiments, the nucleic acid library comprises at least 50 RNAs, each of the at least 50 RNAs encoding a different guide RNA (gRNA) sequence, and at least about 99% of the at least 50 RNAs are each present in the nucleic acid library at an amount within 1.5-fold of the average frequency for the at least 50 RNAs in the library.

[0095] Provided herein is a nucleic acid library comprising at least 100,000 purified RNAs.For example, the nucleic acid library can comprise at least 100,000, 150,000, 200,000, 250,000, 300,000, 350,000, 400,000, 450,000, 500,000, 550,000, 600,000, 650,000, 700,000, 750,000, 800,000, 850,000, 900,000, 950,000, or 1,000,000 purified RNAs. In some examples, the purified RNA comprises RNA sequences, each of which comprises at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 nucleotides in length. In some embodiments, each of the RNA sequences comprises 10-20 nucleotides, 10-30 nucleotides, 10-40 nucleotides, 10-50 nucleotides, 10-60 nucleotides, 10-70 nucleotides, 10-80 nucleotides, 10-90 nucleotides, 10-100 nucleotides, 20-30 nucleotides, 20-40 nucleotides, 20-50 nucleotides, 20-60 nucleotides, 20-70 nucleotides, 20-80 nucleotides, 20-90 nucleotides, 20-100 nucleotides, 30-40 nucleotides, 30-50 nucleotides, 30-60 nucleotides, 30-70 nucleotides, 30-80 nucleotides, and lengths of 30-90 nucleotides, 30-100 nucleotides, 40-50 nucleotides, 40-60 nucleotides, 40-70 nucleotides, 40-80 nucleotides, 40-90 nucleotides, 40-100 nucleotides, 50-60 nucleotides, 50-70 nucleotides, 50-80 nucleotides, 50-90 nucleotides, 50-100 nucleotides, 60-70 nucleotides, 60-80 nucleotides, 60-90 nucleotides, 60-100 nucleotides, 70-80 nucleotides, 70-90 nucleotides, 70-100 nucleotides, 80-90 nucleotides, 80-100 nucleotides, or 90-100 nucleotides.In some embodiments, each of the RNA sequences comprises at most 20, at most 30, at most 40, at most 50, at most 60, at most 70, at most 80, at most 90, or at most 100 nucleotides in length. In some embodiments, each of the RNAs comprises at least 20 nucleotides in length. In some embodiments, each of the RNAs comprises at least 80 nucleotides in length. In some embodiments, each of the RNAs comprises at most 30 nucleotides in length.

[0096] Further provided herein are nucleic acid libraries comprising a plurality of RNAs, each of which comprises the same RNA (e.g., gRNA, miRNA, siRNA, mRNA, piRNA, shRNA, tRNA, RNA aptamer, or ASO) sequence. In some examples, the nucleic acid library comprises two or more groups of RNAs, each of which comprises a different RNA sequence, and each of which comprises a plurality of RNAs. In some examples, the nucleic acid library comprises at least 50, at least 100, at least 200, at least 300, at least 400, or at least 500 different groups of RNAs, each of which comprises a different RNA sequence. In some embodiments, the nucleic acid library comprises at least 200 groups of RNAs, each of which comprises a different RNA sequence. For example, the nucleic acid library may comprise 200 groups of RNAs, each of which comprises a plurality of RNA molecules, and each of which comprises a different RNA sequence; thus, the nucleic acid library may comprise a plurality of RNAs with 200 different RNA sequences.

[0097] The nucleic acid library described herein may further comprise additional components.The additional components may include any element involved in the microfluidic polynucleotide synthesis described herein, such as NHS ester reaction product, dissociated transcription complex or fragment, transcription reaction components, nucleotide factors, ITP and / or CE buffer, microfluidic coating polymer, and UV adjuncts.For example, the nucleic acid library may comprise at least one single-stranded DNA (ssDNA) encoding a truncated RNA polymerase promoter as a template used in microfluidic polynucleotide synthesis. Additional components may include, but are not limited to, an RNA polymerase, or a functional fragment or variant thereof, a fusion protein comprising an RNA polymerase, or a functional fragment or variant thereof, and a DNA-binding protein, or a functional fragment or variant thereof, a plurality of oligonucleotides, a plurality of single nucleotides, Tris-HCl, MgCl2, Mn2+, spermidine, dithiothreitol, a DNA template, NaCl, beta-mercaptoethanol (β-ME), ethylenediaminetetraacetic acid (EDTA), glycerol, pyrophosphatase, Triton X-100, Tween-20, potassium glutamate, tris(2-carboxyethyl)phosphine (TCEP), bovine serum albumin (BSA), polyethylene glycol (PEG) 8000, and acetate.

[0098] Provided herein are nucleic acid libraries further comprising at least one oligonucleotide. For example, the oligonucleotide can be an RNA primer, such as an initiator oligo or a terminator oligo. In some embodiments, at least one oligonucleotide comprises 2 to 10 nucleotides in length. In some embodiments, at least one oligonucleotide comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In some embodiments, at least one oligonucleotide comprises RNA. In some embodiments, at least one oligonucleotide comprises DNA. In some embodiments, at least one oligonucleotide is 80%, 85%, 90%, 95%, or 100% identical to 2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous nucleotides at or near the 5' end of purified RNA, e.g., gRNA, miRNA, siRNA, or mRNA. In some embodiments, at least one oligonucleotide is 80%, 85%, 90%, 95%, or 100% identical to 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive nucleotides at or near the 3' end of a purified RNA, e.g., gRNA, miRNA, siRNA, or mRNA. In a preferred embodiment, at least one oligonucleotide is 100% identical to 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive nucleotides at or near the 5' end of a purified gRNA. In another embodiment, at least one oligonucleotide is 100% identical to 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive nucleotides at or near the 3' end of a purified gRNA. In some embodiments, at least one oligonucleotide may contain a chemical modification described herein (see, e.g., paragraphs

[0107] -

[0110] ). Non-limiting examples of initiator oligos include ApA, ApU, ApG, ApC, UpA, UpU, UpG, UpC, GpA, GpU, GpG, GpC, CpA, CpU, CpG, CpC, where p indicates a phosphodiester bond and the remaining 5' phosphate group.

[0099] Provided herein are nucleic acid libraries comprising additional components described herein, wherein the additional components may be present in the nucleic acid library in an amount of less than 10 pM, 9 pM, 8 pM, 7 pM, 6 pM, 5 pM, 4 pM, 3 pM, 2 pM, 1 pM, 0.5 pM, 0.1 pM, 0.05 pM, or less than 0.01 pM. Further provided herein are nucleic acid libraries comprising additional components described herein, wherein the additional components may be present in the nucleic acid library in an amount of at least 0.01 pM, 0.05 pM, 0.1 pM, 0.5 pM, 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, or at least 10 pM. Further provided herein are nucleic acid libraries comprising the additional components described herein, wherein the additional components may be present in the nucleic acid library in an amount of up to 20 pM, 15 pM, 10 pM, 9 pM, 8 pM, 7 pM, 6 pM, 5 pM, 4 pM, 3 pM, 2 pM, 1 pM, 0.5 pM, 0.1 pM, 0.05 pM, or up to 0.01 pM. For example, a nucleic acid library comprising a plurality of purified RNAs may further comprise at least 1 pM of a microfluidic coating polymer. In some embodiments, the at least 1 pM of a microfluidic coating polymer comprises up to 10 pM of a microfluidic coating polymer. For example, a nucleic acid library comprising a plurality of purified RNAs may further comprise at least 1 pM of a UV-assisted agent. In some embodiments, the at least 1 pM of a UV-assisted agent comprises up to 10 pM of a UV-assisted agent.

[0100] Computer Systems

[0101] Any of the systems described herein may be operably linked to a computer and may be automatically operated locally or remotely via the computer. In various examples, the methods and systems of the present invention may further include software programs on a computer system and their use. Thus, computer control for synchronization of dispense / vacuum / refill functions, such as orchestrating and synchronizing the operation of a material deposition device, dispense actions, and vacuum activation, is within the scope of the present invention. The computer system is programmed to interface between a user-specified template array and the position of the material deposition device to deliver the correct reagent to the specified area of ​​the surface.

[0102] The computer system (700) illustrated in FIG. 7 can be understood as a logical device capable of reading instructions from a network port (705), which may optionally be connected to a server (709) having a medium (711) and / or a fixed medium (712). A system such as that illustrated in FIG. 7 may include a CPU (701), a disk drive (703), optional input devices such as a keyboard (715) and / or a mouse (716), and an optional monitor (707). Data communication may be achieved via the indicated communication medium to a server at a local or remote location. The communication medium may include any means of transmitting and / or receiving data. For example, the communication medium may be a network connection, a wireless connection, or an Internet connection. Such a connection may provide for communication over the World Wide Web. It is envisioned that data related to the present disclosure may be transmitted over such a network or connection for receipt and / or consideration by a party (722) as illustrated in FIG. 7.

[0103] As shown in FIG. 8, a block diagram illustrating a first example architecture of a computer system (800) that may be used in connection with examples of the present invention is provided herein. As depicted in FIG. 8, the exemplary computer system may include a processor (802) for processing instructions. Non-limiting examples of processors include an Intel Xeon™ processor, an AMD Opteron™ processor, a Samsung 8-bit RISC ARM 1176JZ(F)-S v1.0™ processor, an ARM Cortex-A8 Samsung S5PC100™ processor, an ARM Cortex-A8 Apple A4™ processor, a Marvell PXA 930™ processor, or a functionally equivalent processor. Multiple threads of execution may be used for parallel processing. In some examples, multiple processors, or processors with multiple cores, may be used, whether in a single computer system, in a cluster, or distributed across a network of systems including multiple computers, mobile phones, and / or personal digital assistant devices.

[0104] As illustrated in FIG. 8, a high-speed cache (804) may be connected to or incorporated into the processor (802) to provide high-speed memory for instructions or data recently or frequently used by the processor (802). The processor (802) is connected to a northbridge (806) by a processor bus (808). The northbridge (806) is connected to random access memory (RAM) (810) by a memory bus (812) and manages access to the RAM (810) by the processor (802). The northbridge (806) is also connected to a southbridge (88) by a chipset bus (816). The southbridge (814) is then connected to a peripheral bus (818). The peripheral bus may be, for example, PCI, PCI-X, PCI Express, or other peripheral bus. The northbridge and southbridge, often referred to as the processor chipset, manage data transfers between the processor, RAM, and peripheral components over the peripheral bus (818). In some alternative architectures, the northbridge functionality may be incorporated into the processor instead of using a separate northbridge chip. In some examples, the system 800 may include an accelerator card 822 attached to the peripheral bus 818. The accelerator may include a field programmable gate array (FPGA) or other hardware to expedite specific processing. For example, the accelerator may be used for adaptive data reconstruction or to evaluate algebraic expressions used in extended configuration processing.

[0105] Software and data may be stored in external storage 824 and loaded into RAM 810 and / or cache 804 for use by the processor. System 800 includes an operating system for managing system resources, non-limiting examples of which include Linux, Windows™, MACOS™, BlackBerry OS™, iOS™, and other functionally equivalent operating systems, as well as application software running on the operating system for managing data storage and optimization in accordance with examples of the present invention. In this example, system 800 further includes network interface cards (NICs) 820 and 821 connected to the peripheral bus to provide a network interface for external storage devices, such as network-attached storage (NAS), and other computer systems that can be used for distributed parallel processing.

[0106] As shown in Figure 9, a diagram illustrating a network 900 including multiple computer systems 902a and 902b, multiple mobile phones and personal digital assistants 902c, and network-attached storage (NAS) 904a and 904b is provided herein. In an example, systems 902a, 902b, and 902c can manage data storage and optimize data access to data stored in network-attached storage (NAS) 904a and 904b. Mathematical models can be used on this data and evaluated using distributed parallel processing across computer systems 902a and 902b and mobile phones and personal digital assistant systems 902c. The computer systems 902a and 902b, as well as the mobile phone and personal digital assistant system 902c, can also provide parallel processing for adaptive data restructuring of data stored in network-attached storage (NAS) 904a and 904b. Figure 9 shows only one example, and various other computer architectures and systems can be used with various examples of the present invention. For example, blade servers can be used to provide parallel processing. Processor blades can be connected via a backplane to provide parallel processing. Storage can also be connected to the backplane or can be connected as network-attached storage (NAS) via a separate network interface.

[0107] In some examples, the processors may maintain separate memory spaces and communicate data over a network interface, backplane, or other connector for parallel processing by other processors, while in other examples, some or all of the processors may use a shared virtual address memory space.

[0108] A block diagram of a multiprocessor computer system (1000) using a shared virtual address memory space, as illustrated in FIG. 10 , according to an example embodiment, is provided herein. The system includes a processor (1002) including multiple processors (1002a-1002f) that can access a shared memory subsystem (1004). The system incorporates multiple programmable hardware memory algorithm processors (MAPs) (1006a-1006f) in the memory subsystem (1004). Each MAP (1006a-1006f) may include memory (1008a-1008f) and one or more field programmable gate arrays (FPGAs) (1010a-1010f). The MAPs provide configurable functional units, and specific algorithms or portions of algorithms may be provided to the FPGAs (1010a-1010f) for processing in close cooperation with the respective processors. For example, the MAPs may be used to evaluate algebraic expressions related to a data model and perform adaptive data restructuring in the example. In this example, each MAP is globally accessible by all of the processors for such purposes. In one configuration, each MAP can use direct memory access (DMA) to access its associated memory (1008a-1008f), thereby allowing it to perform tasks independently of and asynchronously from its respective microprocessor (1002a-1002f). In this configuration, a MAP can feed results directly to another MAP for pipelining and parallel execution of algorithms.

[0109] The above computer architectures and systems are merely examples, and various other computer, mobile phone, and personal data assistant architectures and systems may be used in connection with the examples, including systems using any combination of general processors, coprocessors, FPGAs and other programmable logic devices, systems-on-chips (SOCs), application-specific integrated circuits (ASICs), and other processing and logic elements. In some examples, all or part of the computer system may be implemented in software or hardware. Various data storage media may be used with the examples, including random access memory, hard drives, flash memory, tape drives, disk arrays, network-attached storage (NAS), and other local or distributed data storage devices and systems.

[0110] In examples, the computer system may be implemented using software modules executing on any of the above or other computer architectures and systems. In other examples, the system's functionality may be implemented partially or fully in firmware, programmable logic devices such as field programmable gate arrays (FPGAs) as referenced in FIG. 10, systems-on-chips (SOCs), application-specific integrated circuits (ASICs), or other processing and logic elements. For example, the set processor and optimizer may be implemented with hardware acceleration through the use of a hardware accelerator card, such as the accelerator card (722) illustrated in FIG. 7.

[0111] Other embodiments

[0112] In some aspects, provided herein are methods for synthesizing RNA, the methods comprising: providing an RNA polymerase immobilized on a surface; and synthesizing a plurality of RNAs at an extension rate of at least 50 nucleotides per hour, wherein each of the plurality of RNAs has a preselected sequence, and the synthesizing comprises extending the sequence one base at a time in a single extension reaction. In some embodiments, each of the plurality of RNAs comprises a guide RNA (gRNA). In some embodiments, each of the plurality of RNAs is selected from the group consisting of messenger RNA (mRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), piwi-interacting RNA (piRNA), RNA aptamer, transfer RNA (tRNA), and antisense oligonucleotide (ASO). In some embodiments, the synthesis is carried out using template DNA. In some embodiments, the template DNA is immobilized on a surface. In some embodiments, the template DNA is immobilized on a surface by a linker, biotin, or streptavidin. In some embodiments, the template DNA comprises single-stranded DNA (ssDNA). In some embodiments, the template DNA comprises double-stranded DNA (dsDNA). In some embodiments, the template DNA comprises a secondary structure. In some embodiments, the secondary structure comprises a hairpin. In some embodiments, the template DNA comprises a promoter sequence. In some embodiments, the promoter sequence is a truncated promoter sequence. In some embodiments, the extension rate is at least 50 nucleotides per minute. In some embodiments, the extension rate is at least 50 nucleotides per second. In some embodiments, the RNA polymerase is selected from the group consisting of a phage RNA polymerase, a bacterial RNA polymerase, and a eukaryotic RNA polymerase. In some embodiments, the RNA polymerase is immobilized on a surface by activating the surface with a standard N-hydroxysuccinimide ester (NHS) functional group. In some embodiments, the RNA polymerase is immobilized on a surface by activating the surface with a trifluoroacetic anhydride (TFAA) functional group.In some embodiments, the RNA polymerase is immobilized on the surface by activating the surface with glutaraldehyde (GA) functional groups. In some embodiments, the RNA polymerase is immobilized on the surface by a linker, biotin, or streptavidin. In some embodiments, the surface is a solid surface. In some embodiments, the surface comprises magnetic beads, agarose beads, fused silica, sol-gel, silica polymer, silica monolith, cellulose, agar, acrylamide, gold beads, or a gel matrix. In some embodiments, each of the plurality of RNAs has the same preselected sequence. In some embodiments, the plurality of RNAs comprises at least two RNAs comprising different preselected sequences. In some embodiments, the plurality of RNAs comprises at least one chemically modified gRNA.

[0113] In some aspects, provided herein are methods for synthesizing RNA, the methods comprising: providing a fusion RNA polymerase, or a functional fragment or variant thereof; and synthesizing a plurality of RNAs at an extension rate of at least 50 nucleotides per hour, wherein each of the plurality of RNAs has a preselected sequence, and the synthesizing comprises extending the RNAs one base at a time in a single extension reaction. In some embodiments, the fusion RNA polymerase comprises an RNA polymerase, or a functional fragment or variant thereof, and a DNA-binding protein, or a functional fragment or variant thereof, wherein the RNA polymerase and the DNA-binding protein are heterologous. In some embodiments, the fusion RNA polymerase further comprises a linker. In some embodiments, the RNA polymerase is selected from the group consisting of a phage RNA polymerase, a bacterial RNA polymerase, and a eukaryotic RNA polymerase. In some embodiments, the DNA-binding protein is fused to the N-terminus of the RNA polymerase. In some embodiments, the DNA-binding protein is fused to the C-terminus of the RNA polymerase. In some embodiments, each of the plurality of RNAs comprises a guide RNA (gRNA). In some embodiments, each of the plurality of RNAs is selected from the group consisting of messenger RNA (mRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), piwi-interacting RNA (piRNA), RNA aptamer, transfer RNA (tRNA), and antisense oligonucleotide (ASO). In some embodiments, the synthesizing step is performed using template DNA. In some embodiments, the template DNA comprises single-stranded DNA (ssDNA). In some embodiments, the template DNA comprises double-stranded DNA (dsDNA). In some embodiments, the template DNA comprises a secondary structure. In some embodiments, the secondary structure comprises a hairpin. In some embodiments, the template DNA comprises a promoter sequence. In some embodiments, the promoter sequence is a truncated promoter sequence. In some embodiments, the extension rate is at least 50 nucleotides per minute.In some embodiments, the extension rate is at least 50 nucleotides per second. In some embodiments, each of the plurality of RNAs has the same preselected sequence. In some embodiments, the plurality of RNAs comprises at least two RNAs comprising different preselected sequences. In some embodiments, the plurality of RNAs comprises at least one chemically modified gRNA.

[0114] In some aspects, provided herein is a nucleic acid library comprising a plurality of purified guide RNAs (gRNAs) and at least one single-stranded DNA (ssDNA) molecule encoding a truncated RNA polymerase promoter region. In some embodiments, each of the purified gRNAs comprises a sequence at least 20 nucleotides in length. In some embodiments, each of the purified gRNAs comprises a sequence at least 80 nucleotides in length. In some embodiments, the plurality of purified gRNAs comprises at least 100,000 purified gRNAs. In some embodiments, the truncated RNA polymerase promoter region comprises a sequence selected from the group consisting of SEQ ID NOs: 3-6. In some embodiments, the plurality of purified gRNAs comprises modified nucleotides.

[0115] In some aspects, provided herein are nucleic acid libraries comprising a plurality of purified guide RNAs (gRNAs) at least 20 nucleotides in length and at least one oligonucleotide 2-10 nucleotides in length. In some embodiments, the plurality of purified guide RNAs (gRNAs) comprises at least 80 nucleotides in length. In some embodiments, the plurality of purified gRNAs comprises at least 100,000 purified gRNAs. In some embodiments, at least one oligonucleotide is 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In some embodiments, at least one oligonucleotide comprises RNA. In some embodiments, at least one oligonucleotide is 100% identical to 2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous nucleotides at or near the 5' end of a purified guide RNA. In some embodiments, at least one oligonucleotide is 100% identical to 2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous nucleotides at or near the 3' end of a purified guide RNA.

[0116] In some embodiments, the plurality of purified gRNAs comprises at least one chemically modified gRNA. In some embodiments, the chemical modification comprises one or more 5' modifications selected from the group consisting of 5' triphosphate, 5' diphosphate, 5' monophosphate, and 5' hydroxyl. In some embodiments, the chemical modification comprises one or more ribose modifications selected from the group consisting of 2'-O-methylation (2'OMe), 2'-O-methoxy-ethyl (2'-MOE), 2'-fluoro (2'F), 2-deoxy-2'-thio, and 2'-azido. In some embodiments, the chemical modification comprises one or more internucleotide linkage modifications selected from the group consisting of phosphorothioate, methylphosphonate, phosphonocarboxylate, phosphonothiocarboxylate, boranophosphonate, alkylphosphonate, and alkylphosphonate. In some embodiments, the chemical modification comprises a modified nucleotide comprising one or more heterocyclic modifications selected from the group consisting of 2,6-diaminopurine, 2-aminopurine, inosine, 2-aminoadenosine, N6-methyladenosine, N6,2'-O-dimethyladenosine, N1-methyladenosine, 2-amino-6-chloropurine riboside, 5-methylcytidine, 5-hydroxymethylcytidine, 8-oxo-7,8-dihydroguanosine, pseudouridine, N4-acetylcytidine, 5-bromouridine, 5-methyluridine, and 5-nitroindole. In some embodiments, the chemical modification comprises a modified nucleotide comprising one or more 5' cap modifications selected from the group consisting of GpppG, 7-methylguanylate (m7GpppG), m2,2,7GpppG, and m7-3'-OGpppG (ARCA). In some embodiments, the chemical modification comprises a modified nucleotide comprising one or more 5' cap modifications selected from the group consisting of attachment chemistry, dyes, cell targeting moieties, activation chemistry, and amino modifiers. In some embodiments, the attachment chemistry comprises biotin. In some embodiments, the dye comprises fluorescein. In some embodiments, the cell targeting moiety comprises digoxigenin. In some embodiments, the activation chemistry comprises an azide, an acrydite, a thiol, or an alkyne. In some embodiments, the amino modifier comprises an aminoallyl.

[0117] In some aspects, provided herein are methods for generating a nucleic acid library comprising at least 50 guide RNAs (gRNAs), the method comprising: synthesizing at least 50 gRNAs using an RNA polymerase, wherein at least one of the at least 50 gRNAs comprises a spacer sequence complementary to a target sequence in a target gene, and the 5'-terminal nucleotide of the spacer sequence is complementary to the 3'-terminal nucleotide of the target sequence. In some embodiments, each of the at least 50 gRNAs comprises a spacer sequence complementary to a target sequence in the target gene, and the 5'-terminal nucleotide of the spacer sequence is complementary to the 3'-terminal nucleotide of the target sequence. In some embodiments, the RNA polymerase is selected from the group consisting of a phage RNA polymerase, a bacterial RNA polymerase, and a eukaryotic RNA polymerase. In some embodiments, the RNA polymerase is T7 RNA polymerase. In some embodiments, each of the at least 50 gRNAs exhibits enhanced editing efficiency of the target sequence compared to a gRNA comprising a 5'-terminal nucleotide that is not complementary to the 3'-terminal nucleotide of the target sequence. In some embodiments, each of the at least 50 gRNAs exhibits reduced off-target editing compared to a gRNA comprising a 5'-terminal nucleotide that is not complementary to the 3'-terminal nucleotide of the target sequence. In some embodiments, the at least 50 gRNAs comprise a gRNA sequence lacking a 5'-terminal guanine (G) nucleotide, the gRNA sequence is at least 40 nucleotides in length, and at least three consecutive nucleotides at the 5'-end of the gRNA sequence are 100% identical to the 3'-end of the target sequence in the genome. In some embodiments, each of the at least 50 gRNAs exhibits enhanced 5'-end pairing with the target sequence compared to a gRNA comprising a gRNA sequence comprising a 5'-terminal G nucleotide, and the 5'-terminal G nucleotide is not present in the protospacer sequence.In some embodiments, each of the at least 50 gRNAs exhibits enhanced editing efficiency of a target sequence compared to a gRNA comprising a gRNA sequence comprising a spacer sequence, wherein the gRNA sequence comprises one or more G nucleotides at the 5' end of the spacer sequence, and wherein the one or more G nucleotides at the 5' end of the spacer sequence are not complementary to one or more nucleotides at the 3' end of the target sequence in the genome. In some embodiments, each of the at least 50 gRNAs exhibits reduced off-target editing compared to a gRNA comprising a gRNA sequence comprising a spacer sequence, wherein the gRNA sequence comprises one or more G nucleotides at the 5' end of the spacer sequence, and wherein the one or more G nucleotides at the 5' end of the spacer sequence are not complementary to one or more nucleotides at the 3' end of the target sequence in the genome. In some embodiments, the at least 50 gRNAs comprise a gRNA sequence lacking a guanine (G) nucleotide in the 5' terminal codon, wherein the gRNA sequence is at least 40 nucleotides in length, and wherein at least three consecutive nucleotides at the 5' end of the gRNA sequence are 100% identical to the 3' end of the target sequence in the genome. In some embodiments, at least 50 gRNAs exhibit enhanced 5'-end pairing with target sequences compared to gRNAs comprising a gRNA sequence comprising a guanine (G) nucleotide in the 5'-end codon, wherein the G nucleotide in the 5'-end codon is absent from the protospacer sequence. In some embodiments, each of the at least 50 gRNAs exhibits enhanced editing efficiency of target sequences compared to gRNAs comprising a gRNA sequence comprising a spacer sequence, wherein the gRNA sequence comprises one or more G nucleotides at the 5'-end of the spacer sequence, wherein the one or more G nucleotides at the 5'-end of the spacer sequence are not complementary to one or more nucleotides at the 3'-end of the target sequence in the genome. In some embodiments, each of the at least 50 gRNAs exhibits reduced off-target editing compared to gRNAs comprising a gRNA sequence comprising a spacer sequence, wherein the gRNA sequence comprises one or more G nucleotides at the 5'-end of the spacer sequence, wherein the one or more G nucleotides at the 5'-end of the spacer sequence are not complementary to one or more nucleotides at the 3'-end of the target sequence in the genome.In some embodiments, the at least 50 gRNAs comprise at least 100,000 purified gRNAs.

[0118] In some aspects, provided herein is a nucleic acid library comprising at least 50 purified gRNAs (gRNAs), wherein the at least 50 purified gRNAs comprise gRNA sequences comprising a 5'-terminal guanine (G) analog. In some embodiments, each of the at least 50 purified gRNAs exhibits enhanced editing efficiency of a target sequence compared to a gRNA comprising a gRNA sequence lacking a 5'-terminal G analog. In some embodiments, each of the at least 50 purified gRNAs exhibits enhanced stability compared to a gRNA comprising a gRNA sequence lacking a 5'-terminal G analog. In some embodiments, the at least 50 purified gRNAs comprise at least 100,000 purified gRNAs.

[0119] In some embodiments, provided herein are nucleic acid libraries, wherein the nucleic acid library comprises at least 50 RNAs, each of the at least 50 RNAs encoding a different guide RNA (gRNA) sequence, and wherein at least about 90% of the at least 50 RNAs are each present in the nucleic acid library at an abundance within 1.5-fold of the average frequency for the at least 50 RNAs in the library. In some embodiments, the nucleic acid library comprises at least 100, at least 500, at least 1,000, at least 5,000, at least 10,000, at least 50,000, or at least 100,000 RNAs, and at least about 90% of the at least 100, at least 500, at least 1,000, at least 5,000, at least 10,000, at least 50,000, or at least 100,000 RNAs are present in the nucleic acid library at an abundance within 1.5-fold of the average frequency for at least 100, at least 500, at least 1,000, at least 5,000, at least 10,000, at least 50,000, or at least 100,000 RNAs in the library, respectively. In some embodiments, the different gRNA sequences comprise at least 80 nucleotides in length. In some embodiments, the different gRNA sequences comprise at least 100 different gRNA sequences. In some embodiments, at least about 95% of the at least 50 RNAs are each present in the nucleic acid library at an amount within 1.5-fold of the average frequency for each of the RNAs in the library. In some embodiments, at least about 99% of the at least 50 RNAs are each present in the nucleic acid library at an amount within 1.5-fold of the average frequency for each of the RNAs in the library.

[0120] In some aspects, modified polypeptide compositions are provided herein, the modified polypeptide compositions comprising a purified RNA polymerase, or a functional fragment or variant thereof, and a purified nucleic acid binding protein, optionally a zinc finger containing protein, or a functional fragment or variant thereof, wherein the purified RNA polymerase and the purified nucleic acid binding protein are heterologous, and the purified RNA polymerase and the purified nucleic acid binding protein are linked. In some embodiments, the purified RNA polymerase and the purified nucleic acid binding protein are linked by conjugation. In some embodiments, the purified RNA polymerase and the purified nucleic acid binding protein are linked by fusion. In some embodiments, the purified nucleic acid binding protein is linked to the N-terminus of the purified RNA polymerase. In some embodiments, the purified nucleic acid binding protein is linked to the C-terminus of the purified RNA polymerase. In some embodiments, the purified RNA polymerase is T7 RNA polymerase. In some embodiments, the purified nucleic acid binding protein comprises a zinc finger domain, a leucine zipper, a helix-turn-helix (HTH) motif, a helix-loop-helix (HLH) motif, a winged helix (WH), a winged-HTH (WHTH) motif, a high-mobility group (HMG) box, a White-Opaque Regulator 3 (Wor3) domain, an oligonucleotide / oligosaccharide-binding (OB) fold domain, an immunoglobulin fold, a B3 domain, a Transcription Activator-Like Effector (TALE), a TALE-like protein, or a clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas protein. In some embodiments, the purified nucleic acid binding protein comprises a zinc finger domain.

[0121] In some aspects, provided herein are compositions comprising a fusion RNA polymerase, or a functional fragment or variant thereof, and a DNA polynucleotide, wherein the fusion RNA polymerase comprises (i) an RNA polymerase, or a functional fragment or variant thereof, and (ii) a DNA-binding protein, or a functional fragment or variant thereof, wherein the RNA polymerase and the DNA-binding protein are heterologous. In some embodiments, the DNA polynucleotide is a single-stranded DNA polynucleotide. In some embodiments, the DNA polynucleotide comprises a 3' secondary structure. In some embodiments, the 3' secondary structure comprises a hairpin.

[0122] In some embodiments, provided herein are modified polypeptides comprising a variant T7 RNA polymerase or a functional fragment thereof, wherein the variant T7 RNA polymerase comprises at least four variations selected from the group consisting of K172L, P266L, H772R, N748X, R756M, Q758X, and E775V, the positions of which are determined by alignment with SEQ ID NO: 1, and wherein X is any amino acid different from the wild-type amino acid. In some embodiments, provided herein are modified polypeptides comprising a variant T7 RNA polymerase or a functional fragment thereof, wherein the variant T7 RNA polymerase comprises at least one variation selected from the group consisting of K172L, P266L, H772R, N748X, R756M, Q758X, and E775V, the position of which is determined by alignment with SEQ ID NO: 1, and wherein X is any amino acid different from the wild-type amino acid, and wherein the modified polypeptide is immobilized on a surface.

[0123] In some embodiments, the variant T7 RNA polymerase or functional fragment thereof is linked to a DNA-binding protein. In some embodiments, the variant T7 RNA polymerase or functional fragment thereof is linked to the DNA-binding protein by fusion. In some embodiments, the variant T7 RNA polymerase or functional fragment thereof is linked to the DNA-binding protein by conjugation. In some embodiments, the DNA-binding protein is linked to the N-terminus of the T7 RNA polymerase. In some embodiments, the DNA-binding protein is linked to the C-terminus of the T7 RNA polymerase. In some embodiments, the variant T7 RNA polymerase or functional fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 1. In some embodiments, the variant T7 RNA polymerase or functional fragment thereof exhibits increased stability during the transcription initiation and / or elongation process compared to a T7 RNA polymerase comprising the sequence set forth in SEQ ID NO: 1. In some embodiments, the variant T7 RNA polymerase or functional fragment thereof exhibits reduced binding affinity for a T7 promoter sequence compared to a T7 RNA polymerase comprising the sequence set forth in SEQ ID NO: 1.

[0124] In some aspects, a device is provided herein, comprising a surface, a T7 RNA polymerase, or a functional fragment or variant thereof, and a DNA template, the DNA template comprising a truncated T7 promoter sequence, wherein the T7 RNA polymerase, functional fragment or variant thereof, or DNA template is linked to the surface. In some embodiments, the device further comprises a nucleic acid binding protein, or a functional fragment or variant thereof, linked to the surface. In some embodiments, the surface is a solid surface. In some embodiments, the surface comprises magnetic beads, agarose beads, fused silica, sol-gel, silica polymer, silica monolith, cellulose, agar, acrylamide, or gold beads. In some embodiments, the solid surface comprises a gel matrix for encapsulation or entrapment of the T7 RNA polymerase, the DNA template, or the nucleic acid binding protein. In some embodiments, the nucleic acid binding protein comprises a streptavidin tag. In some embodiments, the T7 RNA polymerase is immobilized. In some embodiments, the DNA template is immobilized. In some embodiments, the nucleic acid binding protein, or functional fragment or variant thereof, comprises a zinc finger domain, a leucine zipper, a helix-turn-helix (HTH) motif, a helix-loop-helix (HLH) motif, a winged helix (WH), a winged-HTH (WHTH) motif, a high-mobility group (HMG) box, a White-Opaque Regulator 3 (Wor3) domain, an oligonucleotide / oligosaccharide-binding (OB) fold domain, an immunoglobulin fold, a B3 domain, a Transcription Activator-Like Effector (TALE), a TALE-like protein, or a clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas protein. In some embodiments, the nucleic acid binding protein, or functional fragment or variant thereof, binds to nucleic acids by sequence-specific binding.In some embodiments, the nucleic acid-binding protein, or functional fragment or variant thereof, binds to a nucleic acid through one or more chemical modifications on the nucleic acid. In some embodiments, the one or more chemical modifications include biotin or avidin. In some embodiments, the DNA-binding protein comprises a zinc finger domain, a leucine zipper, a helix-turn-helix (HTH) motif, a helix-loop-helix (HLH) motif, a winged helix (WH), a winged HTH (WHTH) motif, a high-mobility group (HMG) box, a white-opaque regulator 3 (Wor3) domain, an oligonucleotide / oligosaccharide-binding (OB) fold domain, an immunoglobulin fold, a B3 domain, a transcription activator-like effector (TALE), a TALE-like protein, or a clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas protein. In some embodiments, the DNA template comprises a sequence comprising SEQ ID NO:7. In some embodiments, the T7 RNA polymerase or variant thereof comprises a sequence having at least 90% identity to SEQ ID NO: 1. In some embodiments, the device further comprises a piezoelectric transducer, a rotary valve, a Peltier heater, a voltage controller, a syringe pump, a UV LED, and a sensor or a vacuum pump.

[0125] array

[0126] SEQ ID NO: 1 (WT T7 RNA polymerase protein sequence) MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQNLKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEML IESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTK GLLTLAKGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTDTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLRLDILESDFAFA

[0127] SEQ ID NO:2(WT T7 RNA POLYメラーーDNA SEQ ID NO.)

[0128] SEQ ID NO: 3 (T7 RNA polymerase promoter sequence) TTAAATTAATACGACTCACTATATATA

[0129] SEQ ID NO: 4 (truncated T7 RNA polymerase promoter sequence) TACGACTCACTATA

[0130] SEQ ID NO: 5 (truncated T7 RNA polymerase promoter sequence) AATACGACTCACTATA

[0131] SEQ ID NO: 6 (truncated T7 RNA polymerase promoter sequence) TTAATACGACTCACTATA

[0132] SEQ ID NO: 7 (hairpin, full length (5'-3') template) TATAGTGAGTCGTATTAATTTACAACAAAATTAATACGACTCA

[0133] SEQ ID NO: 8 (ZFA) NLISLFSGAGGLDLGFQKAGFRIICANEYDKSIWKTYESNHSAKLIKGDISKISSDEFPKCDGIIGGPPCQSWSEGGSLRGIDDPRGKLFYEYIRILKQKKPIFFLAENVKGMMAQRHNKAVQEFIQEFDNAGYDVHIILLNANDYGVAQDRKRVFYIGFRKELN INYLPPIPHLIKPTFKDVIWDLKDNPIPALDKNKTNGNKCIYPNHEYFIGSYSTIFMSRNRVRQWNEPAFTVQASGRQCQLHPQAPVMLKVSKNLNKFVEGKEHLYRRLTVRECARVQGFPDDFIFHYESLNDGYKMIGNAVPVNLA,YEIAKTIKSALEICKGN

[0134] SEQ ID NO: 9 (Rhizovidin, monomeric streptavidin) FDASNFKDFSSIASASSSWQNQHGSTMIIQVDSFGNVSGQYVNRAEGTGCQNSPYPLTGRVNGTFIDFSVKWNNSTENCNSNTQWTGYAQVNGNNTEIVTRWNLKYEGGSGPAIWQGQDTFQYVPTTE

[0135] SEQ ID NO: 10 (Linker 1) GSGGGGSGGGGSGGGGS

[0136] SEQ ID NO: 11 (Linker 2) GSGGGGSGGGGS

[0137] SEQ ID NO: 12 (Linker 3) GSGGGGSGGGGSGGGGSGGGGS

[0138] SEQ ID NO: 13 (XTEN linker) SGSETPGTSESATPES [Example]

[0139] These examples are provided for illustrative purposes only and are not intended to limit the scope of the claims.

[0140] Example 1: RNA purification using capillary electrophoresis

[0141] Using hairpin single-stranded DNA (ssDNA) templates, gRNAs were produced by in vitro transcription and purity was assessed using capillary electrophoresis (CE). Microfluidic LabChip® capillary electrophoresis technology (PerkinElmer) was used according to standard protocols (Small RNA Assay User Guide, 2020, PerkinElmer). CE analysis was used to compare the quality of in vitro transcribed gRNA (green), chemically synthesized gRNA (blue), and gRNA synthesized using a microfluidic polynucleotide synthesis method (orange) (see Figure 11). Peak amplitudes represent nominal significance, and easily separated single peaks with slight peak broadening indicate gRNA purity (Figure 11, orange). Broader spectral tails or multiple peaks indicate gRNA impurities (e.g., multiple gRNA products of various sizes) and low-quality gRNA (Figure 11, green and blue). The results of a CE analysis comparing the quality of gRNAs synthesized by various in vitro transcription methods (green, red, and purple) with that synthesized using a microfluidic polynucleotide synthesis method (blue) are shown in Figure 12. In addition, we used CE analysis to demonstrate that high purity can be reproducibly achieved by comparing the quality of eight different gRNA samples (Table 1 and Figure 13). As can be seen in Table 1, for example, gRNAs approximately 100 nucleotides long analyzed by CE have 100% purity.

[0142] [Table 1]

[0143] Example 2: gRNA editing efficiency

[0144] Cleavage assay

[0145] An in vitro cleavage assay was performed to compare the nuclease activity of S. pyogenes Cas9 on DNA substrates containing target sequences using separately supplied gRNAs. A reaction mixture was prepared by mixing nuclease-free water, buffer, 300 nM gRNA, and 1 μM Cas9. 30 nM substrate DNA was added to the reaction mixture, which was then mixed and pulse-spun in a microcentrifuge. A sample was removed at 0 min (Figure 14, lane 3), and the remaining reaction mixture was incubated at 37 °C for 5 min (Figure 14, lanes 4-6). 1 μl of proteinase K was added to each sample and mixed thoroughly. The samples were pulse-spun in a microcentrifuge and incubated at room temperature for 10 min before proceeding to fragment analysis. As shown in Figure 14, a 1 kb target DNA sequence was cleaved into two small (approximately 500 bp) fragments by Cas9 with commercially available in vitro transcribed gRNA or chemically synthesized gRNA (lanes 4 and 5, respectively), and with gRNA synthesized in vitro using a hairpin ssDNA template (lane 6).

[0146] In vivo nuclease activity

[0147] In vivo CRISPR-Cas9-mediated nuclease assays were performed according to standard protocols (Integrated DNA Technologies' User Guide, Alt-R CRISPR-Cas9 System: Cationic Lipid Delivery of CRISPR Ribonucleoprotein Complexes into Mammalian Cells, Version 4, 2018, and Integrated DNA Technologies' User Guide, Alt-R CRISPR-Cas9 System: Delivery of Ribonucleoprotein Complexes into HEK-293 Cells Using the Amaxa® Nucleofector® System, Version 3.1, 2019) using two different single guide RNAs (sgRNAs) to measure the efficacy of gRNAs for Cas9 nuclease activity against target DNA. Briefly, HEK-293 cells were freshly thawed and subcultured for a minimum of 2–3 days before electroporation, maintaining a confluency of 90% or less. Cas9 and sgRNA were diluted in PBS and incubated at room temperature for 10–20 minutes for ribonucleoprotein (RNP) complex formation. The RNP complex was electroporated into HEK-293 cells. The efficiency and mutation spectrum were analyzed using Tracking Indels (Insertions and Deletions) by Degradation (TIDE) analysis, which quantified editing efficiency while simultaneously identifying the predominant type of indel in the targeted population of cells. TIDE analysis involves three simple steps using standard molecular biology reagents in PCR reactions: 1) a set of standard PCR reactions, 2) a set of standard capillary ("Sanger") sequencing reactions, and 3) analysis of the resulting two raw sequencing files using the TIDE web tool. The TIDE web tool uses an algorithm to accurately reconstruct the spectrum of indels from the sequence traces and reports the identity of detected indels and their frequency. As shown in Figure 15A and Figure 15B (top left corner), both sgRNAs tested demonstrated editing efficiencies greater than 80%.

[0148] Example 3: Microfluidics integration of RNA synthesis

[0149] Microfluidic cartridges are designed to perform guide RNA (gRNA) synthesis and purification. Each microfluidic cartridge contains multiple reaction chambers, each capable of producing gRNAs with the same or different sequences. A transcription complex containing a DNA template and an RNA polymerase enzyme is immobilized on a surface, such as a bead, inside the reaction chamber using surface chemistry. To support RNA attachment and synthesis, the surface of each reaction chamber (e.g., magnetic beads) is functionalized using standard N-hydroxysuccinimide ester (NHS) reaction chemistry. A DNA-binding protein (e.g., strep) is added, followed by quenching the NHS reaction. T7 RNA polymerase is added to bind the DNA-binding protein and form a stable complex. A DNA template is then added, allowing it to bind to the complex by binding to the DNA-binding protein.

[0150] A pneumatic and peristaltic pumping system can be used for programmable fluid manipulation. Mixing groups and other transcription reagents are introduced into the reaction chamber through inlets. Enzymes catalyze the reaction, producing highly pure RNA product.

[0151] Example 4: RNA synthesis

[0152] A DNA template, such as single-stranded DNA (ssDNA), is added to each reaction chamber of the microfluidic cartridge. Transcription reaction reagents containing mixed RNA bases, and optionally initiator and / or terminator oligonucleotides, are then injected into the reaction chamber through an inlet and allowed to flow over the transcription complexes immobilized on the surface. A thermoelectric Peltier element heater is used to maintain the reaction chamber temperature at 37°C. Once RNA is produced, it is removed from the reaction chamber within a few seconds, controlled by the flow rate.

[0153] Example 5: RNA purification using isotachophoresis

[0154] According to Han et al. (Lab Chip, 2019, 19, 2741-2749), isotachophoresis (ITP) can be used for RNA purification. ITP is a robust electrophoretic separation and preconcentration technique that generates a strong electric field gradient, allowing selective focusing and separation of charged species based on their electrophoretic mobility. The chemistry of the ITP electrolyte can be controlled to purify RNA within a target size range.

[0155] Example 6: RNA production by promoters with fusion enzymes and enzyme-binding domains

[0156] A T7 RNA polymerization activity assay was developed based on the complex formation of DNA-transcribed RNA products and their fluorescent aptamers, such as Broccoli-DFHBI-1T. Due to its fluorometric nature, this assay allows for the measurement of RNA transcripts in real time. An annealing protocol was used to generate double-stranded DNA (dsDNA) substrates, in which equal molar amounts of template and non-template strands were mixed and heated to 94°C for 2 minutes in a thermocycler, followed by cooling of the mixture. The continuous assay was initiated by adding T7 RNA polymerase enzyme to a 20-μl reaction mixture containing 100 μM DFHBI-1T, 400 nM dsDNA Broccoli template (for fluorescence), 4 mM NTPs, 0.1 U inorganic pyrophosphatase, and 1x transcription buffer. The reaction mixture was incubated at 37°C for up to 1 hour, and enzyme activity was measured at 37°C by fluorescence reading (excitation wavelength 469 nm and emission wavelength 501 nm) using a Cytation 5 imaging reader (Biotek). Commercially available T7 RNA polymerase was replaced with a fusion enzyme containing an RNA polymerase and a DNA-binding domain (e.g., rhizavidin) described herein to compare RNA production activity (Figure 16A). In addition, promoter structures with enzyme-binding domains were tested to compare RNA production activity with that of conventional promoters lacking the enzyme-binding domain (Figure 16B).

[0157] Example 7: Stronger promoter binding compared to commercial RNA polymerases

[0158] To investigate how the T7 promoter sequence affects RNA polymerization activity, a series of Broccoli dsDNA substrates were synthesized with 0 to 10 upstream sequence deletions of the non-essential promoter region. To further analyze whether additional substrate binding to the T7 RNA polymerase enzyme would enhance RNA synthesis as well as create a synergistic promoter effect, DNA substrates conjugated with biotin at the 5' end of the non-template strand in each pair were prepared. A real-time RNA polymerization assay was initiated by adding a fusion enzyme containing an RNA polymerase and a DNA-binding domain described herein (e.g., resavidin) to a 20 μl reaction mixture containing 100 μM DFHBI-1T, 400 nM truncated dsDNA Broccoli template, 4 mM NTPs, 0.1 U inorganic pyrophosphatase, and 1x transcription buffer. The reaction mixture was incubated at 37°C for up to 1 hour, and enzyme activity was measured using the same method as described in Example 6. The sequences of Broccoli, the essential T7 promoter, and the non-essential T7 promoter are shown below.

[0159] SEQ ID NO: 14 (Broccoli sequence) GAGCCCACACTCTACTCGACAGATACGAATATCTGGACCCGACCGTCTC

[0160] SEQ ID NO: 15 (essential T7 promoter) TATAGTGAGTC

[0161] SEQ ID NO: 16 (non-essential T7 promoter) GTATTAATTT

[0162] The sequences of the DNA templates are shown in Table 2. The results are shown in Figures 17A-17B.

[0163] [Table 2]

[0164] The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes suggested to those skilled in the art are intended to be included within the spirit and scope of this specification and the appended claims.

Claims

1. 1. A method for synthesizing RNA, the method comprising: providing an RNA polymerase immobilized on a surface; synthesizing a plurality of RNAs at an extension rate of at least 50 nucleotides per hour, each of the plurality of RNAs having a preselected sequence, the synthesizing comprising extending each RNA base in a single extension reaction; A method comprising:

2. 2. The method of claim 1, wherein each of the plurality of RNAs comprises a guide RNA (gRNA).

3. 2. The method of claim 1, wherein each of the plurality of RNAs is selected from the group consisting of messenger RNA (mRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), piwi-interacting RNA (piRNA), RNA aptamer, transfer RNA (tRNA), and antisense oligonucleotide (ASO).

4. The method of claim 1 , wherein the synthesizing step is carried out using a template DNA.

5. The method of claim 4 , wherein the template DNA is immobilized on the surface.

6. The method of claim 5 , wherein the template DNA is immobilized on the surface by a linker, biotin, or streptavidin.

7. The method of claim 4, wherein the template DNA comprises single-stranded DNA (ssDNA).

8. The method of claim 4, wherein the template DNA comprises double-stranded DNA (dsDNA).

9. The method of claim 4 , wherein the template DNA comprises a secondary structure.

10. The method of claim 9 , wherein the secondary structure comprises a hairpin.

11. The method of claim 4 , wherein the template DNA comprises a promoter sequence.

12. The method of claim 11 , wherein the promoter sequence is a truncated promoter sequence.

13. 10. The method of claim 1, wherein the extension rate is at least 50 nucleotides per hour.

14. 10. The method of claim 1, wherein the extension rate is at least 50 nucleotides per second.

15. 2. The method of claim 1, wherein the RNA polymerase is selected from the group consisting of a phage RNA polymerase, a bacterial RNA polymerase, and a eukaryotic RNA polymerase.

16. 2. The method of claim 1, wherein the RNA polymerase is immobilized on the surface by activating the surface with a standard N-hydroxysuccinimide ester (NHS) functional group.

17. 10. The method of claim 1, wherein the RNA polymerase is immobilized on the surface by activating the surface with trifluoroacetic anhydride (TFAA) functional groups.

18. 10. The method of claim 1, wherein the RNA polymerase is immobilized on the surface by activating the surface with anhydrous glutaraldehyde (GA) functional groups.

19. The method of claim 1 , wherein the surface is a solid surface.

20. 20. The method of claim 19, wherein the solid surface comprises magnetic beads, agarose beads, fused silica, sol-gel, silica polymer, silica monolith, cellulose, agar, acrylamide, gold beads, or a gel matrix.

21. 10. The method of claim 1, wherein each of the plurality of RNAs has the same preselected sequence.

22. 10. The method of claim 1, wherein the plurality of RNAs comprises at least two RNAs comprising different preselected sequences.

23. 2. The method of claim 1, wherein the plurality of RNAs comprises at least one chemically modified gRNA.

24. 1. A method for synthesizing RNA, the method comprising: providing a fusion RNA polymerase, or a functional fragment or variant thereof; synthesizing a plurality of RNAs at an extension rate of at least 50 nucleotides per hour, each of the plurality of RNAs having a preselected sequence, the synthesizing comprising extending each of the RNAs one base at a time in a single extension reaction; A method comprising:

25. 25. The method of claim 24, wherein the fusion RNA polymerase comprises an RNA polymerase, or a functional fragment or variant thereof, and a DNA binding protein, or a functional fragment or variant thereof, and wherein the RNA polymerase and DNA binding protein are heterologous.

26. The method of claim 24 or 25, wherein the fusion RNA polymerase further comprises a linker.

27. 26. The method of claim 25, wherein the RNA polymerase is selected from the group consisting of a phage RNA polymerase, a bacterial RNA polymerase, and a eukaryotic RNA polymerase.

28. 26. The method of claim 25, wherein the DNA binding protein is fused to the N-terminus of the RNA polymerase.

29. 26. The method of claim 25, wherein the DNA binding protein is fused to the C-terminus of the RNA polymerase.

30. 25. The method of claim 24, wherein each of the plurality of RNAs comprises a guide RNA (gRNA).

31. 25. The method of claim 24, wherein each of the plurality of RNAs is selected from the group consisting of messenger RNA (mRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), piwi-interacting RNA (piRNA), RNA aptamer, transfer RNA (tRNA), and antisense oligonucleotide (ASO).

32. The method of claim 24, wherein the synthesizing step is carried out using a template DNA.

33. 33. The method of claim 32, wherein the template DNA comprises single-stranded DNA (ssDNA).

34. 33. The method of claim 32, wherein the template DNA comprises double-stranded DNA (dsDNA).

35. 33. The method of claim 32, wherein the template DNA comprises a secondary structure.

36. 36. The method of claim 35, wherein the secondary structure comprises a hairpin.

37. 33. The method of claim 32, wherein the template DNA comprises a promoter sequence.

38. 38. The method of claim 37, wherein the promoter sequence is a truncated promoter sequence.

39. 25. The method of claim 24, wherein the extension rate is at least 50 nucleotides per hour.

40. 25. The method of claim 24, wherein the extension rate is at least 50 nucleotides per second.

41. 25. The method of claim 24, wherein each of the plurality of RNAs has the same preselected sequence.

42. 25. The method of claim 24, wherein the plurality of RNAs comprises at least two RNAs comprising different preselected sequences.

43. 25. The method of Claim 24, wherein the plurality of RNAs comprises at least one chemically modified gRNA.

44. 1. A nucleic acid library comprising: A plurality of purified guide RNAs (gRNAs), and At least one single-stranded DNA (ssDNA) encoding a truncated RNA polymerase promoter region A nucleic acid library comprising:

45. 45. The nucleic acid library of Claim 44, wherein each of the purified gRNAs comprises a sequence at least 20 nucleotides in length.

46. 45. The nucleic acid library of Claim 44, wherein each of the purified gRNAs comprises a sequence at least 80 nucleotides in length.

47. 45. The nucleic acid library of Claim 44, wherein the plurality of purified gRNAs comprises at least 100,000 purified gRNAs.

48. 45. The nucleic acid library of claim 44, wherein the truncated RNA polymerase promoter region comprises a sequence selected from the group consisting of SEQ ID NOs: 3 to 6.

49. 45. The nucleic acid library of Claim 44, wherein said plurality of purified gRNAs comprises modified nucleotides.

50. 1. A nucleic acid library comprising: a plurality of purified guide RNAs (gRNAs) at least 20 nucleotides in length; and at least one oligonucleotide of 2 to 10 nucleotides in length A nucleic acid library comprising:

51. 51. The nucleic acid library of Claim 50, wherein said plurality of purified guide RNAs (gRNAs) comprises at least 80 nucleotides in length.

52. 51. The nucleic acid library of Claim 50, wherein said plurality of purified gRNAs comprises at least 100,000 purified gRNAs.

53. 51. The nucleic acid library of claim 50, wherein the at least one oligonucleotide is 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length.

54. 51. The nucleic acid library of claim 50, wherein the at least one oligonucleotide comprises RNA.

55. 51. The nucleic acid library of Claim 50, wherein the at least one oligonucleotide is 100% identical to 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive nucleotides at or near the 5' end of a purified guide RNA.

56. 51. The nucleic acid library of Claim 50, wherein the at least one oligonucleotide is 100% identical to 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive nucleotides at or near the 3' end of the purified guide RNA.

57. 51. The nucleic acid library of Claim 50, wherein the plurality of purified gRNAs comprises at least one chemically modified gRNA.

58. 1. A method for generating a nucleic acid library comprising at least 50 guide RNAs (gRNAs), the method comprising: synthesizing at least 50 gRNAs using an RNA polymerase, wherein at least one of the at least 50 gRNAs comprises a spacer sequence complementary to a target sequence in a target gene, and the 5'-terminal nucleotide of the spacer sequence is complementary to the 3'-terminal nucleotide of the target sequence; A method comprising:

59. 59. The method of Claim 58, wherein each of the at least 50 gRNAs comprises a spacer sequence complementary to a target sequence in a target gene, and the 5'-terminal nucleotide of the spacer sequence is complementary to the 3'-terminal nucleotide of the target sequence.

60. 59. The method of claim 58, wherein the RNA polymerase is selected from the group consisting of a phage RNA polymerase, a bacterial RNA polymerase, and a eukaryotic RNA polymerase.

61. 61. The method of claim 60, wherein the RNA polymerase is T7 RNA polymerase.

62. 59. The method of Claim 58, wherein each of the at least 50 gRNAs exhibits enhanced editing efficiency of the target sequence compared to a gRNA that comprises a 5'-terminal nucleotide that is not complementary to the 3'-terminal nucleotide of the target sequence.

63. 59. The method of Claim 58, wherein each of the at least 50 gRNAs exhibits reduced off-target editing of the target sequence compared to a gRNA that includes a 5'-terminal nucleotide that is not complementary to the 3'-terminal nucleotide of the target sequence.

64. 59. The method of Claim 58, wherein the at least 50 gRNAs comprise gRNA sequences lacking a 5'-terminal guanine (G) nucleotide, the gRNA sequences comprise at least 40 nucleotides in length, and at least three contiguous nucleotides at the 5'-end of the gRNA sequences are 100% identical to the 3'-end of the target sequence in the genome.

65. 65. The method of Claim 64, wherein the at least 50 gRNAs exhibit enhanced 5' ends that pair with the target sequence compared to a gRNA comprising a gRNA sequence that includes a 5' terminal G nucleotide, and wherein the 5' terminal G nucleotide is not present in a protospacer sequence.

66. 65. The method of Claim 64, wherein each of the at least 50 gRNAs exhibits enhanced editing efficiency of a target sequence compared to a gRNA comprising a gRNA sequence comprising a spacer sequence, wherein the gRNA sequence comprises one or more G nucleotides at a 5' end of the spacer sequence, and wherein the one or more G nucleotides at the 5' end of the spacer sequence are not complementary to one or more nucleotides at a 3' end of the target sequence in a genome.

67. 65. The method of Claim 64, wherein each of the at least 50 gRNAs exhibits reduced off-target editing compared to a gRNA comprising a gRNA sequence that comprises a spacer sequence, the gRNA sequence comprising one or more G nucleotides at a 5' end of the spacer sequence, and the one or more G nucleotides at the 5' end of the spacer sequence are not complementary to one or more nucleotides at a 3' end of the target sequence in a genome.

68. 59. The method of Claim 58, wherein the at least 50 gRNAs comprise gRNA sequences lacking a guanine (G) nucleotide in their 5' terminal codon, the gRNA sequences comprise at least 40 nucleotides in length, and at least three consecutive nucleotides at the 5' end of the gRNA sequences are 100% identical to the 3' end of the target sequence in the genome.

69. 69. The method of Claim 68, wherein each of the at least 50 gRNAs exhibits enhanced 5' ends that pair with the target sequence compared to a gRNA comprising a gRNA sequence that includes a G nucleotide in its 5' terminal codon, wherein the G nucleotide in the 5' terminal codon is not present in a protospacer sequence.

70. 69. The method of Claim 68, wherein each of the at least 50 gRNAs exhibits enhanced editing efficiency of a target sequence compared to a gRNA comprising a gRNA sequence comprising a spacer sequence, wherein the gRNA sequence comprises one or more G nucleotides at a 5' end of the spacer sequence, and wherein the one or more G nucleotides at the 5' end of the spacer sequence are not complementary to one or more nucleotides at a 3' end of the target sequence in a genome.

71. 69. The method of Claim 68, wherein each of the at least 50 gRNAs exhibits reduced off-target editing compared to a gRNA comprising a gRNA sequence that comprises a spacer sequence, the gRNA sequence comprising one or more G nucleotides at a 5' end of the spacer sequence, and the one or more G nucleotides at the 5' end of the spacer sequence are not complementary to one or more nucleotides at a 3' end of the target sequence in a genome.

72. 72. The method of any one of claims 58-71, wherein the at least 50 gRNAs comprise at least 100,000 purified gRNAs.

73. 1. A nucleic acid library comprising: At least 50 purified guide RNAs (gRNAs) containing gRNA sequences containing a 5'-terminal guanine (G) analog. A nucleic acid library comprising:

74. 74. The nucleic acid library of Claim 73, wherein each of the at least 50 purified gRNAs exhibits enhanced editing efficiency of a target sequence compared to a gRNA comprising a gRNA sequence lacking a 5' terminal G analog.

75. 74. The nucleic acid library of Claim 73, wherein each of the at least 50 purified gRNAs exhibits enhanced stability compared to a gRNA comprising a gRNA sequence lacking a 5' terminal G analog.

76. 76. The nucleic acid library of any one of claims 73 to 75, wherein the at least 50 purified gRNAs comprise at least 100,000 purified gRNAs.

77. A nucleic acid library, the nucleic acid library comprising: at least 50 RNAs, each of the at least 50 RNAs encoding a different guide RNA (gRNA) sequence, and at least about 90% of the at least 50 RNAs are each present in the nucleic acid library at an amount within 1.5-fold of the average frequency for the at least 50 RNAs in the library; Nucleic acid library.

78. 78. The nucleic acid library of Claim 77, wherein the at least 50 RNAs comprise at least 100, at least 500, at least 1,000, at least 5,000, at least 10,000, at least 50,000, or at least 100,000 RNAs, and at least about 90% of the at least 100, at least 500, at least 1,000, at least 5,000, at least 10,000, at least 50,000, or at least 100,000 RNAs are present in the nucleic acid library at an amount within 1.5-fold of the average frequency for the at least 100, at least 500, at least 1,000, at least 5,000, at least 10,000, at least 50,000, or at least 100,000 RNAs in the library, respectively.

79. 78. The nucleic acid library of Claim 77, wherein the different gRNA sequences comprise at least 80 nucleotides in length.

80. 78. The nucleic acid library of Claim 77, wherein said different gRNA sequences comprise at least 100 different gRNA sequences.

81. 78. The nucleic acid library of Claim 77, wherein at least about 95% of the at least 50 RNAs are each present in the nucleic acid library at an amount within 1.5-fold of the average frequency for each of the RNAs in the library.

82. 78. The nucleic acid library of Claim 77, wherein at least about 99% of the at least 50 RNAs are each present in the nucleic acid library at an amount within 1.5-fold of the average frequency for each of the RNAs in the library.

83. A modified polypeptide composition, said modified polypeptide composition comprising: a purified RNA polymerase, or a functional fragment or variant thereof; a purified nucleic acid binding protein, and optionally a zinc finger containing protein, or a functional fragment or variant thereof; the purified RNA polymerase and the purified nucleic acid binding protein are heterologous, and the purified RNA polymerase and the purified nucleic acid binding protein are linked; composition.

84. 84. The modified polypeptide composition of claim 83, wherein the purified RNA polymerase and the purified nucleic acid binding protein are linked by conjugation.

85. 84. The modified polypeptide composition of claim 83, wherein the purified RNA polymerase and the purified nucleic acid binding protein are linked by fusion.

86. 84. The modified polypeptide composition of claim 83, wherein the purified nucleic acid binding protein is linked to the N-terminus of the purified RNA polymerase.

87. 84. The modified polypeptide composition of claim 83, wherein the purified nucleic acid binding protein is linked to the C-terminus of the purified RNA polymerase.

88. 84. The modified polypeptide composition of claim 83, wherein the purified RNA polymerase is T7 RNA polymerase.

89. 84. The modified polypeptide composition of claim 83, wherein the purified nucleic acid binding protein comprises a zinc finger domain, a leucine zipper, a helix-turn-helix (HTH) motif, a helix-loop-helix (HLH) motif, a winged-helix (WH), a winged-HTH (WHTH) motif, a high-mobility group (HMG) box, a White-Opaque Regulator 3 (Wor3) domain, an oligonucleotide / oligosaccharide-binding (OB) fold domain, an immunoglobulin fold, a B3 domain, a Transcription Activator-Like Effector (TALE), a TALE-like protein, or a clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas protein.

90. 90. The modified polypeptide composition of claim 89, wherein the purified nucleic acid binding protein comprises a zinc finger domain.

91. A composition, the composition comprising: A method for producing a nucleic acid sequence comprising: a fusion RNA polymerase, or a functional fragment or variant thereof, and a DNA polynucleotide; (i) an RNA polymerase, or a functional fragment or variant thereof, and (ii) a DNA-binding protein, or a functional fragment or variant thereof, wherein the RNA polymerase and the DNA-binding protein are heterologous; composition.

92. 92. The composition of claim 91, wherein the DNA polynucleotide is a single-stranded DNA polynucleotide.

93. 93. The composition of claim 91 or 92, wherein the DNA polynucleotide comprises a 3' secondary structure.

94. 94. The composition of claim 93, wherein the 3' secondary structure comprises a hairpin.

95. A modified polypeptide, said modified polypeptide comprising: a variant T7 RNA polymerase or a functional fragment thereof, wherein the variant T7 RNA polymerase comprises at least four variations selected from the group consisting of K172L, P266L, H772R, N748X, R756M, Q758X, and E775V, the positions of which are determined by alignment with SEQ ID NO: 1, and wherein X is any amino acid different from the wild-type amino acid; Modified polypeptides.

96. A modified polypeptide, said modified polypeptide comprising: a variant T7 RNA polymerase or a functional fragment thereof, wherein the variant T7 RNA polymerase comprises at least one variation selected from the group consisting of K172L, P266L, H772R, N748X, R756M, Q758X, and E775V, the position of which is determined by alignment with SEQ ID NO: 1, wherein X is any amino acid different from the wild-type amino acid; and wherein the modified polypeptide is immobilized on a surface. Modified polypeptides.

97. 97. The modified polypeptide of claim 95 or 96, wherein the variant T7 RNA polymerase or functional fragment thereof is linked to a DNA binding protein.

98. 98. The modified polypeptide of claim 97, wherein the variant T7 RNA polymerase or functional fragment thereof is linked to the DNA binding protein by fusion.

99. 98. The modified polypeptide of claim 97, wherein the variant T7 RNA polymerase or functional fragment thereof is linked to the DNA binding protein by conjugation.

100. 98. The modified polypeptide of claim 97, wherein the DNA binding protein is linked to the N-terminus of T7 RNA polymerase.

101. 98. The modified polypeptide of claim 97, wherein the DNA binding protein is linked to the C-terminus of T7 RNA polymerase.

102. 97. The modified polypeptide of claim 95 or 96, wherein the variant T7 RNA polymerase or functional fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO:

1.

103. 97. The modified polypeptide of claim 95 or 96, wherein the variant T7 RNA polymerase or functional fragment thereof exhibits increased stability during the transcription initiation and / or elongation process compared to a T7 RNA polymerase comprising the sequence set forth in SEQ ID NO:

1.

104. 97. The modified polypeptide of claim 95 or 96, wherein the variant T7 RNA polymerase or functional fragment thereof exhibits reduced binding affinity to a T7 promoter sequence compared to a T7 RNA polymerase comprising the sequence set forth in SEQ ID NO:

1.

105. A device, the device comprising: The surface and T7 RNA polymerase, or a functional fragment or variant thereof; a DNA template, the DNA template comprising a truncated T7 promoter sequence; The T7 RNA polymerase, a functional fragment or variant thereof, or a DNA template is linked to the surface. device.

106. 106. The device of claim 105, further comprising a nucleic acid binding protein, or a functional fragment or variant thereof, linked to the surface.

107. 107. The device of claim 105 or 106, wherein the surface is a solid surface.

108. 108. The device of claim 107, wherein the solid surface comprises magnetic beads, agarose beads, fused silica, sol-gel, silica polymer, silica monolith, cellulose, agar, acrylamide, or gold beads.

109. 108. The device of claim 107, wherein the solid surface comprises a gel matrix for encapsulation or entrapment of the T7 RNA polymerase, the DNA template, or the nucleic acid binding protein.

110. The device of claim 106, wherein the nucleic acid binding protein comprises a streptavidin tag.

111. 106. The device of claim 105, wherein the T7 RNA polymerase is immobilized.

112. 106. The device of claim 105, wherein the DNA template is immobilized.

113. 106. The device of claim 105, wherein the nucleic acid binding protein, or the functional fragment or variant thereof, comprises a zinc finger domain, a leucine zipper, a helix-turn-helix (HTH) motif, a helix-loop-helix (HLH) motif, a winged-helix (WH), a winged-HTH (WHTH) motif, a high-mobility group (HMG) box, a White-Opaque Regulator 3 (Wor3) domain, an oligonucleotide / oligosaccharide-binding (OB) fold domain, an immunoglobulin fold, a B3 domain, a Transcription Activator-Like Effector (TALE), a TALE-like protein, or a clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas protein.

114. The device of claim 106, wherein the nucleic acid binding protein, or functional fragment or variant thereof, binds to nucleic acids by sequence-specific binding.

115. The device of claim 106, wherein the nucleic acid binding protein, or functional fragment or variant thereof, binds to the nucleic acid through one or more chemical modifications on the nucleic acid.

116. 116. The device of claim 115, wherein the one or more chemical modifications include biotin or avidin.

117. 106. The device of claim 105, wherein the DNA binding protein comprises a zinc finger domain, a leucine zipper, a helix-turn-helix (HTH) motif, a helix-loop-helix (HLH) motif, a winged helix (WH), a winged-HTH (WHTH) motif, a high-mobility group (HMG) box, a White-Opaque Regulator 3 (Wor3) domain, an oligonucleotide / oligosaccharide-binding (OB) fold domain, an immunoglobulin fold, a B3 domain, a Transcription Activator-Like Effector (TALE), a TALE-like protein, or a clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas protein.

118. The device of claim 105, wherein the DNA template comprises a sequence comprising SEQ ID NO:

7.

119. 106. The device of claim 105, wherein the variant T7 RNA polymerase or variant thereof comprises a sequence having at least 90% identity to SEQ ID NO:

1.

120. 106. The device of claim 105, further comprising a piezoelectric vibrator, a rotary valve, a Peltier heater, a voltage controller, a syringe pump, an ultraviolet LED, and a sensor or a vacuum pump.

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