Method for preparing large-scale plasmid libraries

Abstract

This document provides a method for preparing plasmid libraries in accordance with current Good Manufacturing Practice (cGMP) guidelines.

Description

Technical Field

[0001] [Related Application] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 503,921, filed May 23, 2023, entitled "Method for Producing a Large-Scale Plasmid Library", the entire contents of which are hereby incorporated by reference.

Background Art

[0002] Plasmid production according to current Good Manufacturing Practice (cGMP) standards involves a series of strict processes to ensure the quality, safety, and consistency of the final product. This typically includes the selection of high-quality bacterial strains for plasmid propagation, controlled fermentation under optimal conditions to maximize plasmid yield, and purification using validated methods to remove impurities and endotoxins.

Summary of the Invention

[0003] To date, protocols compliant with current Good Manufacturing Practice (cGMP) guidelines for plasmid production have been limited to the production of homogeneous plasmids (e.g., a plasmid pool in which each plasmid contains the same genetic material). Thus, it has not been possible to perform large-scale library-based assays while complying with cGMP requirements for clinical products.

[0004] Accordingly, provided herein in some embodiments is a method for preparing a current good manufacturing (cGMP) plasmid library comprising several different plasmids, the method comprising the steps of: transforming several competent Escherichia coli with several different plasmids, wherein the plasmids comprise nucleic acids encoding antibiotic resistance genes; contacting the transformed competent Escherichia coli with a first culture system to achieve a minimum number of colonies for each of the different plasmids, wherein the first culture system comprises an antibiotic corresponding to the antibiotic resistance gene; removing the colonies from the first culture system; growing the removed colonies in a second culture system according to cGMP guidelines, wherein the second culture system comprises an antibiotic corresponding to the antibiotic resistance gene, and the antibiotic is present at a concentration of at least twice the standard concentration of the antibiotic; and removing the plasmids from the second culture system according to cGMP guidelines to prepare a cGMP plasmid library.

[0005] In some embodiments, the minimum number of colonies is approximately 100 colonies for each different plasmid. In some embodiments, the minimum number of colonies is approximately 1000 colonies for each different plasmid.

[0006] In some embodiments, the plasmid library includes plasmids encoding RNA or proteins. In some embodiments, the plasmid library includes plasmids encoding therapeutic RNA or therapeutic proteins.

[0007] In some embodiments, the plasmid library contains at least about 5 different plasmids. In some embodiments, the plasmid library contains at least about 50 different plasmids. In some embodiments, the plasmid library contains at least about 100 different plasmids. In some embodiments, the plasmid library contains at least about 250 different plasmids. In some embodiments, the plasmid library contains at least about 500 different plasmids. In some embodiments, the plasmid library contains at least about 1000 different plasmids.

[0008] In some embodiments, competent E. coli includes electrocompetent E. coli. In some embodiments, competent E. coli includes chemical competent E. coli.

[0009] In some embodiments, the antibiotic is one of the following: ampicillin, carbenicillin, kanamycin, specinomycin, streptomycin, bleomycin, erythromycin, chloramphenicol, tetracycline, zeosin, D-cycloserine, gentamicin, G418, nalidixic acid, rifampicin, or trimethoprim. In some embodiments, the antibiotic is a beta-lactam antibiotic. In some embodiments, the beta-lactam antibiotic is a penicillin antibiotic. In some embodiments, the penicillin antibiotic is a carboxypenicillin antibiotic. In some embodiments, the carboxypenicillin antibiotic is carbenicillin. In some embodiments, the carbenicillin concentration is at least about 150 μg / mL. In some embodiments, the carbenicillin concentration is at least about 200 μg / mL.

[0010] In some embodiments, the antibiotics and their corresponding concentrations are: ampicillin: at least about 200 μg / mL; carbenicillin: at least about 200 μg / mL; kanamycin: at least about 200 μg / mL; spectinomycin: at least about 100 μg / mL; streptomycin: at least about 200 μg / mL; bleomycin: at least about 200 μg / mL; erythromycin: at least about 200 μg / mL; chloramphenicol The dosages are: 1.50 μg / mL for ru: at least approximately 50 μg / mL; 2.50 μg / mL for tetracycline: at least approximately 20 μg / mL; 3.50 μg / mL for zeosin: at least approximately 200 μg / mL; 4.50 μg / mL for D-cycloserine: at least approximately 400 μg / mL; 4.50 μg / mL for gentamicin: at least approximately 30 μg / mL; 4.50 μg / mL for G418: at least approximately 800 μg / mL; 4.50 μg / mL for nalidixic acid: at least approximately 60 μg / mL; 5.50 μg / mL for rifampicin: at least approximately 100 μg / mL; or 4.50 μg / mL for trimethoprim: at least approximately 400 μg / mL.

[0011] In some embodiments, the Disclosure provides a method for preparing a current good manufacturing (cGMP) plasmid library comprising several different plasmids, the method comprising the steps of: transforming several electrocompetent Escherichia coli with several different plasmids, wherein the plasmids comprise nucleic acids encoding carbenicillin resistance genes; contacting the transformed competent Escherichia coli with a first culture system to achieve a minimum number of colonies for each of the different plasmids, wherein the first culture system comprises carbenicillin; removing the colonies from the first culture system; growing the removed colonies in a second culture system according to cGMP guidelines, wherein the second culture system comprises at least about 200 μg / mL of carbenicillin; and removing the plasmids from the second culture system according to cGMP guidelines to prepare a cGMP plasmid library.

[0012] In some embodiments, the Disclosure provides a method for preparing a current good manufacturing (cGMP) plasmid library comprising several different plasmids, the method comprising the steps of: transforming several electrocompetent Escherichia coli with several different plasmids, wherein the plasmids comprise nucleic acids encoding carbenicillin resistance genes; contacting the transformed electrocompetent Escherichia coli with a first culture system to achieve a minimum number of colonies for each of the different plasmids, wherein the first culture system comprises carbenicillin; removing the colonies from the first culture system; growing the removed colonies in a second culture system according to cGMP guidelines, wherein the second culture system comprises approximately 200-400 μg / mL of carbenicillin; and removing the plasmids from the second culture system according to cGMP guidelines to prepare a cGMP plasmid library.

[0013] In some embodiments, the Disclosure provides a method for preparing a current good manufacturing (cGMP) plasmid library comprising several different plasmids, the method comprising the steps of: transforming several electrocompetent Escherichia coli with several different plasmids, wherein the plasmids comprise nucleic acids encoding carbenicillin resistance genes; contacting the transformed competent Escherichia coli with a first culture system to achieve a minimum number of colonies for each of the different plasmids, wherein the first culture system comprises carbenicillin; removing the colonies from the first culture system; growing the removed colonies in a second culture system according to cGMP guidelines, wherein the second culture system comprises approximately 200 μg / mL of carbenicillin; and removing the plasmids from the second culture system according to cGMP guidelines to prepare a cGMP plasmid library.

[0014] In some embodiments, the Disclosure provides a method for preparing a current good manufacturing (cGMP) plasmid library comprising several different plasmids, the method comprising the steps of: transforming several electrocompetent Escherichia coli with several different plasmids, wherein the plasmids comprise nucleic acids encoding carbenicillin resistance genes; contacting the transformed competent Escherichia coli with an agar plate to achieve at least about 1000 colonies for each of the different plasmids, wherein the agar plate contains carbenicillin; removing the colonies from the agar plate; growing the removed colonies in a liquid culture system according to cGMP guidelines, wherein the liquid culture system contains about 200 μg / mL of carbenicillin; and removing the plasmids from the liquid culture system according to cGMP guidelines to prepare a cGMP plasmid library.

[0015] In some embodiments, the minimum number of colonies is approximately 100 colonies for each different plasmid. In some embodiments, the minimum number of colonies is approximately 1000 colonies for each different plasmid. [Brief explanation of the drawing]

[0016] [Figure 1] Figure 1 shows the growth curves of bacteria transformed with library plasmids. [Figure 2] Figure 2 shows that the gRNA plasmid library yield is low when using a standard protocol that conforms to the current Good Manufacturing Practices (cGMP) guidelines. [Figure 3] Figure 3 shows the yield of gRNA plasmid libraries after treatment with various antibiotic concentrations. [Figure 4A] Figures 4A-4C show the distribution of single guide RNA (sgRNA) in gRNA library plasmid preparations cultured from glycerol stocks at various concentrations of carbenicillin, compared to the original plasmid library. Figure 4A shows the distribution of gRNA frequencies. [Figure 4B-4C] Figures 4B-4C show a comparison of the original DNA (x axis) with DNA (y axis) from various preparations cultured directly from glycerol or with 100 μg / mL antibiotic (Figure 4B), 200 μg / mL antibiotic, or 400 μg / mL antibiotic (Figure 4C). [Figure 5] Figure 5 shows the improved cGMP gRNA plasmid library yield achieved using the method developed here. The gel shows the cGMP gRNA plasmid library after treatment with restriction enzymes ScaI and EcoRI. [Figure 6] Figure 6 shows histograms of sgRNA distribution from non-cGMP standard gRNA plasmid library preparations (H01-H04) or cGMP large-scale contract research organization (CRO) gRNA plasmid libraries (H05-H08). [Figure 7] Figure 7 shows a histogram of the sgRNA distribution in primary human T cells into which viruses prepared from non-cGMP standard gRNA plasmid library preparations (GPPs) or large-scale CRO cGMP gRNA plasmid library preparations have been introduced. [Modes for carrying out the invention]

[0017] Regulatory authorities in the United States, such as the Food and Drug Administration (FDA), have developed guidelines known as Good Manufacturing Practices (cGMP) to ensure the quality of products consumed or used by humans. Such guidelines apply to facilities and institutions that manufacture these products and are strictly enforced to protect consumers. To date, cGMP-compliant protocols have only allowed for the production of homogeneous plasmids (e.g., plasmids encoding the same sequence) at one time, and there is no method for constructing plasmid libraries (containing two or more different plasmids, e.g., two or more plasmids encoding different sequences) in accordance with cGMP guidelines, such as for the transduction of immune effector cells for therapeutic purposes in humans. This paper provides a methodology for constructing plasmid libraries containing multiple different plasmids, enabling large-scale preparations for cGMP-compliant library-based approaches and ultimately for constructing plasmid libraries for clinical use.

[0018] [Current Good Manufacturing Practices (cGMP)] Current Good Manufacturing Practices (cGMP) are a set of guidelines enforced by regulatory authorities (e.g., the Food and Drug Administration (FDA) in the United States) to ensure the quality, safety, and efficacy of various products, including clinical products. These guidelines cover various aspects of manufacturing, including personnel, facilities, equipment, documentation, and processes, and compliance with the guidelines guarantees the identity, quality, purity, and potency of clinical products. The FDA inspects manufacturing facilities (such as contract research organizations (CROs)) to ensure they are fully compliant with cGMP requirements.

[0019] The cGMP industry guidance regarding the use of microbial vectors for gene therapy ensures that materials used in clinical products (e.g., DNA) comply with FDA regulations. In some embodiments, a gene therapy product refers to a product that exerts its effect by changing the genetic material or the expression of genetic material within a cell and affects the cell phenotype. The term "gene" can refer to a nucleic acid sequence (DNA) that is transcribed into RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. The cGMP guidelines provided by the FDA regarding the use of microbial vectors (e.g., bacteria such as Escherichia coli) for the manufacture of gene therapy drugs include specific culture procedures, cell growth conditions, collection conditions, purification processes, process timing, and intermediate storage.

[0020] Parameters measured in the purified final product to determine compliance with cGMP guidelines include, for example, the following: (i) Sterile filtration of the sample and sample appearance, e.g., colorless, transparent, no visible particulates; (ii) The A260:A280 ratio, which needs to be within the range of approximately 1.7 - 2.0 to confirm the purity of DNA and the absence of RNA in the sample; (iii) For plasmid-derived products administered orally, the presence of endotoxin (a pathogenic component of gram-negative bacteria), which needs to be less than 30 EU / mg and less than 5 EU / kg / dose; (iv) Plasmid size (compared to the starting plasmid being purified); (v) DNA homogeneity, determined by the minimum percentage of supercoiled DNA (at least 75% is supercoiled DNA); (vi) Residual RNA, residual single-stranded DNA, residual linear DNA, and residual chromosomal DNA, which should not be detected when testing 1 μg of the final plasmid preparation; (vii) The restriction enzyme fragment sizes of DNA containing inverted terminal sequences to confirm DNA identity; (viii) Protein concentration, as the purified sample should not contain detectable residual host cell proteins; (ix) When prepared with TE buffer (10 mM Tris-HCl, 1 mM EDTA), the pH of the final product should be in the range of 7.5 to 8.5; and (x) Bioburden, which should not exist, i.e., unexpected proliferation of other microbial species after a certain period of time (e.g., 5 days).

[0021] Adherence to the exemplary standards of the cGMP guidelines described above ensures the suitability of plasmids for human use. Examples of standards for plasmid preparation in accordance with the cGMP guidelines are as set forth herein, are readily accessible, and are known in the art. See, for example, the U.S. Department of Health and Human Services, Food and Drug Administration, Center for Biologics Evaluation and Research, “Recommendations for Microbial Vectors Used for Gene Therapy, 2016” (https: / / www.fda.gov / files / vaccines,%20blood%20&%20biologics / published / Recommendations-for-Microbial-Vectors-Used-for-Gene-Therapy--Guidance-for-Industry.pdf), which also includes non-binding recommendations and suggested procedures for plasmid preparation, which are incorporated herein by reference in their entirety with attribution.

[0022] Current protocols compliant with cGMP guidelines focus on producing only one gene therapy product at a time (e.g., a single guide RNA targeting an intracellular gene for human use). Provided herein is a method for constructing a cGMP plasmid library containing multiple different plasmids (e.g., encoding different sequences, such as different guide RNAs). Plasmid libraries constructed according to the method provided herein enable the construction of cGMP libraries for use, for example, in large-scale transduction or transformation of immune effector cells (such as T cells) suitable for human use. Such a method can be used to construct any cGMP plasmid library for products suitable for human use and therefore represents a significant contribution to the field.

[0023] In some embodiments, this disclosure provides a method for constructing a cGMP plasmid library comprising several different plasmids, comprising the steps of: transforming several competent Escherichia coli with several different plasmids, wherein the plasmids comprise nucleic acids encoding antibiotic resistance genes; contacting the transformed competent Escherichia coli with a first culture system to achieve a minimum number of colonies for each of the different plasmids, wherein the first culture system comprises an antibiotic corresponding to the antibiotic resistance gene; removing the colonies from the first culture system; growing the removed colonies in a second culture system according to cGMP guidelines, wherein the second culture system comprises an antibiotic corresponding to the antibiotic resistance gene, and the antibiotic is present at a concentration at least twice the standard concentration of the antibiotic; and removing the plasmids from the second culture system according to cGMP guidelines to construct a cGMP plasmid library.

[0024] In this context, "to contact" refers to mixing cells with a culture medium, such as an agar plate or a liquid culture system (e.g., broth). "To extract" colonies refers to isolating colonies from a culture medium, for example by scraping them off an agar plate or by centrifuging a liquid culture system, and separating colonies from a liquid medium for isolation. "To extract" plasmids refers to isolating plasmids from a culture system (e.g., an E. coli culture system).

[0025] "First culture system" refers to a first culture medium that promotes the growth of E. coli, such as an agar plate or a liquid culture system. "Second culture system" refers to a second culture medium that promotes the growth of E. coli, such as an agar plate or a liquid culture system. In some embodiments, the first culture system includes an agar plate, and the second culture system includes an agar plate. In some embodiments, the first culture system includes an agar plate, and the second culture system includes a liquid culture system. In some embodiments, the first culture system includes a liquid culture system, and the second culture system includes a liquid culture system. In some embodiments, the first culture system includes a liquid culture system, and the second culture system includes an agar plate.

[0026] In some embodiments, a method for preparing a cGMP plasmid library containing multiple different plasmids includes the steps of: transforming multiple electrocompetent Escherichia coli with multiple different plasmids, wherein the plasmids contain nucleic acids encoding a carbenicillin resistance gene; contacting the transformed electrocompetent Escherichia coli with a first culture system to achieve a minimum number of colonies for each of the different plasmids, wherein the first culture system contains carbenicillin; removing the colonies from the first culture system; growing the removed colonies in a second culture system according to cGMP guidelines, wherein the second culture system contains at least about 200 μg / mL of carbenicillin and the antibiotic is present at a concentration of at least twice the standard concentration of the antibiotic; and removing the plasmids from the second culture system according to cGMP guidelines to prepare a cGMP plasmid library.

[0027] In some embodiments, a method for preparing a cGMP plasmid library containing multiple different plasmids includes the steps of: transforming multiple electrocompetent Escherichia coli with multiple different plasmids, wherein the plasmids contain nucleic acids encoding carbenicillin resistance genes; contacting the transformed competent Escherichia coli with a first culture system to achieve a minimum number of colonies for each of the different plasmids, wherein the first culture system contains carbenicillin; removing the colonies from the first culture system; growing the removed colonies in a second culture system according to cGMP guidelines, wherein the second culture system contains approximately 200-400 μg / mL of carbenicillin and the antibiotic is present at a concentration of at least twice the standard concentration of the antibiotic; and removing the plasmids from the second culture system according to cGMP guidelines to prepare a cGMP plasmid library.

[0028] In some embodiments, a method for preparing a cGMP plasmid library containing multiple different plasmids includes the steps of: transforming multiple electrocompetent Escherichia coli with multiple different plasmids, wherein the plasmids contain nucleic acids encoding carbenicillin resistance genes; contacting the transformed electrocompetent Escherichia coli with a first culture system to achieve a minimum number of colonies for each of the different plasmids, wherein the first culture system contains carbenicillin; removing the colonies from the first culture system; growing the removed colonies in a second culture system according to cGMP guidelines, wherein the second culture system contains approximately 200 μg / mL of carbenicillin; and removing the plasmids from the second culture system according to cGMP guidelines to prepare a cGMP plasmid library.

[0029] In some embodiments, a method for preparing a cGMP plasmid library containing multiple different plasmids includes the steps of: transforming multiple electrocompetent Escherichia coli with multiple different plasmids, wherein the plasmids contain nucleic acids encoding a carbenicillin resistance gene; contacting the transformed electrocompetent Escherichia coli with an agar plate to achieve at least 1000 colonies for each of the different plasmids, wherein the agar plate contains carbenicillin; removing the colonies from the agar plate; growing the removed colonies in a liquid culture system according to cGMP guidelines, wherein the liquid culture system contains approximately 200 μg / mL of carbenicillin; and removing the plasmids from the liquid culture system according to cGMP guidelines to prepare a cGMP plasmid library.

[0030] In some embodiments, a method for preparing a cGMP plasmid library containing multiple different plasmids includes the steps of: transforming multiple electrocompetent Escherichia coli with multiple different plasmids, wherein the plasmids contain nucleic acids encoding carbenicillin resistance genes; contacting the transformed electrocompetent Escherichia coli with an agar plate to achieve at least 1000 colonies for each of the different plasmids, wherein the agar plate contains carbenicillin; removing the colonies from the agar plate; growing the removed colonies in a liquid culture system according to cGMP guidelines, wherein the liquid culture system contains 200 μg / mL of carbenicillin; and removing the plasmids from the liquid culture system according to cGMP guidelines to prepare a cGMP plasmid library.

[0031] [Plasmids and Plasmid Libraries] As used herein, “plasmid library” refers to a group of different plasmids (e.g., those encoding different sequences). “Current Good Manufacturing Practice (cGMP) plasmid library” refers to a plasmid library prepared in accordance with cGMP guidelines (e.g., as described herein). In some embodiments, “plasmid” as described herein refers to a circular DNA piece containing sequence elements for the replication and expression of polynucleotides (e.g., guide RNA). In some embodiments, the plasmid contains nucleic acids encoding antibiotic resistance genes.

[0032] In some embodiments, the plasmid library contains at least about 5 different plasmids (for example, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 35, at least about 40, at least about 45, etc.). In some embodiments, the plasmid library contains at least about 50 different plasmids (for example, at least about 51, at least about 52, at least about 53, at least about 54, at least about 55, at least about 56, at least about 57, at least about 58, at least about 59, at least about 60, at least about 61, at least about 62, at least about 63, at least about 64, at least about 65, at least about 66, at least about 67, at least about 68, at least about 69, at least about 70, at least about 71, at least about 72, at least about 73, at least about 74, at least about 75, at least about 76, at least about 77, at least about 78, at least about 79, at least about 80, at least about 85, at least about 90, at least about 95, etc.). In some embodiments, the plasmid library contains at least about 100 different plasmids (e.g., at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, at least about 200, at least about 210, at least about 220, at least about 230, at least about 240, etc.). In some embodiments, the plasmid library contains at least about 500 different plasmids (e.g., at least about 550, at least about 600, at least about 650, at least about 700, at least about 750, at least about 800, at least about 850, at least about 900, at least about 950, etc.).In some embodiments, the plasmid library contains at least about 1000 different plasmids (e.g., at least about 1100, at least about 1200, at least about 1300, at least about 1400, at least about 1500, at least about 1600, at least about 1700, at least about 1800, at least about 1900, at least about 2000, at least about 2500, etc.).

[0033] In some embodiments, the plasmid library includes at least 5 different plasmids (e.g., at least 6, 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 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 35, at least 40, at least 45, etc.). In some embodiments, the plasmid library includes at least 50 different plasmids (e.g., at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 85, at least 90, at least 95, etc.). In some embodiments, the plasmid library includes at least 100 different plasmids (e.g., at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, etc.). In some embodiments, the plasmid library contains at least 500 different plasmids (e.g., at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 950, etc.).In some embodiments, the plasmid library contains at least 1000 different plasmids (e.g., at least 1100, at least 1200, at least 1300, at least 1400, at least 1500, at least 1600, at least 1700, at least 1800, at least 1900, at least 2000, at least 2500, etc.).

[0034] In some embodiments, the plasmid library includes plasmids encoding RNA or proteins. The term “protein” refers to a polymer of amino acids. In some embodiments, the plasmid library includes plasmids encoding therapeutic RNA or therapeutic proteins. “Therapeutic RNA” or “therapeutic protein” refers to RNA or proteins that can be used to treat, improve, inhibit, slow the progression of, or halt a disease or disorder (e.g., cancer, plasma cell disease, or autoimmune disease). In some embodiments, the therapeutic RNA or therapeutic protein improves the symptoms or side effects of the disease or disorder.

[0035] In some embodiments, the plasmid library includes plasmids encoding variants of a gene that result in increased expression of the protein encoded by the gene. In some embodiments, the plasmid library includes plasmids encoding variants of a gene that result in decreased expression of the protein encoded by the gene.

[0036] [Guide RNA (gRNA)] This disclosure provides, in several embodiments, a method for providing a current Good Manufacturing Practice (cGMP) plasmid library comprising multiple different plasmids. For example, the plasmid library may comprise multiple different plasmids, each encoding a different guide RNA (gRNA). As used herein, the terms “guide RNA (gRNA)” or “guide RNA (gRNA) polynucleotide” refer to an RNA polynucleotide or DNA encoding a guide RNA (gRNA). The guide RNA polynucleotide comprises a sequence that clusters and binds to a regularly arranged short palindromic sequence repeat (CRISPR) protein or CRISPR-related protein, and an additional sequence (i.e., homologous region) complementary to the target polynucleotide. As used herein, the term “CRISPR” may refer to a gene editing system comprising a guide RNA component (e.g., the guide RNA encoded by the plasmid provided herein) and a CRISPR-related (Cas) protein component. The guide RNA polynucleotide may comprise a homologous region complementary to the target gene and a stem-loop region capable of binding to the Cas protein. The Cas protein may comprise a guide RNA binding site and nuclease activity. Methods for designing guide RNAs (e.g., selecting homologous region sequences to target specific genes) are also well known in the art, as described in their entirety in Computational and Structural Biotechnology Journal 18(2020):35-44, which is cited in their entirety. In some embodiments, the gRNA (encoded by gRNA polynucleotides) is designed using CRISPick (portals.broadinstitute.org / gppx / crispick / public), which functions as described in Doench et al., Nature Biotechnology, 34(2), 184-191 (2016) and Sanson et al., Nature Communications, 9(1), 5416 (2018), both of which are cited in their entirety.

[0037] [Bacteria and their transformations] Provided herein is a method for constructing a current Good Manufacturing Practice (cGMP) library containing multiple different plasmids, the method comprising transforming multiple competent Escherichia coli (Escherichia coli) cells with the multiple different plasmids. Bacterial transformation involves the introduction of heterologous DNA into bacterial cells, such as linear DNA or plasmids described herein.

[0038] Escherichia coli is the most widely used bacterium for transformation due to its rapid growth rate, well-established culture methods, versatility, and the safety of its laboratory strains. E. coli can be cultured using several types of culture media, such as agar plates or liquid media, such as Luria Bertani (LB) agar plates or broth, Terrific Buffer (TB) agar plates or broth, catabolite-suppressed Super Optimal Broth (SOC) liquid medium (e.g., for recovering E. coli after heat shock), MacConkey agar plates, X-Gal / IPTG agar plates, or other solid or liquid substrates. In some embodiments, the methods provided herein involve contacting transformed competent E. coli with a first culture system to achieve a minimum number of colonies for each of several different plasmids. In some embodiments, achieving a minimum number of colonies for each of the different plasmids results in the presentation of each of the different plasmids in the multiple plasmids when the plasmids are isolated and a cGMP plasmid library is constructed. In some embodiments, the minimum number of colonies is the approximate minimum number of colonies.

[0039] In some embodiments, the minimum number of colonies is approximately 100 colonies for each different plasmid. In some embodiments, the minimum number of colonies is approximately 200 colonies for each different plasmid. In some embodiments, the minimum number of colonies is approximately 300 colonies for each different plasmid. In some embodiments, the minimum number of colonies is approximately 400 colonies for each different plasmid. In some embodiments, the minimum number of colonies is approximately 500 colonies for each different plasmid. In some embodiments, the minimum number of colonies is approximately 600 colonies for each different plasmid. In some embodiments, the minimum number of colonies is approximately 700 colonies for each different plasmid. In some embodiments, the minimum number of colonies is approximately 800 colonies for each different plasmid. In some embodiments, the minimum number of colonies is approximately 900 colonies for each different plasmid. In some embodiments, the minimum number of colonies is approximately 1000 colonies for each different plasmid. In some embodiments, the minimum number of colonies is approximately 1100 colonies for each different plasmid. In some embodiments, the minimum number of colonies is approximately 1200 colonies for each different plasmid. In some embodiments, the minimum number of colonies is approximately 1300 colonies for each different plasmid. In some embodiments, the minimum number of colonies is approximately 1400 colonies for each different plasmid. In some embodiments, the minimum number of colonies is approximately 1500 colonies for each different plasmid. In some embodiments, the minimum number of colonies is approximately 2000 colonies for each different plasmid.

[0040] In some embodiments, the minimum number of colonies is 100 colonies for each different plasmid. In some embodiments, the minimum number of colonies is 200 colonies for each different plasmid. In some embodiments, the minimum number of colonies is 300 colonies for each different plasmid. In some embodiments, the minimum number of colonies is 400 colonies for each different plasmid. In some embodiments, the minimum number of colonies is 500 colonies for each different plasmid. In some embodiments, the minimum number of colonies is 600 colonies for each different plasmid. In some embodiments, the minimum number of colonies is 700 colonies for each different plasmid. In some embodiments, the minimum number of colonies is 800 colonies for each different plasmid. In some embodiments, the minimum number of colonies is 900 colonies for each different plasmid. In some embodiments, the minimum number of colonies is 1000 colonies for each different plasmid. In some embodiments, the minimum number of colonies is 1100 colonies for each different plasmid. In some embodiments, the minimum number of colonies is 1200 colonies for each different plasmid. In some embodiments, the minimum number of colonies is 1300 colonies for each different plasmid. In some embodiments, the minimum number of colonies is 1400 colonies for each different plasmid. In some embodiments, the minimum number of colonies is 1500 colonies for each different plasmid. In some embodiments, the minimum number of colonies is 2000 colonies for each different plasmid.

[0041] In some embodiments, transformation involves transforming competent E. coli. Competent E. coli are treated to enhance the uptake of foreign DNA compared to non-competent E. coli. Examples of competent E. coli include chemically competent E. coli transformed using, for example, heat shock, and electrocompetent E. coli transformed using, for example, electroporation. Chemically competent E. coli cells are commercially available and can also be prepared, for example, by treating E. coli with a series of salt washes to disrupt the cell membrane. Electrocompetent E. coli are also commercially available and can also be prepared, for example, by washing cells with cold deionized water and glycerol. In some embodiments, competent E. coli includes electrocompetent E. coli. In some embodiments, competent E. coli includes chemically competent E. coli. In some embodiments, competent E. coli includes DH10B, TOP10, GB10B TM GB5-α TM BL21, TG1, RR1, JM109, HB101, DH5a-E, DH12S, or Stbl4 TM Contains competent E. coli cells. In some embodiments, the competent E. coli is Stbl4 TM Contains competent E. coli cells.

[0042] The method provided herein may also use other bacterial species (e.g., Bacillus subtilis).

[0043] [Antibiotics and Antibiotic Resistance] By incorporating antibiotic resistance genes into plasmids that transform bacterial cells, it becomes possible to select cells that have successfully taken up the plasmid. An "antibiotic resistance gene" is a gene that protects cells (e.g., bacterial cells) from the cytotoxic effects of the corresponding antibiotic. For example, by incorporating a gene that confers carbenicillin resistance into a plasmid, it becomes possible to select E. coli cells that have taken up the plasmid by introducing carbenicillin into the culture medium (e.g., agar plates or liquid culture system) in which E. coli cells are cultured.

[0044] In some embodiments, the antibiotic is any antibiotic effective in killing E. coli cells. This includes, but is not limited to, antibiotics belonging to the classes of antibiotics such as β-lactams, fluoroquinolones, aminoglycosides, sulfonamides, and trimethoprims, tetracyclines, and macrolides. In some embodiments, the antibiotic is a β-lactam antibiotic. In some embodiments, the β-lactam antibiotic is a penicillin antibiotic. In some embodiments, the penicillin antibiotic is a carboxypenicillin antibiotic. In some embodiments, the carboxypenicillin antibiotic is carbenicillin.

[0045] The "standard concentration" of an antibiotic is the concentration at which the antibiotic is used in a cell culture system to prevent the growth of susceptible bacteria (e.g., the effective concentration). Standard concentrations of antibiotics typically range from approximately 1 μg / mL to approximately 1000 μg / mL. Standard concentrations of antibiotics for use in cell culture systems are known in the art and are therefore readily apparent to those skilled in the art. Table 1 shows example antibiotics and their standard concentrations.

[0046] In some embodiments, the antibiotic is carbenicillin. In some embodiments, the antibiotic is ampicillin. In some embodiments, the antibiotic is bleomycin. In some embodiments, the antibiotic is chloramphenicol. In some embodiments, the antibiotic is D-cycloserine. In some embodiments, the antibiotic is erythromycin. In some embodiments, the antibiotic is gentamicin. In some embodiments, the antibiotic is G418. In some embodiments, the antibiotic is kanamycin. In some embodiments, the antibiotic is nalidixic acid. In some embodiments, the antibiotic is novobiocin. In some embodiments, the antibiotic is rifampicin. In some embodiments, the antibiotic is spectinomycin. In some embodiments, the antibiotic is streptomycin. In some embodiments, the antibiotic is tetracycline. In some embodiments, the antibiotic is trimethoprim. In some embodiments, the antibiotic is zeosin.

[0047] In some embodiments, provided herein are methods for preparing a current Good Manufacturing Practice (cGMP) plasmid library containing several different plasmids, which partially include growing transformed bacterial cells in a liquid culture system containing an antibiotic present at a concentration of at least about twice (or at least 2×) the standard concentration of the antibiotic. In some embodiments, the antibiotic is present at a concentration of at least about 1.5× the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least about 1.6× the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least about 1.7× the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least about 1.8× the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least about 1.9× the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least about 2.1× the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least about 2.2× the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least about 2.3× the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least about 2.4 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least about 2.5 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least about 2.6 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least about 2.7 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least about 2.8 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least about 2.9 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least about 3 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least about 3.1 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least about 3.2 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least about 3.3 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least about 3.4 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least about 3.5 × of the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least about 3.6 × of the standard concentration.In some embodiments, the antibiotic is present at a concentration of at least about 3.7 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least about 3.8 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least about 3.9 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least about 4 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least about 4.5 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least about 5 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least about 5.5 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least about 6 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least about 6.5 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least about 7 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least about 7.5 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least about 8 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least about 8.5 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least about 9 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least about 9.5 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least about 10 × the standard concentration.

[0048] In some embodiments, the antibiotic is present at a concentration of at least 1.5 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least 1.6 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least 1.7 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least 1.8 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least 1.9 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least 2.1 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least 2.2 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least 2.3 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least 2.4 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least 2.5 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least 2.6 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least 2.7 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least 2.8 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least 2.9 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least 3 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least 3.1 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least 3.2 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least 3.3 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least 3.4 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least 3.5 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least 3.6 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least 3.7 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least 3.8 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least 3.9 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least 4x of the standard concentration.In some embodiments, the antibiotic is present at a concentration of at least 4.5 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least 5 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least 5.5 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least 6 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least 6.5 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least 7 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least 7.5 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least 8 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least 8.5 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least 9 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least 9.5 × the standard concentration. In some embodiments, the antibiotic is present at a concentration of at least 10 × the standard concentration.

[0049] In some embodiments, the antibiotic is present at a concentration at least about 50% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 60% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 70% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 80% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 90% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 100% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 110% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 120% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 130% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 140% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 150% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 160% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 170% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 180% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 190% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 200% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 210% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 220% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 230% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 240% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 250% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 260% higher than the standard concentration. In some embodiments, antibiotics are present at concentrations at least approximately 270% higher than standard concentrations.In some embodiments, the antibiotic is present at a concentration at least about 280% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 290% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 300% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 350% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 400% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 450% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 500% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 550% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 600% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 650% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 700% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 750% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 800% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 850% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 900% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 950% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least about 1000% higher than the standard concentration.

[0050] In some embodiments, the antibiotic is present at a concentration at least 50% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 60% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 70% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 80% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 90% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 100% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 110% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 120% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 130% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 140% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 150% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 160% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 170% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 180% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 190% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 200% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 210% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 220% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 230% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 240% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 250% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 260% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 270% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 280% higher than the standard concentration.In some embodiments, the antibiotic is present at a concentration at least 290% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 300% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 350% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 400% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 450% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 500% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 550% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 600% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 650% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 700% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 750% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 800% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 850% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 900% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 950% higher than the standard concentration. In some embodiments, the antibiotic is present at a concentration at least 1000% higher than the standard concentration.

[0051] In some embodiments, the agar plate or liquid culture system contains an antibiotic present at a concentration at least twice the standard concentration of the antibiotic. In some embodiments, the antibiotics and their corresponding concentrations are: ampicillin: at least about 200 μg / mL; carbenicillin: at least about 200 μg / mL; kanamycin: at least about 200 μg / mL; spectinomycin: at least about 100 μg / mL; streptomycin: at least about 200 μg / mL; bleomycin: at least about 200 μg / mL; erythromycin: at least about 200 μg / mL; chloramphenicol The concentrations are: 1 / 2: at least about 50 μg / mL; 2 / 2: at least about 20 μg / mL; 3 / 2: at least about 200 μg / mL; 4 / 2: at least about 400 μg / mL; 5 / 2: at least about 30 μg / mL; 6 / 2: at least about 800 μg / mL; 700 μg / mL: at least about 60 μg / mL; 800 μg / mL: at least about 60 μg / mL; 900 μg / mL: at least about 100 μg / mL; or 1 / 2: at least about 400 μg / mL. In some embodiments, the antibiotic and corresponding concentration is ampicillin: at least about 200 μg / mL. In some embodiments, the antibiotic and corresponding concentration is carbenicillin: at least about 200 μg / mL. In some embodiments, the antibiotic and corresponding concentration is kanamycin: at least about 200 μg / mL. In some embodiments, the antibiotic and corresponding concentration is spectinomycin: at least about 100 μg / mL. In some embodiments, the antibiotic and its corresponding concentration is streptomycin: at least about 200 μg / mL. In some embodiments, the antibiotic and its concentration is bleomycin: at least about 200 μg / mL. In some embodiments, the antibiotic and its concentration is erythromycin: at least about 200 μg / mL. In some embodiments, the antibiotic and its corresponding concentration is chloramphenicol: at least about 50 μg / mL. In some embodiments, the antibiotic and its corresponding concentration is tetracycline: at least about 20 μg / mL. In some embodiments, the antibiotic and its corresponding concentration is zeosin: at least about 200 μg / mL. In some embodiments, the antibiotic and its corresponding concentration is D-cycloserine: at least about 400 μg / mL.In some embodiments, the antibiotic and corresponding concentration is gentamicin: at least about 30 μg / mL. In some embodiments, the antibiotic and corresponding concentration is G418: at least about 800 μg / mL. In some embodiments, the antibiotic and corresponding concentration is nalidixic acid: at least about 60 μg / mL. In some embodiments, the antibiotic and corresponding concentration is rifampicin: at least about 100 μg / mL. In some embodiments, the antibiotic and corresponding concentration is trimethoprim: at least about 400 μg / mL. In some embodiments, the antibiotic and corresponding concentration is trimethoprim: at least about 400 μg / mL. In some embodiments, the antibiotic is carbenicillin, and the corresponding concentration is about 200-400 μg / mL. In some embodiments, the antibiotic is carbenicillin, and the corresponding concentration is at least about 150 μg / mL. In some embodiments, the antibiotic is carbenicillin, and the corresponding concentration is about 200 μg / mL.

[0052] In some embodiments, the agar plate or liquid culture system contains an antibiotic present at a concentration at least twice the standard concentration of the antibiotic. In some embodiments, the antibiotic and corresponding concentration are ampicillin: at least 200 μg / mL; carbenicillin: at least 200 μg / mL; kanamycin: at least 200 μg / mL; spectinomycin: at least 100 μg / mL; streptomycin: at least 200 μg / mL; bleomycin: at least 200 μg / mL; erythromycin: at least 200 μg / mL; chloramphenicol: at least 50 μg / mL; tetracycline: at least 20 μg / mL; zeosin: at least 200 μg / mL; D-cycloserine: at least 400 μg / mL; gentamicin: at least 30 μg / mL; G418: at least 800 μg / mL; nalidixic acid: at least 60 μg / mL; rifampicin: at least 100 μg / mL; or trimethoprim: at least 400 μg / mL. In some embodiments, the antibiotic and corresponding concentration is ampicillin: at least 200 μg / mL. In some embodiments, the antibiotic and corresponding concentration is carbenicillin: at least 200 μg / mL. In some embodiments, the antibiotic and corresponding concentration is kanamycin: at least 200 μg / mL. In some embodiments, the antibiotic and corresponding concentration is spectinomycin: at least 100 μg / mL. In some embodiments, the antibiotic and corresponding concentration is streptomycin: at least 200 μg / mL. In some embodiments, the antibiotic and corresponding concentration is bleomycin: at least 200 μg / mL. In some embodiments, the antibiotic and corresponding concentration is erythromycin: at least 200 μg / mL. In some embodiments, the antibiotic and corresponding concentration is chloramphenicol: at least 50 μg / mL. In some embodiments, the antibiotic and corresponding concentration is tetracycline: at least 20 μg / mL. In some embodiments, the antibiotic and corresponding concentration is zeosin: at least 200 μg / mL. In some embodiments, the antibiotic and corresponding concentration is D-cycloserine: at least 400 μg / mL.In some embodiments, the antibiotic and corresponding concentration is gentamicin: at least 30 μg / mL. In some embodiments, the antibiotic and corresponding concentration is G418: at least 800 μg / mL. In some embodiments, the antibiotic and corresponding concentration is nalidixic acid: at least 60 μg / mL. In some embodiments, the antibiotic and corresponding concentration is rifampicin: at least 100 μg / mL. In some embodiments, the antibiotic and corresponding concentration is trimethoprim: at least 400 μg / mL. In some embodiments, the antibiotic and corresponding concentration is trimethoprim: at least 400 μg / mL. In some embodiments, the antibiotic and corresponding concentration is trimethoprim: at least 400 μg / mL. In some embodiments, the antibiotic is carbenicillin, and the corresponding concentration is 200-400 μg / mL. In some embodiments, the antibiotic is carbenicillin, and the corresponding concentration is at least 150 μg / mL. In some embodiments, the antibiotic is carbenicillin, with a corresponding concentration of 200 μg / mL. Exemplary antibiotics and their corresponding concentrations are shown in Table 1.

[0053] TIFF2026518048000002.tif133170

[0054] [General definition] The terms “reduce,” “reduced,” “decrease,” or “inhibit” are all used here to mean a reduction of a statistically significant amount. In some embodiments, “reduce,” “decrease,” or “decrease” or “inhibit” typically mean a reduction of at least 10% compared to a baseline level (e.g., in the absence of a given treatment or drug), and may include reductions of, for example, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or greater. “Complete inhibition” is 100% inhibition compared to a baseline level. Where applicable, reduction may preferably be a reduction to a level that is acceptable as within the normal range for a given non-disabled individual. The terms “increased,” “enhance,” “enhance,” or “activate” are all used here to mean an increase of a statistically significant amount. In some embodiments, the terms “increased,” “enhance,” or “activate” may mean an increase of at least 10% compared to a reference level, for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to 100% compared to a reference level, or any increase between 10% and 100% compared to a reference level, or an increase of at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times compared to a reference level, or any increase between 2 times and 10 times or more compared to a reference level. In the context of markers or symptoms, “increase” is a statistically significant increase of such a level.

[0055] In some embodiments, the term “modified” and its grammatical synonyms as used herein may refer to one or more human-designed modifications of nucleic acids, such as nucleic acids within the genome of an organism. In other embodiments, “modified” may refer to changes, additions, and / or deletions of genes. In some embodiments, “modified cell” refers to a cell in which genes have been added, deleted, and / or altered (for example, using guide RNA produced by the methods provided herein). In some embodiments, “modified nucleic acid,” “modified nucleic acid molecule,” or “modified polynucleotide” refers to a nucleic acid, nucleic acid molecule, or polynucleotide containing changes designed by one or more humans.

[0056] The terms “cell” or “modified cell” used herein, and their grammatical synonyms, may refer to cells of human or non-human animal origin.

[0057] The term “polynucleotide” is used here interchangeably with “nucleic acid molecule” and refers to a polymer of nucleosides. Typically, polynucleotides are composed of naturally occurring nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) linked to DNA or RNA by phosphodiester bonds. However, the term encompasses molecules containing nucleosides or nucleoside analogs, including chemically or biologically modified bases, modified skeletons, etc., whether or not they are present in naturally occurring nucleic acids, and such molecules may be preferred in a given application. When polynucleotides are referred to in this application, it is understood that both DNA and RNA, and in each case both single-stranded and double-stranded forms (and complementary strands of each single-stranded molecule) are provided. As used herein, “polynucleotide sequence” may refer to the polynucleotide material itself and / or sequence information (i.e., a series of letters used as abbreviations for bases) that biochemically characterizes a particular nucleic acid. In some embodiments, the nucleic acid molecule is a heterogeneous nucleic acid molecule. As used herein, the term “heterogeneous nucleic acid molecule” refers to a nucleic acid molecule that does not naturally exist within a given cell.

[0058] As used herein, the term “vector” refers to a nucleic acid construct designed for delivery to or transport between different host cells. As used herein, a vector can be viral or nonviral. The term “vector” encompasses any genetic element that, when bound to an appropriate regulatory element, is replicable and can introduce a gene sequence into a cell. Vectors may include, but are not limited to, cloning vectors, expression vectors, plasmids, phages, transposons, cosmids, artificial chromosomes, viruses, and virions.

[0059] Where used herein, the term “expression vector” may refer to a vector that directs the expression of RNA or protein from a sequence ligated to a transcriptional regulatory sequence on the vector. The term “expression” refers to cellular processes involved in the production of RNA and protein, and, if applicable, the secretion of proteins, and, where applicable, includes, but is not limited to, transcription, transcriptional processing, translation and protein folding, modification and processing. “Expression products” include RNA transcribed from genes and polypeptides obtained by translation of mRNA transcribed from genes. Where used herein, the term “viral vector” may refer to a nucleic acid vector construct that includes at least one viral element and is packaged into viral vector particles. Viral vectors may include nucleic acids encoding the polypeptides described herein, instead of non-essential viral genes. Vectors and / or particles may be used for the purpose of introducing nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art. Unless otherwise stated in the examples or elsewhere, all numerical values ​​representing the amounts of components or reaction conditions used herein should be understood in all cases as being modified by the term “approximately”.

[0060] When used in relation to a value, the terms "approximately" or "about" may mean that the value or any description of that value encompasses a range of ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, ±10%, ±1-5%, ±2-7%, ±3-8%, ±4-9%, or ±5-10% of that value.

[0061] The singular terms “a,” “an,” and “the” include plural referents unless the context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. Methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this disclosure, but suitable methods and materials are described below. The abbreviation “e.g.” “exempli gratia” is derived from the Latin “exempli gratia” and is used here to indicate non-restrictive examples. Thus, the abbreviation “eg” is synonymous with the term “for example.”

[0062] Other terms are defined in the descriptions of the various aspects and embodiments of this technology, as set forth herein. [Examples]

[0063] Example 1. Preparation of a large-scale cGMP plasmid library Adherence to current Good Manufacturing Practice (cGMP) guidelines ensures the safety of clinical products and guarantees the claimed ingredients and efficacy. However, standard cGMP guidelines for plasmid preparation are limited to single plasmids and do not permit large-scale transduction or transformation using plasmid libraries. This paper provides a novel process for maintaining the presentation of the entire library while adhering to cGMP guidelines.

[0064] [Methods and Results] A plasmid library was prepared containing 1180 unique plasmids, each containing 1180 distinctive clustered and regularly arranged short palindromic repeat (CRISPR) guide RNAs (gRNAs) and carbenicillin resistance genes. First, the plasmids were transformed into chemically competent Escherichia coli according to a standard cGMP-compliant protocol, and the E. coli were grown on agar plates containing carbenicillin. Glycerol stocks were prepared from bacterial colonies isolated from the agar plates. Initial plasmid yield analysis was performed by scraping the glycerol stocks and culturing each scrape in 15 mL of Terrific Buffer (TB) containing 100 ug / mL of carbenicillin at 30°C. Analysis of the growth curve of E. coli revealed that the E. coli culture had just begun to exit the logarithmic growth phase after 16 hours (measured by optical density, OD) (Figure 1), resulting in very low plasmid yield (Figure 2), and ultimately making it unsuitable for large-scale transduction or transformation of immunoeffector cells.

[0065] Next, several parameters were adjusted to improve plasmid yield. The plasmids were transformed into electrocompetent E. coli and plated onto agar plates containing carbenicillin. After incubating the agar plates until bacterial growth reached an average colony coverage of at least 1000 colonies per plasmid, a glycerol stock pool containing the colonies was prepared. The glycerol stock was scraped off and cultured overnight in 250 mL of TB containing 100 μg / mL; 200 μg / mL; or 400 μg / mL of carbenicillin. Both cultures with 200 μg / mL and 400 μg / mL of carbenicillin showed a significant improvement in DNA yield (Figure 3).

[0066] To determine whether the DNA yield correlated with the complete coverage of the gRNA library, plasmid preparations were sequenced and the results were compared with those of the original plasmid library. The plasmid preparations were found to achieve similar guide abundances compared to the original library (Figures 4A-4C).

[0067] To determine whether the above preparations would achieve similar results using cGMP guidelines, glycerol stocks were prepared and sent to a contract research organization (CRO). Approximately 20 μL of glycerol scrapings were cultured in 15 mL of Terrific Buffer (TB) containing 200 μg / mL carbenicillin at 37°C for 6–8 hours. The liquid culture system was then transferred to 12 L of TB containing 200 μg / mL carbenicillin, from which plasmids were extracted according to cGMP guidelines.

[0068] The pooled gRNA library plasmid preparations were treated with Scal and ExoRI restriction enzymes to obtain DNA fragments of the expected size (Figure 5). Comparison of the gRNA distribution with that prepared according to cGMP guidelines showed a similar distribution of guide abundances compared to the plasmid pool preparation prepared as described above (Figure 6). Critically, the gRNA distribution in primary human T cells transduced with virus using the method provided here in accordance with cGMP guidelines, and also from pool plasmids prepared in a standard laboratory (non-GMP), both showed similar guide abundances and frequencies.

[0069] In summary, the data demonstrate that the method presented here enables large-scale plasmid library preparation in accordance with cGMP guidelines, and allows for large-scale transduction or transformation of immunoeffector cells (such as T cells) for human use, which was previously limited by standard protocols compliant with cGMP guidelines for the preparation of homogeneous plasmids (e.g., plasmids encoding the same sequence). This method can also be used to prepare large-scale preparations using other library-based approaches for CRISPR screening or in vivo testing.

Claims

1. In a method for preparing a cGMP-compliant plasmid library containing multiple different plasmids, A step of transforming multiple competent Escherichia coli with multiple different plasmids, wherein the plasmids contain nucleic acids encoding antibiotic resistance genes; A step of bringing transformed competent Escherichia coli into contact with a first culture system to achieve a minimum number of colonies for each of different plasmids, wherein the first culture system contains an antibiotic corresponding to the antibiotic resistance gene; Steps to remove colonies from the first culture system; A step of growing the isolated colonies in a second culture system in accordance with cGMP guidelines, wherein the second culture system contains an antibiotic corresponding to the antibiotic resistance gene, and the antibiotic is present at a concentration of at least twice the standard concentration of the antibiotic; and Steps to extract plasmids from the second culture system according to cGMP guidelines and prepare a cGMP plasmid library. Methods that include...

2. The method according to claim 1, wherein the minimum number of colonies is approximately 100 colonies for each of the different plasmids.

3. The method according to claim 1, wherein the minimum number of colonies is approximately 1,000 colonies for each of the different plasmids.

4. The method according to any one of claims 1 to 3, wherein the plasmid library comprises plasmids encoding RNA or proteins.

5. The method according to any one of claims 1 to 4, wherein the plasmid library comprises plasmids encoding therapeutic RNA or therapeutic proteins.

6. The method according to any one of claims 1 to 5, wherein the plasmid library comprises at least about five different plasmids.

7. The method according to any one of claims 1 to 5, wherein the plasmid library comprises at least about 50 different plasmids.

8. The method according to any one of claims 1 to 5, wherein the plasmid library comprises at least about 100 different plasmids.

9. The method according to any one of claims 1 to 5, wherein the plasmid library comprises at least about 250 different plasmids.

10. The method according to any one of claims 1 to 5, wherein the plasmid library comprises at least about 500 different plasmids.

11. The method according to any one of claims 1 to 5, wherein the plasmid library comprises at least about 1,000 different plasmids.

12. The method according to any one of claims 1 to 11, wherein the competent Escherichia coli includes electrocompetent Escherichia coli.

13. The method according to any one of claims 1 to 11, wherein the competent Escherichia coli includes chemically competent Escherichia coli.

14. The method according to any one of claims 1 to 13, wherein the antibiotic is one of ampicillin, carbenicillin, kanamycin, specinomycin, streptomycin, bleomycin, erythromycin, chloramphenicol, tetracycline, zeosin, D-cycloserine, gentamicin, G418, nalidixic acid, rifampicin, or trimethoprim.

15. The method according to any one of claims 1 to 13, wherein the antibiotic is a beta-lactam antibiotic.

16. The method according to claim 15, wherein the beta-lactam antibiotic is a penicillin antibiotic.

17. The method according to claim 16, wherein the penicillin antibiotic is a carboxypenicillin antibiotic.

18. The method according to claim 17, wherein the carboxypenicillin antibiotic is carbenicillin.

19. The method according to claim 18, wherein the carbenicillin concentration is at least about 150 μg / mL.

20. The method according to claim 19, wherein the carbenicillin concentration is at least about 200 μg / mL.

21. The antibiotics and their corresponding concentrations are: ampicillin: at least about 200 μg / mL; carbenicillin: at least about 200 μg / mL; kanamycin: at least about 200 μg / mL; spectinomycin: at least about 100 μg / mL; streptomycin: at least about 200 μg / mL; bleomycin: at least about 200 μg / mL; erythromycin: at least about 200 μg / mL; chloramphenicol: at least about 50 μg / mL; The method according to any one of claims 1 to 14, wherein the dosage is: tetracycline: at least about 20 μg / mL; zeosin: at least about 200 μg / mL; D-cycloserine: at least about 400 μg / mL; gentamicin: at least about 30 μg / mL; G418: at least about 800 μg / mL; nalidixic acid: at least about 60 μg / mL; rifampicin: at least about 100 μg / mL; or trimethoprim: at least about 400 μg / mL.

22. In a method for preparing a cGMP-compliant plasmid library containing multiple different plasmids, A step of transforming multiple electrocompetent Escherichia coli with multiple different plasmids, wherein the plasmids contain nucleic acids encoding a carbenicillin resistance gene; A step of bringing transformed competent Escherichia coli into contact with a first culture system to achieve a minimum number of colonies for each of different plasmids, wherein the first culture system contains carbenicillin; Steps to remove colonies from the first culture system; A step of growing the isolated colonies in a second culture system in accordance with cGMP guidelines, wherein the second culture system contains at least about 200 μg / mL of carbenicillin; and Steps to extract plasmids from the second culture system according to cGMP guidelines and prepare a cGMP plasmid library. Methods that include...

23. In a method for preparing a cGMP-compliant plasmid library containing multiple different plasmids, A step of transforming multiple electrocompetent Escherichia coli with multiple different plasmids, wherein the plasmids contain nucleic acids encoding a carbenicillin resistance gene; A step of bringing transformed electrocompetent Escherichia coli into contact with a first culture system to achieve a minimum number of colonies for each of different plasmids, wherein the first culture system contains carbenicillin; Steps to remove colonies from the first culture system; A step of growing the isolated colonies in a second culture system in accordance with cGMP guidelines, wherein the second culture system contains approximately 200-400 μg / mL of carbenicillin; and Steps to extract plasmids from the second culture system according to cGMP guidelines and prepare a cGMP plasmid library. Methods that include...

24. In a method for preparing a cGMP-compliant plasmid library containing multiple different plasmids, A step of transforming multiple electrocompetent Escherichia coli with multiple different plasmids, wherein the plasmids contain nucleic acids encoding a carbenicillin resistance gene; A step of bringing transformed electrocompetent Escherichia coli into contact with a first culture system to achieve a minimum number of colonies for each of different plasmids, wherein the first culture system contains carbenicillin; Steps to remove colonies from the first culture system; A step of growing the isolated colonies in a second culture system in accordance with cGMP guidelines, wherein the second culture system contains approximately 200 μg / mL of carbenicillin; and Steps to extract plasmids from the second culture system according to cGMP guidelines and prepare a cGMP plasmid library. Methods that include...

25. In a method for preparing a cGMP-compliant plasmid library containing multiple different plasmids, A step of transforming multiple electrocompetent Escherichia coli with multiple different plasmids, wherein the plasmids contain nucleic acids encoding a carbenicillin resistance gene; A step of bringing transformed competent Escherichia coli into contact with an agar plate to achieve at least about 1000 colonies for each of the different plasmids, wherein the agar plate contains carbenicillin; Steps to remove colonies from agar plates; A step of growing the isolated colonies in a liquid culture system in accordance with cGMP guidelines, wherein the liquid culture system contains approximately 200 μg / mL of carbenicillin; and Steps to extract plasmids from liquid culture systems according to cGMP guidelines and prepare a cGMP plasmid library. Methods that include...

26. The method according to any one of claims 22 to 24, wherein the minimum number of colonies is about 100 colonies for each of the different plasmids.

27. The method according to any one of claims 22 to 24, wherein the minimum number of colonies is approximately 1,000 colonies for each of the different plasmids.

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