Patent Application
20230112702
The present disclosure relates to methods and compositions for modulation of gene expression using nuclease-mediated systems, as well as automated multi-module instruments for performing these methods and using these compositions.
In the following discussion certain articles and methods will be described for background and introductory purposes. Nothing contained herein is to be construed as an “admission” of prior art. Applicant expressly reserves the right to demonstrate, where appropriate, that the articles and methods referenced herein do not constitute prior art under the applicable statutory provisions.
A variety of organisms, such as bacteria, fungi, and plants, have become an increasingly important platform for the industrial production of bioproducts, including biopharmaceuticals, biochemicals, amino acids, and biofuel precursors. To maximize biosynthesis of such products, expression of multiple metabolic pathway genes in these organisms must typically be regulated. Accordingly, multiplexed control of multiple metabolic pathway genes has become a central focus for current synthetic biology and genome engineering efforts. Yet, despite recent advances in these fields, current multiplexed methods are still time-consuming and inefficient.
There is therefore a need in the art for improved methods, compositions, modules, and instruments for increasing the efficiency of gene expression modulation, and in particular, multiplexed gene expression modulation. The present disclosure addresses this need.
This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will be apparent from the following written Detailed Description including those aspects illustrated in the accompanying drawings and defined in the appended claims.
The present disclosure relates to methods, compositions, and automated multi-module cell processing instruments for modulation of gene expression (i.e., gene regulation) utilizing nuclease-mediated systems, and in particular, inactive (“dead”) nuclease-mediated CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) systems. With the present compositions and methods, efficiency of gene expression modulation is improved utilizing an inactive nuclease lacking catalytic activity but retaining strong specific DNA binding function, thus enabling robust, programmable, sequence-specific gene repression (e.g., CRISPRi), or activation (e.g., CRISPRa), in diverse microbial species with minimal toxicity.
In some aspects, the inactive nuclease may be introduced into cells using a DNA molecule coding for the inactive nuclease separately or covalently-linked to an expression cassette comprising one or more gRNAs, or the inactive nuclease may be introduced into cells using a DNA molecule coding for the inactive nuclease separately or covalently-linked to an engine vector, or the inactive nuclease may be introduced separately in polypeptide/protein form or as part of a complex, or the inactive nuclease may be introduced into cells using an mRNA coding for the inactive nuclease. In addition to the inactive nuclease, the expression cassette comprising one or more gRNAs is utilized. The one or more gRNAs may target one or more target regions (i.e., target loci) in a cell genome corresponding to coding regions of genes to be regulated. In specific aspects, the genes include endogenous or heterologous metabolic pathway genes.
In some aspects, the inactive nuclease includes dMAD7 or a variant thereof, such as dMAD7 D877A, dMAD7 E962A, dMAD7 D1213A, and/or combinations thereof.
In some aspects, the inactive nuclease includes one or more of dMAD2007, dMAD2017, dMAD2019, dMAD297, dMAD298, dMAD299, other inactive MADzymes®, variants (e.g., orthologues) thereof, and/or combinations hereof.
In some aspects, a region of complementarity between one of the one or more gRNAs and the target locus is between 4-120 nucleotides in length, or between 5-80 nucleotides in length, or between 6-60 nucleotides in length. The one or more gRNAs may be designed to bind with the template or non-template strand of double stranded DNA.
In some aspects, there is provided a gRNA-containing expression cassette for performing nuclease-mediated modulation of gene expression, the expression cassette comprising from 5′ to 3′ an optional melting temperature booster, which is a short protective DNA sequence that increases the “landing pad” for the forward PCR primer used to amplify the cassette, a repeat region of the gRNA, a spacer region of the gRNA, an optional barcode, and a subpool primer binding sequence. In addition, in specific aspects, the expression cassettes comprise regions of homology to a vector for gap-repair insert of the expression cassettes into the vector.
In some aspects, there is provided a multiplex gRNA-containing expression cassette for performing nuclease-mediated modulation of gene expression, the multiplex expression cassette comprising two or more gRNAs, wherein each of the two or more gRNAs are separated from other gRNAs by a linker or spacer. In some aspects, there is provided a multi-pack gRNA-containing expression cassette for performing nuclease-mediated modulation of gene expression, the multi-pack expression cassette comprising two or more single-pack expression cassettes, wherein each of the two or more single-pack expression cassettes are separated from other single-pack expression cassettes by a linker or spacer.
In some aspects, there is provided an expression vector for performing nuclease-mediated modulation of gene expression, the expression vector comprising one or more expression cassettes. In some aspects, the expression vector further includes a selectable marker component, e.g., an antibiotic resistance gene or a fluorescent protein gene. In some aspects, the expression vector includes an inactive nuclease. In some aspects, the expression vector further includes one or more promoters positioned to drive transcription of the gRNAs in the one or more expression cassettes, the selectable marker component, and/or the nuclease. The one or more promoters may be constitutive or inducible.
In some aspects, there is provided an engine vector or combined engine/expression vector for performing nuclease-mediated modulation of gene expression. In some aspects, the vector is an engine vector comprising an inactive nuclease, e.g., a dMAD nuclease, an optional selectable marker, e.g., an antibiotic resistance gene, and an optional barcode. In some aspects, the vector further comprises a promoter positioned to drive transcription of the nuclease and/or the assembled expression cassette.
In some aspects, there is provided a library of vector backbones and a library of expression cassettes to be transformed into cells, the library of expression cassettes including a first expression cassette comprising a gRNA and an optional barcode. In some aspects, the library of vector backbones includes a first vector backbone comprising an inactive nuclease, an optional selectable marker, and an optional barcode. In some aspects, the utilization of a library of expression cassettes, and in certain cases, a library of vector backbones, enables combinatorial or multiplex modulation of expression of a plurality of genes in the cells. In specific aspects, the expression of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more genes is modulated utilizing the methods describe herein.
In some aspects, one or more expression cassettes in the library of expression cassettes each comprise a different gRNA targeting a different target region within the cell genomes. In some aspects, one or more expression cassettes in the library of expression cassettes each comprise a plurality of gRNAs, wherein at least one of the plurality of gRNAs is different from the plurality of gRNAs of another expression cassette.
In some aspects, the binding of the inactive nuclease at the target locus attenuates or prevents transcription of the target locus. In specific aspects, binding of the inactive nuclease at the target locus blocks initiation of transcription. In other aspects, binding of the inactive nuclease at the target locus blocks transcription elongation.
In some aspects, a repressor, e.g., a repressor domain, is fused to the inactive nuclease to increase a degree of transcription repression. In specific aspects, the repressor includes a Kruppel-associated box (KRAB) domain. In specific aspects, the repressor includes MAX interactor 1 (MXI1) or MAX dimerization protein 1 (MXD1), or Sin3 interacting domain (SID). In specific aspects, the repressor includes GAL80 or LexA.
In some aspects, an activator, e.g., an activator domain, is fused to the inactive nuclease to facilitate gene activation, or CRISPRa. In specific aspects, the activator includes VP64-p65-Rta (VPR), synergistic activation mediator (SAM), or SunTag activators. In specific aspects, the activator includes superoxide response transcriptional regulator SoxS, multiple antibiotic resistance activator (MarA), DNA-binding transcriptional activator Rob, or catabolite activator protein (CAP). In specific aspects, the activator includes a bacteriophage or transposon effector, such as TetD, lamda transcription activator II (λCII), RNA polymerase-associated protein GP33, N4-coded single-stranded DNA-binding protein (N4SSB), or anti-sigma factor AsiA. In specific aspects, the activator includes an RNA polymerase subunit, such as RpoZ (ω), RpoD (σ70), or the N-terminal domain of RpoA (aNTD).
In some aspects, the gene expression modulation systems described herein are combined with an inducible promoter systems, thus enabling tunable repression to alter expression levels of essential genes.
The present disclosure includes methods of using nuclease-mediated CRISPRi/a in cell populations, e.g., bacterial and fungal cells, for modulation of expression of one or more genes thereof. The cells that can be used with the methods of the present disclosure include any prokaryotic, archaeal, or eukaryotic cells. For example, prokaryotic cells for use with the present illustrative embodiments can include gram-positive bacterial cells, e.g., Bacillus subtilis, or gram-negative bacterial cells, e.g., E. coli cells. Eukaryotic cells for use with the automated multi-module cell processing instruments of the illustrative embodiments include any plant cells and any animal cells, e.g., fungal cells (including yeast), insect cells, amphibian cells, and the like. In specific aspects, methods of the present disclosure are used with common research microbial species such as E. coli or S. cerevisiae. Other model organisms that can be used with the methods and compositions of the present disclosure include Streptomyces spp., Pseudomonas spp., Corynebactierum spp., Bacillus spp., Aspergillus spp., Vibrio spp., Yarrowia lypolytica, and Pichia pastoris.
In some aspects, the present disclosure provides methods of modulating the expression of one or more metabolic pathway genes in cells to modulate the biosynthesis of one or more desired bioproducts. In specific aspects, the modulation of the expression of the one or more metabolic pathway genes increases or optimizes the biosynthesis of the one or more desired bioproducts by the cells. In such aspects, the cells used with the methods of the present disclosure include microbial cells, which may serve as mini “factories” to produce a desired bioproduct. Accordingly, the present disclosure provides efficient methods for metabolic engineering.
In some aspects, the present disclosure provides methods of regulating mechanisms of transcription. Such transcriptional regulation methods may be utilized to assess the relationship between an epigenetic network and cellular function, and enable a cost-effective and efficient platform for such studies as compared to zinc fingers (ZFs) and transcription activator-like effectors (TALEs), which require extensive protein engineering for DNA targeting. In some aspects, the methods described herein may be utilized for epigenetic diagnosis and related studies.
In some aspects, the microbes used with the methods of the present disclosure include bacterial cells. In some aspects, the microbes used with the methods of the present disclosure include fungal cells.
In some aspects, the one or more genes regulated by the methods of the present disclosure include genes naturally occurring in the cells. In some aspects, the one or more genes regulated by the methods of the present disclosure include genes heterologously introduced from another organism or species.
In some aspects, automated methods are used for nuclease-mediated modulation of expression of one or more genes in multiple cells for modification of the ability of these cells to produce a bioproduct, the methods being performed in automated multi-module cell processing instruments. The automated methods carried out using the automated multi-module cell processing instruments described herein can be used with a variety of nuclease-mediated gene modulation techniques, as well as genome editing techniques, and can be used with or without use of one or more selectable markers.
The present disclosure thus provides, in selected embodiments, modules, instruments, and systems for automated multi-module cell processing for nuclease-mediated modulation of expression of one or more genes in multiple cells. Automated systems for cell processing that may be used for can be found, e.g., in U.S. Pat. Nos. 10,253,316; 10,329,559; 10,323,242; 10,421,959; 10,465,185; 10,519,437; 10,584,333; 10,584,334; 10,647,982; 10,689,645; 10,738,301; and 10,738,663.
In some aspects, the automated multi-module cell processing instruments of the present disclosure are further designed for nucleic acid-guided genome editing, such as recursive genome editing, e.g., sequentially introducing multiple edits into genomes inside one or more cells of a cell population through two or more editing operations within the instruments.
In certain embodiments, a method for controlling expression of a target nucleic acid in a cell is provided, the method comprising: introducing into the cell: (a) a guide RNA (gRNA) comprising a first nucleotide sequence that is at least partially complementary to a coding sequence of the target nucleic acid and a second nucleotide sequence configured to interact with an inactive dMAD7 nuclease; and (b) an inactive dMAD7 nuclease polypeptide and/or a nucleotide sequence encoding an inactive MAD7® nuclease polypeptide, wherein the gRNA guides the inactive dMAD7 nuclease polypeptide to the target nucleic acid, and wherein binding of the inactive dMAD7 nuclease polypeptide to the target nucleic acid attenuates or prevents transcription of the target nucleic acid.
In certain embodiments, a method for simultaneously controlling expression of a plurality of target nucleic acids in a cell is provided, the method comprising: introducing into the cell: (a) a plurality of guide RNAs (gRNAs), each gRNA comprising a first nucleotide sequence that is at least partially complementary to a coding sequence of a corresponding target nucleic acid of the plurality of target nucleic acids and a second nucleotide sequence configured to interact with an inactive dMAD7 nuclease; and (b) one or more inactive dMAD7 nuclease polypeptides and/or nucleotide sequences each encoding an inactive MAD7 x nuclease polypeptide, wherein each gRNA of the plurality of gRNAs guides one of the one or more inactive dMAD7 nuclease polypeptides to the corresponding target nucleic acid of the plurality of target nucleic acids, and wherein binding of each inactive dMAD7 nuclease polypeptide to the corresponding target nucleic acid of the plurality of target nucleic acids attenuates or prevents transcription of the corresponding one of the plurality of target nucleic acids.
In certain embodiments, a system for controlling expression of a target nucleic acid in a cell is provided, the system comprising: a guide RNA (gRNA) comprising a first nucleotide sequence that is at least partially complementary to a coding sequence of the target nucleic acid and a second nucleotide sequence configured to interact with an inactive dMAD7 nuclease; and an inactive dMAD7 nuclease polypeptide and/or a nucleotide sequence encoding an inactive MAD7® nuclease polypeptide, wherein the gRNA is configured to guide the inactive dMAD7 nuclease polypeptide to the target nucleic acid, and wherein binding of the inactive dMAD7 nuclease polypeptide to the target nucleic acid attenuates or prevents transcription of the target nucleic acid.
In certain embodiments, a system for simultaneously controlling expression of a plurality of target nucleic acids in a cell is provided, the system comprising: a plurality of guide RNAs (gRNAs), each gRNA comprising a first nucleotide sequence that is at least partially complementary to a coding sequence of a corresponding target nucleic acid of the plurality of target nucleic acids and a second nucleotide sequence configured to interact with an inactive dMAD7 nuclease; and one or more inactive dMAD7 nuclease polypeptides and/or nucleotide sequences each encoding an inactive MAD7® nuclease polypeptide, wherein each gRNA of the plurality of gRNAs is configured to guide one of the one or more inactive dMAD7 nuclease polypeptides to the corresponding target nucleic acid of the plurality of target nucleic acids, and wherein binding of each inactive dMAD7 nuclease polypeptide to the corresponding target nucleic acid of the plurality of target nucleic acids attenuates or prevents transcription of the corresponding one of the plurality of target nucleic acids.
These aspects and other features and advantages of the invention are described below in more detail.
All of the functionalities described in connection with one embodiment of the methods, devices or instruments described herein are intended to be applicable to the additional embodiments of the methods, devices and instruments described herein except where expressly stated or where the feature or function is incompatible with the additional embodiments. For example, where a given feature or function is expressly described in connection with one embodiment but not expressly mentioned in connection with an alternative embodiment, it should be understood that the feature or function may be deployed, utilized, or implemented in connection with the alternative embodiment unless the feature or function is incompatible with the alternative embodiment.
The practice of the techniques described herein may employ, unless otherwise indicated, conventional techniques and descriptions of molecular biology (including recombinant techniques), cell biology, biochemistry, and genetic engineering technology, which are within the skill of those who practice in the art. Such conventional techniques and descriptions can be found in standard laboratory manuals such as Green and Sambrook, Molecular Cloning: A Laboratory Manual. 4th, ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (2014); Current Protocols in Molecular Biology, Ausubel, et al. eds., (2017); Neumann, et al., Electroporation and Electrofusion in Cell Biology, Plenum Press, New York, 1989; and Chang, et al., Guide to Electroporation and Electrofusion, Academic Press, Calif. (1992), all of which are herein incorporated in their entirety by reference for all purposes. Nucleic acid-guided nuclease techniques can be found in, e.g., Genome Editing and Engineering from TALENs and CRISPRs to Molecular Surgery, Appasani and Church (2018); and CRISPR: Methods and Protocols, Lindgren and Charpentier (2015); both of which are herein incorporated in their entirety by reference for all purposes.
Note that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” refers to one or more cells, and reference to “the system” includes reference to equivalent steps, methods and devices known to those skilled in the art, and so forth. Additionally, it is to be understood that terms such as “left,” “right,” “top,” “bottom,” “front,” “rear,” “side,” “height,” “length,” “width,” “upper,” “lower,” “interior,” “exterior,” “inner,” “outer” that may be used herein merely describe points of reference and do not necessarily limit embodiments of the present disclosure to any particular orientation or configuration. Furthermore, terms such as “first,” “second,” “third,” etc., merely identify one of a number of portions, components, steps, operations, functions, and/or points of reference as disclosed herein, and likewise do not necessarily limit embodiments of the present disclosure to any particular configuration or orientation.
Unless defined otherwise, 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 invention belongs. All publications mentioned herein are incorporated by reference for all purposes, including but not limited to describing and disclosing devices, formulations and methodologies that may be used in connection with the presently described invention.
Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in 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 included in the invention.
In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention. The terms used herein are intended to have the plain and ordinary meaning as understood by those of ordinary skill in the art.
The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs that function similarly to naturally occurring amino acids.
The terms “cassette” and “expression cassette” refer to a nucleic acid molecule comprising a coding sequence for transcription of a guide nucleic acid or gRNA.
The term “complementary” as used herein refers to Watson-Crick base pairing between nucleotides and specifically refers to nucleotides hydrogen-bonded to one another with thymine or uracil residues linked to adenine residues by two hydrogen bonds and cytosine and guanine residues linked by three hydrogen bonds. In general, a nucleic acid includes a nucleotide sequence described as having a “percent complementarity” or “percent homology” to a specified second nucleotide sequence. For example, a nucleotide sequence may have 80%, 90%, or 100% complementarity to a specified second nucleotide sequence, indicating that 8 of 10, 9 of 10 or 10 of 10 nucleotides of a sequence are complementary to the specified second nucleotide sequence. For instance, the nucleotide sequence 3′-TCGA-5′ is 100% complementary to the nucleotide sequence 5′-AGCT-3′; and the nucleotide sequence 3′-TCGA-5′ is 100% complementary to a region of the nucleotide sequence 5′-TTAGCTGG-3′.
The term DNA “control sequences” refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites, nuclear localization sequences, enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these types of control sequences need to be present so long as a selected coding sequence is capable of being replicated, transcribed and—for some components—translated in an appropriate host cell.
The term “gene” refers to a segment of DNA involved in producing a polypeptide chain. It may include regions preceding and following a coding region (leader and trailer, respectively), as well as intervening sequences (introns) between individual coding segments (exons).
The terms “guide nucleic acid” or “guide RNA” or “gRNA” refer to a polynucleotide comprising 1) a guide sequence capable of hybridizing to a genomic target locus, and 2) a scaffold sequence capable of interacting or complexing with a nucleic acid-guided nuclease.
The term “heterologous” refers to the relationship between two or more nucleic acids or protein sequences from different sources, or the relationship between a protein (or nucleic acid) and a host cell from different sources. For example, if the combination of a nucleic acid and a host cell is usually not naturally occurring, the nucleic acid is heterologous to the host cell. A particular sequence is “heterologous” to the cell or organism into which it is inserted.
“Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or, more often in the context of the present disclosure, between two nucleic acid molecules. The term “homologous region” or “homology arm” refers to a region on a donor DNA with a certain degree of homology with a target genomic DNA sequence. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.
The terms “intermediate compound” and “intermediates” refer to a product of a synthesis pathway that is not the terminal product, but which is useful for the production of the final intended product. The term “naturally occurring” when used in reference to a bioproduct refers to a chemical compound or substance produced by a living organism. In the broadest sense, bioproducts include any substance produced by life, including substrates, enzymes, cofactors, and terminal products (e.g. final intended pesticides) and pathway intermediates of terminal products. The term also encompasses complex extracts and isolated compounds derived from those extracts. In the broadest sense, a chemical or product that is “naturally occurring” includes any substance or combination of substances produced by life. In addition, the term is intended to encompass a substance that forms the structural basis for commercial bioproducts, such as an intermediary product.
As used herein, the terms gene expression “modulation,” gene expression “alteration,” “gene regulation,” and “cell modification” are used interchangeably and refer to mechanisms that act to induce (“turn on”) or repress (“turn off”) expression of one or more genes. In certain examples, modulation of gene expression may be accomplished via “transcriptional regulation,” or the regulation of the conversion of DNA to RNA (transcription).
The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless otherwise indicated, the terms encompass nucleic acids containing known analogues or natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, in addition to the sequence specifically stated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologues, SNPs, and complementary sequences. The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
“Operably linked” refers to an arrangement of elements where the components so described are configured so as to perform their usual function. Thus, control sequences operably linked to a coding sequence are capable of effecting the transcription, and in some cases, the translation, of a coding sequence. The control sequences need not be contiguous with the coding sequence so long as they function to direct the expression of the coding sequence. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence. In fact, such sequences need not reside on the same contiguous DNA molecule (i.e. chromosome) and may still have interactions resulting in altered regulation.
A “promoter” or “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a polynucleotide or polypeptide coding sequence such as messenger RNA, ribosomal RNA, small nuclear or nucleolar RNA, guide RNA, or any kind of RNA transcribed by any class of any RNA polymerase I, II or III. Promoters may be constitutive or inducible.
As used herein, the terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein and refer to a polymer of amino acid residues. Proteins may or may not be made up entirely of amino acids.
As used herein the term “selectable marker” refers to a gene introduced into a cell, which confers a trait suitable for artificial selection. General use selectable markers are well-known to those of ordinary skill in the art. For example, selectable markers can use means that deplete a cell population to enrich for editing or gene regulation, and include ampicillin/carbenicillin, kanamycin, chloramphenicol, nourseothricin N-acetyl transferase, erythromycin, tetracycline, gentamicin, bleomycin, streptomycin, puromycin, hygromycin, blasticidin, and G418 or other selectable markers may be employed. In addition, selectable markers include physical markers that confer a phenotype that can be utilized for physical or computations cell enrichment, e.g., optical selectable markers such as fluorescent proteins (e.g., green fluorescent protein, blue fluorescent protein) and cell surface handles.
The term “specifically binds” as used herein includes an interaction between two molecules, e.g., an engineered peptide antigen and a binding target, with a binding affinity represented by a dissociation constant of about 10−7 M, about 10−8 M, about 10−9 M, about 10−10 M, about 10−11 M, about 10−12 M about 10−13 M, about 10−14 M or about 10−15 M
The terms “target genomic DNA sequence”, “cellular target sequence”, “target sequence”, or “genomic target locus,” for purposes of the present disclosure, refer to any locus in vitro or in vivo, or in a nucleic acid (e.g., genome or episome) of a cell or population of cells, having a gene or gene element of which a change in expression is desired. The target sequence can be a genomic locus or extrachromosomal locus.
The term “variant” may refer to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions). A variant of a polypeptide may be a conservatively modified variant. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code (e.g., a non-natural amino acid). A variant of a polypeptide may be naturally occurring, such as an allelic variant, or it may be a variant that is not known to occur naturally.
A “vector” is any of a variety of nucleic acids that comprise a desired sequence or sequences to be delivered to and/or expressed in a cell. Vectors are typically composed of DNA, although RNA vectors are also available. Vectors include, but are not limited to, plasmids, fosmids, phagemids, virus genomes, synthetic chromosomes, and the like. In the present disclosure, the term “combined engine/expression vector” may include a coding sequence for a nuclease and an expression cassette comprising a gRNA sequence to be transcribed. In other embodiments, however, two vectors— a recombinant engine vector comprising the coding sequence for a nuclease, and an expression cassette, comprising the gRNA sequence to be transcribed— may be used.
The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings in which:
This disclosure is directed to the modulation, i.e., regulation, of gene expression in cells to increase or decrease the production of specific gene products (e.g., proteins and RNA). In particular embodiments, this disclosure provides compositions and methods, including automated methods, for performing clustered regularly interspaced short palindromic repeats interference (CRISPRi) and activation (CRISPRa) utilizing, e.g., an inactive dMAD7 nuclease variant. The inactive dMAD7 lacks catalytic activity but retains strong specific DNA binding function, thus enabling robust, programmable, and sequence-specific gene expression modulation in diverse species with minimal toxicity. Further, when utilized in combination with expression cassettes engineered with barcode sequences and one or more target-specific gRNAs, the dMAD7 systems described herein facilitate trackable gene modulation on a genome-wide scale, providing an alternative genome engineering approach that reveals epigenetic genotype-phenotype relationships.
In certain aspects, the compositions and methods described herein may be utilized for metabolic engineering. For example, to bypass the toxicity associated with double-stranded breaks (DSBs) during CRISPR editing, CRISPRi/a with dMAD7 may be utilized to re-direct metabolic flux toward a target biosynthesis pathway for production of, e.g., diverse chemicals and materials, including production of biofuel precursors (bisabolene, butanol, and isopentenol) and food pigment (anthocyanin an carotene) in, e.g., E. coli and S. cerevisiae, amino acids (L-lysine and L-glutamate) in, e.g., C. glutamicum, and increased carbon storage in cyanobacteria.
In certain aspects, the compositions and methods described herein may be utilized for epigenetic studies. For example, transcriptional regulation methods described herein may be utilized to assess the relationship between an epigenetic network and cellular function, and enable a cost-effective and efficient platform for such studies as compared to zinc fingers (ZFs) and transcription activator-like effectors (TALEs), which require extensive protein engineering for DNA targeting. In some aspects, the methods described herein may be utilized for epigenetic diagnosis and related studies.
The compositions and methods described herein are employed to perform nuclease-mediated modulation of gene expression to induce or repress desired genes in a population of microbial cells. In some embodiments, the expression of one or more genes, e.g., metabolic pathway genes, is modulated simultaneously, i.e., in a single round of processing or “epigenetic genome engineering”.
Methods described herein utilize modified versions of nucleic acid-guided nucleases that lack nuclease activity (e.g., “inactive” or “dead” nucleases) but retain specific binding capacity. Such inactive nucleases may be synthesized by introducing point mutations in the nuclease domains thereof, thereby “deactivating” the nucleases while leaving their specific binding capacities unaffected.
A synthetic nucleic acid-guided nuclease lacking cleavage activity, when complexed with an appropriate synthetic guide nucleic acid in a cell, targets and binds to a specific location of the cell genome without cleaving the DNA. The guide nucleic acid helps the inactive nuclease recognize the DNA at a specific target sequence (e.g., a sequence within a target gene-coding sequence). By manipulating the nucleotide sequence of the guide nucleic acid, the nucleic acid-guided inactive nuclease may be programmed to target any DNA sequence for binding as long as an appropriate protospacer adjacent motif (PAM) is nearby. In certain aspects, the nuclease-mediated system may use two separate guide nucleic acid molecules that combine to function as a guide nucleic acid, e.g., a CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA). In other aspects and preferably, the guide nucleic acid is a single guide nucleic acid construct that includes both 1) a guide sequence capable of hybridizing to a genomic target locus, and 2) a scaffold sequence capable of interacting or complexing with an inactive nuclease.
In general, the guide nucleic acid (e.g., gRNA) complexes with a compatible inactive nuclease and can then hybridize with a target sequence, thereby directing the inactive nuclease to the target sequence. A guide nucleic acid can be DNA or RNA; alternatively, a guide nucleic acid may comprise both DNA and RNA. In some embodiments, a guide nucleic acid may comprise modified or non-naturally occurring nucleotides. In cases where the guide nucleic acid comprises RNA, the gRNA may be encoded by a DNA sequence on a polynucleotide molecule such as a plasmid, linear construct, or the coding sequence may and preferably does reside within an expression cassette. For additional information regarding cassettes in the context of gene editing, see U.S. Pat. Nos. 9,982,278; 10,266,849; and 10,240,167, and U.S. Pat. Nos. 15/948,785; 16/056,310; 16,275,439; and 16/275,465, all of which are incorporated by reference herein.
A guide nucleic acid comprises a guide sequence, where the guide sequence is a polynucleotide sequence having sufficient complementarity with a target sequence to hybridize with the target sequence and direct sequence-specific binding of a complexed inactive nuclease to the target sequence. The degree of complementarity between a guide sequence and the corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences. In some embodiments, a guide sequence is about or more than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20 nucleotides in length. Preferably the guide sequence is 10-30 or 15-20 nucleotides long, or 15, 16, 17, 18, 19, or 20 nucleotides in length.
In general, to modulate the expression of a target gene, the gRNA/inactive nuclease complex binds to a target sequence within a sequence encoding the target gene, as determined by the guide RNA. During binding, the inactive nuclease recognizes a protospacer adjacent motif (PAM) sequence adjacent to the target sequence, which allows the inactive nuclease to bind. In certain examples, the binding of the inactive nuclease attenuates or blocks transcription of the target gene by preventing transcription factors and RNA polymerase from accessing the gene, thereby resulting in decreased expression of target gene (repression). In such examples, a repressor may be attached to the inactive nuclease to further enhance transcriptional repression. In certain other examples, however, an activator may be attached to the inactive nuclease for positive control of transcription, thereby resulting in increased expression of the target genet (activation).
The guide nucleic acid may be, and preferably is, part of an expression cassette that may also encode, e.g., a barcode, described in further detail below. The expression cassette can be inserted or assembled into a vector backbone, which may already have a sequence coding for the inactive nuclease assembled or inserted therein. Alternatively, the inactive nuclease may be inserted into the vector backbone after insertion of the expression cassette. In other cases, the inactive nuclease is part of the expression cassette, and may be simultaneously inserted or assembled via gap repair into a linear plasmid or vector backbone to create a cassette expression vector. In still other cases, the guide nucleic acid may be part of an expression cassette and the inactive nuclease may be part of an engine vector which are transformed simultaneously into the desired cell.
The target sequence is associated with a proto-spacer mutation (PAM), which is a short nucleotide sequence recognized by the gRNA/inactive nuclease complex. The precise preferred PAM sequence and length requirements for different nucleic acid-guided nucleases vary; however, PAMs typically are 2-7 base-pair sequences adjacent or in proximity to the target sequence and, depending on the inactive nuclease, can be 5′ or 3′ to the target sequence. Engineering of the PAM-interacting domain of an inactive nuclease may allow for alteration of PAM specificity, improve target site recognition fidelity, decrease target site recognition fidelity, or increase the versatility of a nucleic acid-guided inactive nuclease.
Returning now to the nuclease component of the nuclease-mediated gene modulation system, a polynucleotide sequence encoding an inactive nuclease can be codon optimized for expression in particular microbial cell types, such as stem cells. The choice of inactive nuclease to be employed depends on many factors, such as whether an appropriate PAM is located close to the desired target sequence. Inactive nucleases which may be used with the methods described herein include but are not limited to dMAD7, dMAD2, other inactive MADzymes®, variants thereof, such as dMAD7 D877A, dMAD7 E962A, and dMAD7 D1213A, and combinations thereof. In certain cases, methods described herein may also be utilized with dCas9, dCas 12/ddCpfI, etc. As with the guide nucleic acid, the inactive nuclease is encoded by a DNA sequence on a vector or cassette and optionally is under the control of a constitutive or inducible promoter. In some embodiments, the promoter may be separate from but the same as the promoter controlling transcription of the guide nucleic acid; that is, a separate promoter drives the transcription of the inactive nuclease and guide nucleic acid sequences but the two promoters may be the same type of promoter. Alternatively, the promoter controlling expression of the inactive nuclease may be different from the promoter controlling transcription of the guide nucleic acid; that is, e.g., the inactive nuclease may be under the control of, e.g., the pTEF promoter, and the guide nucleic acid may be under the control of the, e.g., pCYC1 promoter.
In addition to the guide nucleic acid, an expression cassette may comprise one or more primer sites. The primer sites can be used to amplify the expression cassette by using oligonucleotide primers, for example, if the primer sites flank one or more of the other components of the expression cassette. As described above, the expression cassette may further comprise a barcode. A barcode is a unique DNA sequence that corresponds to the guide nucleic acid and/or the expression cassette such that the barcode facilitates tracking/identification of gene regulation events in corresponding cells. The barcode typically comprises four or more nucleotides. In some embodiments, the expression cassettes comprise a plurality or library gRNAs representing, e.g., genome-wide libraries of gRNAs for modulating expression of a plurality of genes. The library of expression cassettes is cloned into vector backbones where, e.g., each different guide nucleic acid may be associated with a different barcode. Also, in preferred embodiments, a cassette expression vector or engine vector encoding components of the nucleic acid-guided inactive nuclease system further encodes a nucleic acid-guided inactive nuclease comprising one or more nuclear localization sequences (NLSs), such as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs, particularly as an element of the inactive nuclease sequence. In some embodiments, the engineered inactive nuclease comprises NLSs at or near the amino-terminus, NLSs at or near the carboxy-terminus, or a combination.
In certain embodiments, the cassettes and/or vectors may further comprise one or more selectable markers to enable artificial selection of cells undergoing gene regulation events. For example, in certain embodiments, the cassettes and/or vectors encode for one or more antibiotic resistance genes, such as ampicillin/carbenicillin and chloramphenicol resistance genes, thereby facilitating enrichment for cells undergoing gene modulation events via depletion of the cell population. In other examples, the cassettes and/or vectors may include an integrated GFP gene to enable phenotypic detection of gene modulation events by flow cytometry, fluorescent cell imaging, etc.
A variety of delivery systems may be used to introduce (e.g., transform, transfect, or transduce) the gene expression modulation system components into a host cell 108. These delivery systems include the use of yeast systems, lipofection systems, microinjection systems, biolistic systems, virosomes, liposomes, immunoliposomes, polycations, lipid:nucleic acid conjugates, virions, artificial virions, viral vectors, electroporation, cell permeable peptides, nanoparticles, nanowires, exosomes. Alternatively, molecular trojan horse liposomes may be used to deliver nucleic acid-guided nuclease components across the blood brain barrier. Of particular interest is the use of electroporation, particularly flow-through electroporation (either as a stand-alone instrument or as a module in an automated multi-module system) as described in, e.g., U.S. Pat. No. 10,253,316, issued 9 Apr. 2019; U.S. Pat. No. 10,329,559, issued 25 Jun. 2019; U.S. Pat. No. 10,323,242, issued 18 Jun. 2019; U.S. Pat. No. 10,421,959, issued 24 Sep. 2019; U.S. Pat. No. 10,465,185, issued 5 Nov. 2019; U.S. Pat. No. 10,519,437, issued 31 Dec. 2019; U.S. Pat. No. 10,584,333, issued 10 Mar. 2020; U.S. Pat. No. 10,584,334, issued 10 Mar. 2020; U.S. Pat. No. 10,647,982, issued 12 May 2020; U.S. Pat. No. 10,689,645, issued 23 Jun. 2020; U.S. Pat. No. 10,738,301, issued 11 Aug. 2020; U.S. Pat. No. 10,738,663, issued 29 Sep. 2020; and U.S. Pat. No. 10,894,958, issued 19 Jan. 2021 all of which are herein incorporated by reference in their entirety.
Once transformed 106, the cells can then be subjected to selection using selection medium 108. Selectable markers and selection medium are employed to select for cells that have received the vector backbone. Commonly used selectable markers include drug selectable markers such as ampicillin/carbenicillin, kanamycin, chloramphenicol, erythromycin, tetracycline, gentamicin, bleomycin, streptomycin, puromycin, hygromycin, blasticidin, and GF18.
Once the cells that have been properly transformed are selected 108, conditions for nuclease-mediated modulation of gene expression may optionally be provided 110. “Providing conditions” includes incubation of the cells in appropriate medium and may also include providing conditions to facilitate, or even induce via an inducible promoter, transcription of the gRNA and inactive nuclease. Once the modification is complete, the cells are allowed to recover and may then be utilized in research, for bioproduction systems, or may be subjected to further processing 112, including another round of gene expression modulation, or nucleic-acid guided editing.
Expression cassettes 151 and 152 may be described as “single-pack” expression cassettes, meaning that each of expression cassettes 151 and 152 comprise a single gRNA (e.g., both CR and SR sequences), as well as associated T1, BC, and P2 sequences. In certain embodiments, a plurality of single-pack expression cassettes (hereinafter, “single-pack cassettes”) may be “stitched” or assembled together to form a “multi-pack” cassette for simultaneous, multi-loci modulation of gene expression. Further, although not shown in
As shown, dMAD7 associates with the gRNA to form a recognition complex that specifically binds to the target locus complementary to the gRNA via Watson-Crick base pairing. In certain embodiments, the gRNA is designed to bind to the template DNA strand. In other embodiments, the gRNA is designed to bind to the non-template DNA strand. By binding to the target locus, the dMAD7-gRNA complex sterically prevents the association of the promoter or transcription factors with their trans-acting sequences, or blocks transcription elongation. Accordingly, the gRNA may be designed to bind to the coding region of the target gene to block transcription elongation, as shown in the top portion of
In some implementations, the reagent cartridges 210 are disposable kits comprising reagents and cells for use in the automated multi-module cell processing instrument 200. For example, a user may open and position each of the reagent cartridges 210 comprising various desired inserts and reagents within the chassis of the automated multi-module cell processing instrument 200 prior to activating cell processing. Further, each of the reagent cartridges 210 may be inserted into receptacles in the chassis having different temperature zones appropriate for the reagents contained therein.
Also illustrated in
Inserts or components of the reagent cartridges 210, in some implementations, are marked with machine-readable indicia (not shown), such as bar codes, for recognition by the robotic handling system 258. For example, the robotic liquid handling system 258 may scan one or more inserts within each of the reagent cartridges 210 to confirm contents. In other implementations, machine-readable indicia may be marked upon each reagent cartridge 210, and a processing system (not shown, but see element 237 of
Inside the chassis 290, in some implementations, will be most or all of the components described in relation to
The drive engagement mechanism 312 engages with a motor (not shown) to rotate the vial. In some embodiments, the motor drives the drive engagement mechanism 312 such that the rotating growth vial 300 is rotated in one direction only, and in other embodiments, the rotating growth vial 300 is rotated in a first direction for a first amount of time or periodicity, rotated in a second direction (i.e., the opposite direction) for a second amount of time or periodicity, and this process may be repeated so that the rotating growth vial 300 (and the cell culture contents) are subjected to an oscillating motion. Further, the choice of whether the culture is subjected to oscillation and the periodicity therefor may be selected by the user. The first amount of time and the second amount of time may be the same or may be different. The amount of time may be 1, 2, 3, 4, 5, or more seconds, or may be 1, 2, 3, 4 or more minutes. In another embodiment, in an early stage of cell growth the rotating growth vial 400 may be oscillated at a first periodicity (e.g., every 60 seconds), and then a later stage of cell growth the rotating growth vial 300 may be oscillated at a second periodicity (e.g., every one second) different from the first periodicity.
The rotating growth vial 300 may be reusable or, preferably, the rotating growth vial is consumable. In some embodiments, the rotating growth vial is consumable and is presented to the user pre-filled with growth medium, where the vial is hermetically sealed at the open end 304 with a foil seal. A medium-filled rotating growth vial packaged in such a manner may be part of a kit for use with a stand-alone cell growth device or with a cell growth module that is part of an automated multi-module cell processing system. To introduce cells into the vial, a user need only pipette up a desired volume of cells and use the pipette tip to punch through the foil seal of the vial. Open end 304 may optionally include an extended lip 302 to overlap and engage with the cell growth device. In automated systems, the rotating growth vial 300 may be tagged with a barcode or other identifying means that can be read by a scanner or camera (not shown) that is part of the automated system.
The volume of the rotating growth vial 300 and the volume of the cell culture (including growth medium) may vary greatly, but the volume of the rotating growth vial 300 must be large enough to generate a specified total number of cells. In practice, the volume of the rotating growth vial 300 may range from 1-250 mL, 2-100 mL, from 5-80 mL, 10-50 mL, or from 12-35 mL. Likewise, the volume of the cell culture (cells+growth media) should be appropriate to allow proper aeration and mixing in the rotating growth vial 400. Proper aeration promotes uniform cellular respiration within the growth media. Thus, the volume of the cell culture should be approximately 5-85% of the volume of the growth vial or from 20-60% of the volume of the growth vial. For example, for a 30 mL growth vial, the volume of the cell culture would be from about 1.5 mL to about 26 mL, or from 6 mL to about 18 mL.
The rotating growth vial 300 preferably is fabricated from a bio-compatible optically transparent material—or at least the portion of the vial comprising the light path(s) is transparent. Additionally, material from which the rotating growth vial is fabricated should be able to be cooled to about 4° C. or lower and heated to about 55° C. or higher to accommodate both temperature-based cell assays and long-term storage at low temperatures. Further, the material that is used to fabricate the vial must be able to withstand temperatures up to 55° C. without deformation while spinning. Suitable materials include cyclic olefin copolymer (COC), glass, polyvinyl chloride, polyethylene, polyamide, polypropylene, polycarbonate, poly(methyl methacrylate (PMMA), polysulfone, polyurethane, and co-polymers of these and other polymers. Preferred materials include polypropylene, polycarbonate, or polystyrene. In some embodiments, the rotating growth vial is inexpensively fabricated by, e.g., injection molding or extrusion.
The motor 338 engages with drive mechanism 312 and is used to rotate the rotating growth vial 300. In some embodiments, motor 338 is a brushless DC type drive motor with built-in drive controls that can be set to hold a constant revolution per minute (RPM) between 0 and about 3000 RPM. Alternatively, other motor types such as a stepper, servo, brushed DC, and the like can be used. Optionally, the motor 338 may also have direction control to allow reversing of the rotational direction, and a tachometer to sense and report actual RPM. The motor is controlled by a processor (not shown) according to, e.g., standard protocols programmed into the processor and/or user input, and the motor may be configured to vary RPM to cause axial precession of the cell culture thereby enhancing mixing, e.g., to prevent cell aggregation, increase aeration, and optimize cellular respiration.
Main housing 336, end housings 352 and lower housing 332 of the cell growth device 330 may be fabricated from any suitable, robust material including aluminum, stainless steel, and other thermally conductive materials, including plastics. These structures or portions thereof can be created through various techniques, e.g., metal fabrication, injection molding, creation of structural layers that are fused, etc. Whereas the rotating growth vial 300 is envisioned in some embodiments to be reusable, but preferably is consumable, the other components of the cell growth device 330 are preferably reusable and function as a stand-alone benchtop device or as a module in a multi-module cell processing system.
The processor (not shown) of the cell growth device 330 may be programmed with information to be used as a “blank” or control for the growing cell culture. A “blank” or control is a vessel containing cell growth medium only, which yields 100% transmittance and 0 OD, while the cell sample will deflect light rays and will have a lower percent transmittance and higher OD. As the cells grow in the media and become denser, transmittance will decrease and OD will increase. The processor (not shown) of the cell growth device 330—may be programmed to use wavelength values for blanks commensurate with the growth media typically used in cell culture (whether, e.g., mammalian cells, bacterial cells, animal cells, yeast cells, etc.). Alternatively, a second spectrophotometer and vessel may be included in the cell growth device 330, where the second spectrophotometer is used to read a blank at designated intervals.
In use, cells are inoculated (cells can be pipetted, e.g., from an automated liquid handling system or by a user) into pre-filled growth media of a rotating growth vial 300 by piercing though the foil seal or film. The programmed software of the cell growth device 330 sets the control temperature for growth, typically 30 ° C., then slowly starts the rotation of the rotating growth vial 300. The cell/growth media mixture slowly moves vertically up the wall due to centrifugal force allowing the rotating growth vial 300 to expose a large surface area of the mixture to a normal oxygen environment. The growth monitoring system takes either continuous readings of the OD or OD measurements at pre-set or pre-programmed time intervals. These measurements are stored in internal memory and if requested the software plots the measurements versus time to display a growth curve. If enhanced mixing is required, e.g., to optimize growth conditions, the speed of the vial rotation can be varied to cause an axial precession of the liquid, and/or a complete directional change can be performed at programmed intervals. The growth monitoring can be programmed to automatically terminate the growth stage at a pre-determined OD, and then quickly cool the mixture to a lower temperature to inhibit further growth.
One application for the cell growth device 330 is to constantly measure the optical density of a growing cell culture. One advantage of the described cell growth device is that optical density can be measured continuously (kinetic monitoring) or at specific time intervals; e.g., every 5, 10, 15, 20, 30 45, or 60 seconds, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes. While the cell growth device 330 has been described in the context of measuring the optical density (OD) of a growing cell culture, it should, however, be understood by a skilled artisan given the teachings of the present specification that other cell growth parameters can be measured in addition to or instead of cell culture OD. As with optional measure of cell growth in relation to the solid wall device or module described supra, spectroscopy using visible, UV, or near infrared (NIR) light allows monitoring the concentration of nutrients and/or wastes in the cell culture and other spectroscopic measurements may be made; that is, other spectral properties can be measured via, e.g., dielectric impedance spectroscopy, visible fluorescence, fluorescence polarization, or luminescence. Additionally, the cell growth device 330 may include additional sensors for measuring, e.g., dissolved oxygen, carbon dioxide, pH, conductivity, and the like. For additional details regarding rotating growth vials and cell growth devices see U.S. Pat. No. 10,435,662, issued 8 Oct. 2019; U.S. Pat. No. 10,443,031, issued 15 Oct. 2019; and U.S. Pat. No. 16/552,981, filed 27 Aug. 2019 and 16/780,640, filed 3 Feb. 2020.
As described above in relation to the rotating growth vial and cell growth module, in order to obtain an adequate number of cells for transformation or transfection, cells typically are grown to a specific optical density in medium appropriate for the growth of the cells of interest; however, for effective transformation or transfection, it is desirable to decrease the volume of the cells as well as render the cells competent via buffer or medium exchange. Thus, one sub-component or module that is desired in cell processing systems to perform the methods described herein is a module or component that can grow, perform buffer exchange, and/or concentrate cells and render them competent so that they may be transformed or transfected with the nucleic acids needed for modulating the expression of the cell's genes.
Permeate/filtrate member 420 is seen in the middle of
On the left of
A membrane or filter is disposed between the retentate and permeate members, where fluids can flow through the membrane but cells cannot and are thus retained in the flow channel disposed in the retentate member. Filters or membranes appropriate for use in the TFF device/module are those that are solvent resistant, are contamination free during filtration, and are able to retain the types and sizes of cells of interest. For example, in order to retain small cell types such as bacterial cells, pore sizes can be as low as 0.2 μm, however for other cell types, the pore sizes can be as high as 20 μm. Indeed, the pore sizes useful in the TFF device/module include filters with sizes from 0.20 μm, 0.21 μm, 0.22 μm, 0.23 μm, 0.24 μm, 0.25 μm, 0.26 μm, 0.27 μm, 0.28 μm, 0.29 μm, 0.30 μm, 0.31 μm, 0.32 μm, 0.33 μm, 0.34 μm, 0.35 μm, 0.36 μm, 0.37 μm, 0.38 μm, 0.39 μm, 0.40 μm, 0.41 μm, 0.42 μm, 0.43 μm, 0.44 μm, 0.45 μm, 0.46 μm, 0.47 μm, 0.48 μm, 0.49 μm, 0.50 μm and larger. The filters may be fabricated from any suitable non-reactive material including cellulose mixed ester (cellulose nitrate and acetate) (CME), polycarbonate (PC), polyvinylidene fluoride (PVDF), polyethersulfone (PES), polytetrafluoroethylene (PTFE), nylon, glass fiber, or metal substrates as in the case of laser or electrochemical etching.
The length of the channel structure 402 may vary depending on the volume of the cell culture to be grown and the optical density of the cell culture to be concentrated. The length of the channel structure typically is from 60 mm to 300 mm, or from 70 mm to 200 mm, or from 80 mm to 100 mm. The cross-section configuration of the flow channel 402 may be round, elliptical, oval, square, rectangular, trapezoidal, or irregular. If square, rectangular, or another shape with generally straight sides, the cross section may be from about 10 μm to 1000 μm wide, or from 200 μm to 800 μm wide, or from 300 μm to 700 μm wide, or from 400 μm to 600 μm wide; and from about 10 μm to 1000 μm high, or from 200 μm to 800 μm high, or from 300 μm to 700 μm high, or from 400 μm to 600 μm high. If the cross section of the flow channel 402 is generally round, oval or elliptical, the radius of the channel may be from about 50 μm to 1000 μm in hydraulic radius, or from 5 μm to 800 μm in hydraulic radius, or from 200 μm to 700 μm in hydraulic radius, or from 300 μm to 600 μm wide in hydraulic radius, or from about 200 to 500 μm in hydraulic radius. Moreover, the volume of the channel in the retentate 422 and permeate 420 members may be different depending on the depth of the channel in each member.
The TFF device may be fabricated from any robust material in which channels (and channel branches) may be milled including stainless steel, silicon, glass, aluminum, or plastics including cyclic-olefin copolymer (COC), cyclo-olefin polymer (COP), polystyrene, polyvinyl chloride, polyethylene, polyamide, polyethylene, polypropylene, acrylonitrile butadiene, polycarbonate, polyetheretheketone (PEEK), poly(methyl methylacrylate) (PMMA), polysulfone, and polyurethane, and co-polymers of these and other polymers. If the TFF device/module is disposable, preferably it is made of plastic. In some embodiments, the material used to fabricate the TFF device/module is thermally-conductive so that the cell culture may be heated or cooled to a desired temperature. In certain embodiments, the TFF device is formed by precision mechanical machining, laser machining, electro discharge machining (for metal devices); wet or dry etching (for silicon devices); dry or wet etching, powder or sandblasting, photostructuring (for glass devices); or thermoforming, injection molding, hot embossing, or laser machining (for plastic devices) using the materials mentioned above that are amenable to this mass production techniques.
The overall work flow for cell growth comprises loading a cell culture to be grown into a first retentate reservoir, optionally bubbling air or an appropriate gas through the cell culture, passing or flowing the cell culture through the first retentate port then tangentially through the TFF channel structure while collecting medium or buffer through one or both of the permeate ports 406, collecting the cell culture through a second retentate port 404 into a second retentate reservoir, optionally adding additional or different medium to the cell culture and optionally bubbling air or gas through the cell culture, then repeating the process, all while measuring, e.g., the optical density of the cell culture in the retentate reservoirs continuously or at desired intervals. Measurements of optical densities (OD) at programmed time intervals are accomplished using a 600 nm Light Emitting Diode (LED) that has been columnated through an optic into the retentate reservoir(s) containing the growing cells. The light continues through a collection optic to the detection system which consists of a (digital) gain-controlled silicone photodiode. Generally, optical density is shown as the absolute value of the logarithm with base 10 of the power transmission factors of an optical attenuator: OD=−log 10 (Power out/Power in). Since OD is the measure of optical attenuation—that is, the sum of absorption, scattering, and reflection—the TFF device OD measurement records the overall power transmission, so as the cells grow and become denser in population, the OD (the loss of signal) increases. The OD system is pre-calibrated against OD standards with these values stored in an on-board memory accessible by the measurement program.
In the channel structure, the membrane bifurcating the flow channels retains the cells on one side of the membrane (the retentate side 422) and allows unwanted medium or buffer to flow across the membrane into a filtrate or permeate side (e.g., permeate member 420) of the device. Bubbling air or other appropriate gas through the cell culture both aerates and mixes the culture to enhance cell growth. During the process, medium that is removed during the flow through the channel structure is removed through the permeate/filtrate ports 406. Alternatively, cells can be grown in one reservoir with bubbling or agitation without passing the cells through the TFF channel from one reservoir to the other.
The overall work flow for cell concentration using the TFF device/module involves flowing a cell culture or cell sample tangentially through the channel structure. As with the cell growth process, the membrane bifurcating the flow channels retains the cells on one side of the membrane and allows unwanted medium or buffer to flow across the membrane into a permeate/filtrate side (e.g., permeate member 420) of the device. In this process, a fixed volume of cells in medium or buffer is driven through the device until the cell sample is collected into one of the retentate ports 404, and the medium/buffer that has passed through the membrane is collected through one or both of the permeate/filtrate ports 406. All types of prokaryotic and eukaryotic cells—both adherent and non-adherent cells—can be grown in the TFF device. Adherent cells may be grown on beads or other cell scaffolds suspended in medium that flow through the TFF device.
The medium or buffer used to suspend the cells in the cell concentration device/module may be any suitable medium or buffer for the type of cells being transformed or transfected, such as LB, SOC, TPD, YPG, YPAD, MEM, DMEM, IMDM, RPMI, Hanks', PBS and Ringer's solution, where the media may be provided in a reagent cartridge as part of a kit. For culture of adherent cells, cells may be disposed on beads, microcarriers, or other type of scaffold suspended in medium. Most normal mammalian tissue-derived cells—except those derived from the hematopoietic system—are anchorage dependent and need a surface or cell culture support for normal proliferation. In the rotating growth vial described herein, microcarrier technology is leveraged. Microcarriers of particular use typically have a diameter of 100-300 μm and have a density slightly greater than that of the culture medium (thus facilitating an easy separation of cells and medium for, e.g., medium exchange) yet the density must also be sufficiently low to allow complete suspension of the carriers at a minimum stirring rate in order to avoid hydrodynamic damage to the cells. Many different types of microcarriers are available, and different microcarriers are optimized for different types of cells. There are positively charged carriers, such as Cytodex 1 (dextran-based, GE Healthcare), DE-52 (cellulose-based, Sigma-Aldrich Labware), DE-53 (cellulose-based, Sigma-Aldrich Labware), and HLX 11-170 (polystyrene-based); collagen- or ECM-(extracellular matrix) coated carriers, such as Cytodex 3 (dextran-based, GE Healthcare) or HyQ-sphere Pro-F 102-4 (polystyrene-based, Thermo Scientific); non-charged carriers, like HyQ-sphere P 102-4 (Thermo Scientific); or macroporous carriers based on gelatin (Cultisphere, Percell Biolytica) or cellulose (Cytopore, GE Healthcare).
In both the cell growth and concentration processes, passing the cell sample through the TFF device and collecting the cells in one of the retentate ports 404 while collecting the medium in one of the permeate/filtrate ports 406 is considered “one pass” of the cell sample. The transfer between retentate reservoirs “flips” the culture. The retentate and permeate ports collecting the cells and medium, respectively, for a given pass reside on the same end of TFF device/module with fluidic connections arranged so that there are two distinct flow layers for the retentate and permeate/filtrate sides, but if the retentate port 404 resides on the retentate member of device/module (that is, the cells are driven through the channel above the membrane and the filtrate (medium) passes to the portion of the channel below the membrane), the permeate/filtrate port 406 will reside on the permeate member of device/module and vice versa (that is, if the cell sample is driven through the channel below the membrane, the filtrate (medium) passes to the portion of the channel above the membrane). Due to the high pressures used to transfer the cell culture and fluids through the flow channel of the TFF device, the effect of gravity is negligible.
At the conclusion of a “pass” in either of the growth and concentration processes, the cell sample is collected by passing through the retentate port 404 and into the retentate reservoir (not shown). To initiate another “pass”, the cell sample is passed again through the TFF device, this time in a flow direction that is reversed from the first pass. The cell sample is collected by passing through the retentate port 404 and into retentate reservoir (not shown) on the opposite end of the device/module from the retentate port 404 that was used to collect cells during the first pass. Likewise, the medium/buffer that passes through the membrane on the second pass is collected through the permeate port 406 on the opposite end of the device/module from the permeate port 406 that was used to collect the filtrate during the first pass, or through both ports. This alternating process of passing the retentate (the concentrated cell sample) through the device/module is repeated until the cells have been grown to a desired optical density, and/or concentrated to a desired volume, and both permeate ports (i.e., if there are more than one) can be open during the passes to reduce operating time. In addition, buffer exchange may be effected by adding a desired buffer (or fresh medium) to the cell sample in the retentate reservoir, before initiating another “pass”, and repeating this process until the old medium or buffer is diluted and filtered out and the cells reside in fresh medium or buffer. Note that buffer exchange and cell growth may (and typically do) take place simultaneously, and buffer exchange and cell concentration may (and typically do) take place simultaneously. For further information and alternative embodiments on TFFs see, e.g., U.S. Ser. No. 62/728,365, filed 7 Sep. 2018; 62/857,599, filed 5 Jun. 2019; and 62/867,415, filed 27 Jun. 2019.
In one embodiment, the reagent reservoirs or reservoirs 504 of reagent cartridge 500 are configured to hold various size tubes, including, e.g., 250 ml tubes, 25 ml tubes, 10 ml tubes, 5 ml tubes, and Eppendorf or microcentrifuge tubes. In yet another embodiment, all reservoirs may be configured to hold the same size tube, e.g., 5 ml tubes, and reservoir inserts may be used to accommodate smaller tubes in the reagent reservoir. In yet another embodiment—particularly in an embodiment where the reagent cartridge is disposable—the reagent reservoirs hold reagents without inserted tubes. In this disposable embodiment, the reagent cartridge may be part of a kit, where the reagent cartridge is pre-filled with reagents and the receptacles or reservoirs sealed with, e.g., foil, heat seal acrylic or the like and presented to a consumer where the reagent cartridge can then be used in an automated multi-module cell processing instrument. As one of ordinary skill in the art will appreciate given the present disclosure, the reagents contained in the reagent cartridge will vary depending on work flow; that is, the reagents will vary depending on the processes to which the cells are subjected in the automated multi-module cell processing instrument, e.g., protein production, cell transformation and culture, gene expression modulation, etc.
Reagents such as cell samples, enzymes, buffers, nucleic acid vectors, expression cassettes, proteins or peptides, reaction components (such as, e.g., MgCl2, dNTPs, nucleic acid assembly reagents, gap repair reagents, and the like), wash solutions, ethanol, and magnetic beads for nucleic acid purification and isolation, etc. may be positioned in the reagent cartridge at a known position. In some embodiments of cartridge 500, the cartridge comprises a script (not shown) readable by a processor (not shown) for dispensing the reagents. Also, the cartridge 500 as one component in an automated multi-module cell processing instrument may comprise a script specifying two, three, four, five, ten or more processes to be performed by the automated multi-module cell processing instrument. In certain embodiments, the reagent cartridge is disposable and is pre-packaged with reagents tailored to performing specific cell processing protocols, e.g., gene expression modulation, genome editing, or protein production. Because the reagent cartridge contents vary while components/modules of the automated multi-module cell processing instrument or system may not, the script associated with a particular reagent cartridge matches the reagents used and cell processes performed. Thus, e.g., reagent cartridges may be pre-packaged with reagents for gene expression modulation and a script that specifies the process steps for performing gene expression modulation in an automated multi-module cell processing instrument, or, e.g., reagents for protein expression and a script that specifies the process steps for performing protein expression in an automated multi-module cell processing instrument.
For example, the reagent cartridge may comprise a script to pipette competent cells from a reservoir, transfer the cells to a transformation module, pipette a nucleic acid solution comprising a vector with expression cassette from another reservoir in the reagent cartridge, transfer the nucleic acid solution to the transformation module, initiate the transformation process for a specified time, then move the transformed cells to yet another reservoir in the reagent cassette or to another module such as a cell growth module in the automated multi-module cell processing instrument. In another example, the reagent cartridge may comprise a script to transfer a nucleic acid solution comprising a vector from a reservoir in the reagent cassette, nucleic acid solution comprising expression cassettes in a reservoir in the reagent cassette, and a nucleic acid assembly mix from another reservoir to the nucleic acid assembly/desalting module, if present. The script may also specify process steps performed by other modules in the automated multi-module cell processing instrument. For example, the script may specify that the nucleic acid assembly/desalting reservoir be heated to 50° C. for 30 min to generate an assembled product; and desalting and resuspension of the assembled product via magnetic bead-based nucleic acid purification involving a series of pipette transfers and mixing of magnetic beads, ethanol wash, and buffer.
As described in relation to
Additional details of the FTEP devices are illustrated in
In the FTEP devices of the disclosure, the toxicity level of the transformation results in greater than 30% viable cells after electroporation, preferably greater than 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or even 99% viable cells following transformation, depending on the cell type and the nucleic acids being introduced into the cells.
The housing of the FTEP device can be made from many materials depending on whether the FTEP device is to be reused, autoclaved, or is disposable, including stainless steel, silicon, glass, resin, polyvinyl chloride, polyethylene, polyamide, polystyrene, polyethylene, polypropylene, acrylonitrile butadiene, polycarbonate, polyetheretheketone (PEEK), polysulfone and polyurethane, co-polymers of these and other polymers. Similarly, the walls of the channels in the device can be made of any suitable material including silicone, resin, glass, glass fiber, polyvinyl chloride, polyethylene, polyamide, polyethylene, polypropylene, acrylonitrile butadiene, polycarbonate, polyetheretheketone (PEEK), polysulfone and polyurethane, co-polymers of these and other polymers. Preferred materials include crystal styrene, cyclo-olefin polymer (COP) and cyclic olephin co-polymers (COC), which allow the device to be formed entirely by injection molding in one piece with the exception of the electrodes and, e.g., a bottom sealing film if present.
The FTEP devices described herein (or portions of the FTEP devices) can be created or fabricated via various techniques, e.g., as entire devices or by creation of structural layers that are fused or otherwise coupled. For example, for metal FTEP devices, fabrication may include precision mechanical machining or laser machining; for silicon FTEP devices, fabrication may include dry or wet etching; for glass FTEP devices, fabrication may include dry or wet etching, powderblasting, sandblasting, or photostructuring; and for plastic FTEP devices fabrication may include thermoforming, injection molding, hot embossing, or laser machining. The components of the FTEP devices may be manufactured separately and then assembled, or certain components of the FTEP devices (or even the entire FTEP device except for the electrodes) may be manufactured (e.g., using 3D printing) or molded (e.g., using injection molding) as a single entity, with other components added after molding. For example, housing and channels may be manufactured or molded as a single entity, with the electrodes later added to form the FTEP unit. Alternatively, the FTEP device may also be formed in two or more parallel layers, e.g., a layer with the horizontal channel and filter, a layer with the vertical channels, and a layer with the inlet and outlet ports, which are manufactured and/or molded individually and assembled following manufacture.
In specific aspects, the FTEP device can be manufactured using a circuit board as a base, with the electrodes, filter and/or the flow channel formed in the desired configuration on the circuit board, and the remaining housing of the device containing, e.g., the one or more inlet and outlet channels and/or the flow channel formed as a separate layer that is then sealed onto the circuit board. The sealing of the top of the housing onto the circuit board provides the desired configuration of the different elements of the FTEP devices of the disclosure. Also, two to many FTEP devices may be manufactured on a single substrate, then separated from one another thereafter or used in parallel. In certain embodiments, the FTEP devices are reusable and, in some embodiments, the FTEP devices are disposable. In additional embodiments, the FTEP devices may be autoclavable.
The electrodes 508 can be formed from any suitable metal, such as copper, stainless steel, titanium, aluminum, brass, silver, rhodium, gold or platinum, or graphite. One preferred electrode material is alloy 303 (UNS330300) austenitic stainless steel. An applied electric field can destroy electrodes made from of metals like aluminum. If a multiple-use (i.e., non-disposable) flow-through FTEP device is desired-as opposed to a disposable, one-use flow-through FTEP device-the electrode plates can be coated with metals resistant to electrochemical corrosion. Conductive coatings like noble metals, e.g., gold, can be used to protect the electrode plates.
As mentioned, the FTEP devices may comprise push-pull pneumatic means to allow multi-pass electroporation procedures; that is, cells to electroporated may be “pulled” from the inlet toward the outlet for one pass of electroporation, then be “pushed” from the outlet end of the flow-through FTEP device toward the inlet end to pass between the electrodes again for another pass of electroporation. This process may be repeated one to many times.
Depending on the type of cells to be electroporated (e.g., bacterial, yeast, mammalian) and the configuration of the electrodes, the distance between the electrodes in the flow channel can vary widely. For example, where the flow channel decreases in width, the flow channel may narrow to between 10 μm and 5 mm, or between 25 μm and 3 mm, or between 50 μm and 2 mm, or between 75 μm and 1 mm. The distance between the electrodes in the flow channel may be between 1 mm and 10 mm, or between 2 mm and 8 mm, or between 3 mm and 7 mm, or between 4 mm and 6 mm. The overall size of the FTEP device may be from 3 cm to 15 cm in length, or 4 cm to 12 cm in length, or 4.5 cm to 10 cm in length. The overall width of the FTEP device may be from 0.5 cm to 5 cm, or from 0.75 cm to 3 cm, or from 1 cm to 2.5 cm, or from 1 cm to 1.5 cm.
The region of the flow channel that is narrowed is wide enough so that at least two cells can fit in the narrowed portion side-by-side. For example, a typical bacterial cell is 1 μm in diameter; thus, the narrowed portion of the flow channel of the FTEP device used to transform such bacterial cells will be at least 2 μm wide. In another example, if a mammalian cell is approximately 50 μm in diameter, the narrowed portion of the flow channel of the FTEP device used to transform such mammalian cells will be at least 100 μm wide. That is, the narrowed portion of the FTEP device will not physically contort or “squeeze” the cells being transformed.
In embodiments of the FTEP device where reservoirs are used to introduce cells and exogenous material into the FTEP device, the reservoirs range in volume from 100 μL to 10 mL, or from 500 μL to 75 mL, or from 1 mL to 5 mL. The flow rate in the FTEP ranges from 0.1 mL to 5 mL per minute, or from 0.5 mL to 3 mL per minute, or from 1.0 mL to 2.5 mL per minute. The pressure in the FTEP device ranges from 1-30 psi, or from 2-10 psi, or from 3-5 psi.
To avoid different field intensities between the electrodes, the electrodes should be arranged in parallel. Furthermore, the surface of the electrodes should be as smooth as possible without pin holes or peaks. Electrodes having a roughness Rz of 1 to 10 μm are preferred. In another embodiment of the invention, the flow-through electroporation device comprises at least one additional electrode which applies a ground potential to the FTEP device.
After processing 6053 (e.g., gene expression modulation), some cells in the colonies of cells may die, e.g., by fitness effects from processing events, and there may be a lag in growth for the cells that survive but must recover following gene expression modulation (microwells 6058), where cells that do not undergo gene expression modulation may thrive (microwells 6059) (vi). All cells are allowed to continue grow to establish colonies and normalize, where the colonies of modified cells in microwells 6058 catch up in size and/or cell number with the cells in microwells 6059 that do not undergo gene expression modulation (vii). Once the cell colonies are normalized, either pooling 6060 of all cells in the microwells can take place, in which case the cells are enriched for modified cells by eliminating the bias from non-modified cells and fitness effects; alternatively, colony growth in the microwells is monitored after processing, and slow growing colonies (e.g., the cells in microwells 6058) are identified and selected 6061 (e.g., “cherry picked”) resulting in even greater enrichment of modified cells.
In growing the cells, the medium used will depend, of course, on the type of cells being processed—e.g., bacterial, yeast or mammalian. For example, medium for yeast cell growth includes LB, SOC, TPD, YPG, YPAD, MEM and DMEM.
A module useful for performing the method depicted in
The SWIIN module 650 in
In this
In this embodiment of a SWIIN module, the perforated member includes through-holes to accommodate ultrasonic tabs disposed on the permeate member. Thus, in this embodiment the perforated member is fabricated from 316 stainless steel, and the perforations form the walls of microwells while a filter or membrane is used to form the bottom of the microwells. Typically, the perforations (microwells) are approximately 150 μm-200 μm in diameter, and the perforated member is approximately 125 μm deep, resulting in microwells having a volume of approximately 2.5 nl, with a total of approximately 200,000 microwells. The distance between the microwells is approximately 279 μm center-to-center. Though here the microwells have a volume of approximately 2.5 nl, the volume of the microwells may be from 1 to 25 nl, or preferably from 2 to 10 nl, and even more preferably from 2 to 4 nl. As for the filter or membrane, like the filter described previously, filters appropriate for use are solvent resistant, contamination free during filtration, and are able to retain the types and sizes of cells of interest. For example, in order to retain small cell types such as bacterial cells, pore sizes can be as low as 0.10 however for other cell types (e.g., such as for mammalian cells), the pore sizes can be as high as 10.0 μm-20.0 μm or more. Indeed, the pore sizes useful in the cell concentration device/module include filters with sizes from 0.10 μm, 0.11 μm, 0.12 μm, 0.13 μm, 0.14 μm, 0.15 μm, 0.16 μm, 0.17 μm, 0.18 μm, 0.19 μm, 0.20 μm, 0.21 μm, 0.22 μm, 0.23 μm, 0.24 μm, 0.25 μm, 0.26 μm, 0.27 μm, 0.28 μm, 0.29 μm, 0.30 μm, 0.31 μm, 0.32 μm, 0.33 μm, 0.34 μm, 0.35 μm, 0.36 μm, 0.37 μm, 0.38 μm, 0.39 μm, 0.40 μm, 0.41 μm, 0.42 μm, 0.43 μm, 0.44 μm, 0.45 μm, 0.46 μm, 0.47 μm, 0.48 μm, 0.49 μm, 0.50 μm and larger. The filters may be fabricated from any suitable material including cellulose mixed ester (cellulose nitrate and acetate) (CME), polycarbonate (PC), polyvinylidene fluoride (PVDF), polyethersulfone (PES), polytetrafluoroethylene (PTFE), nylon, or glass fiber.
The cross-section configuration of the mated serpentine channel may be round, elliptical, oval, square, rectangular, trapezoidal, or irregular. If square, rectangular, or another shape with generally straight sides, the cross section may be from about 2 mm to 15 mm wide, or from 3 mm to 12 mm wide, or from 5 mm to 10 mm wide. If the cross section of the mated serpentine channel is generally round, oval or elliptical, the radius of the channel may be from about 3 mm to 20 mm in hydraulic radius, or from 5 mm to 15 mm in hydraulic radius, or from 8 mm to 12 mm in hydraulic radius.
Serpentine channels 660a and 660b can have approximately the same volume or a different volume. For example, each “side” or portion 660a, 660b of the serpentine channel may have a volume of, e.g., 2 mL, or serpentine channel 660a of permeate member 608 may have a volume of 2 mL, and the serpentine channel 660b of retentate member 604 may have a volume of, e.g., 3 mL. The volume of fluid in the serpentine channel may range from about 2 mL to about 80 mL, or about 4 mL to 60 mL, or from 5 mL to 40 mL, or from 6 mL to 20 mL (note these volumes apply to a SWIIN module comprising a, e.g., 50-500K perforation member). The volume of the reservoirs may range from 5 mL to 50 mL, or from 7 mL to 40 mL, or from 8 mL to 30 mL or from 10 mL to 20 mL, and the volumes of all reservoirs may be the same or the volumes of the reservoirs may differ (e.g., the volume of the permeate reservoirs is greater than that of the retentate reservoirs).
The serpentine channel portions 660a and 660b of the permeate member 608 and retentate member 604, respectively, are approximately 200 mm long, 130 mm wide, and 4 mm thick, though in other embodiments, the retentate and permeate members can be from 75 mm to 400 mm in length, or from 100 mm to 300 mm in length, or from 150 mm to 250 mm in length; from 50 mm to 250 mm in width, or from 75 mm to 200 mm in width, or from 100 mm to 150 mm in width; and from 2 mm to 15 mm in thickness, or from 4 mm to 10 mm in thickness, or from 5 mm to 8 mm in thickness. Embodiments the retentate (and permeate) members may be fabricated from PMMA (poly(methyl methacrylate) or other materials may be used, including polycarbonate, cyclic olefin co-polymer (COC), glass, polyvinyl chloride, polyethylene, polyamide, polypropylene, polysulfone, polyurethane, and co-polymers of these and other polymers. Preferably at least the retentate member is fabricated from a transparent material so that the cells can be visualized (see, e.g.,
Because the retentate member preferably is transparent, colony growth in the SWIIN module can be monitored by automated devices such as those sold by JoVE (ScanLag™ system, Cambridge, Mass.) (also see Levin-Reisman, et al., Nature Methods, 7:737-39 (2010)). Cell growth for, e.g., mammalian cells may be monitored by, e.g., the growth monitor sold by IncuCyte (Ann Arbor, Mich.) (see also, Choudhry, PLos One, 11(2):e0148469 (2016)). Further, automated colony pickers may be employed, such as those sold by, e.g., TECAN (Pickolo™ system, Mannedorf, Switzerland); Hudson Inc. (RapidPick™, Springfield, N.J.); Molecular Devices (QPix 400™ system, San Jose, Calif.); and Singer Instruments (PIXL™ system, Somerset, UK).
Due to the heating and cooling of the SWIIN module, condensation may accumulate on the retentate member which may interfere with accurate visualization of the growing cell colonies. Condensation of the SWIIN module 650 may be controlled by, e.g., moving heated air over the top of (e.g., retentate member) of the SWIIN module 650, or by applying a transparent heated lid over at least the serpentine channel portion 660b of the retentate member 604. See, e.g.,
In SWIIN module 650 cells and medium—at a dilution appropriate for Poisson or substantial Poisson distribution of the cells in the microwells of the perforated member—are flowed into serpentine channel 660b from ports in retentate member 604, and the cells settle in the microwells while the medium passes through the filter into serpentine channel 660a in permeate member 608. The cells are retained in the microwells of perforated member 601 as the cells cannot travel through filter 603. Appropriate medium may be introduced into permeate member 608 through permeate ports 611. The medium flows upward through filter 603 to nourish the cells in the microwells (perforations) of perforated member 601. Additionally, buffer exchange can be effected by cycling medium through the retentate and permeate members. In operation, the cells are deposited into the microwells, are grown for an initial, e.g., 2-100 doublings, and in certain embodiments, gene expression modulation may be induced by, e.g., raising or lower the temperature of the SWIIN to induce a temperature inducible promoter, or by removing growth medium from the permeate member and replacing the growth medium with a medium comprising a chemical component that induces an inducible promoter. In certain other embodiments, no induction as necessary, as relevant components of the nuclease-mediated gene modulation system may be under the control of constitutive promoters
Once gene expression modulation has taken place, in certain embodiments, the temperature of the SWIIN may be modified to stop gene expression modulation, or the inducing medium may be removed and replaced with fresh medium lacking the chemical component thereby de-activating inducible promoters. For example, in certain embodiments, increasing the temperature of the SWINN to 30 ° C. may halt or slow down gene expression modulation. The modified cells then continue to grow in the SWIIN module 650 until the growth of the cell colonies in the microwells is normalized. For the normalization protocol, once the colonies are normalized, the colonies are flushed from the microwells by applying fluid or air pressure (or both) to the permeate member serpentine channel 660a and thus to filter 603 and pooled. Alternatively, if cherry picking is desired, the growth of the cell colonies in the microwells is monitored, and slow-growing colonies are directly selected; or, fast-growing colonies are eliminated.
Imaging of cell colonies growing in the wells of the SWIIN is desired in most implementations for, e.g., monitoring both cell growth and device performance and imaging is necessary for cherry-picking implementations. Real-time monitoring of cell growth in the SWIIN requires backlighting, retentate plate (top plate) condensation management and a system-level approach to temperature control, air flow, and thermal management. In some implementations, imaging employs a camera or CCD device with sufficient resolution to be able to image individual wells. For example, in some configurations a camera with a 9-pixel pitch is used (that is, there are 9 pixels center-to-center for each well). Processing the images may, in some implementations, utilize reading the images in grayscale, rating each pixel from low to high, where wells with no cells will be brightest (due to full or nearly-full light transmission from the backlight) and wells with cells will be dim (due to cells blocking light transmission from the backlight). After processing the images, thresholding is performed to determine which pixels will be called “bright” or “dim”, spot finding is performed to find bright pixels and arrange them into blocks, and then the spots are arranged on a hexagonal grid of pixels that correspond to the spots. Once arranged, the measure of intensity of each well is extracted, by, e.g., looking at one or more pixels in the middle of the spot, looking at several to many pixels at random or pre-set positions, or averaging X number of pixels in the spot. In addition, background intensity may be subtracted. Thresholding is again used to call each well positive (e.g., containing cells) or negative (e.g., no cells in the well). The imaging information may be used in several ways, including taking images at time points for monitoring cell growth. Monitoring cell growth can be used to, e.g., remove the “muffin tops” of fast-growing cells followed by removal of all cells or removal of cells in “rounds” as described above, or recover cells from specific wells (e.g., slow-growing cell colonies); alternatively, wells containing fast-growing cells can be identified and areas of UV light covering the fast-growing cell colonies can be projected (or rastered with shutters) onto the SWIIN to irradiate or inhibit growth of those cells. Imaging may also be used to assure proper fluid flow in the serpentine channel 660.
After recovery, the cells may be transferred to a storage module 712, where the cells can be stored at, e.g., 4° C. or −20° C. for later processing, or the cells may be diluted and transferred to a SWIIN module 720. In the SWIIN 720, the cells are arrayed such that there is an average of one to twenty or fifty or so cells per microwell. The arrayed cells may be in selection medium to select for cells that have been transformed or transfected with the expression vector(s). Once singulated, the cells grow through 2-50 doublings and establish colonies. Once colonies are established, modulation of gene expression is allowed to proceed by providing conditions (e.g., temperature) to facilitate, and in certain embodiments, induce, such modulation. The modified cells are allowed to grow to terminal size (e.g., normalization of the colonies) in the microwells and may then be treated to conditions that cure the expression vector the samples. Once cured, the cells can be flushed out of the microwells and pooled, then transferred to the storage (or recovery) unit 712 or can be transferred back to the growth module 704 for another round of processing. In between pooling and transfer to a growth module, there typically is one or more additional steps, such as cell recovery, medium exchange (rendering the cells electrocompetent), cell concentration (typically concurrently with medium exchange by, e.g., filtration. Note that the selection/singulation/growth/induction/normalization and curing modules may be the same module, where all processes are performed in, e.g., a solid wall device, or selection and/or dilution may take place in a separate vessel before the cells are transferred to the solid wall SWIIN. Similarly, the cells may be pooled after normalization, transferred to a separate vessel, and cured in the separate vessel. Once the modified cells are pooled, they may be subjected to further processing, including another round of gene expression modulation.
The multi-module cell processing instrument exemplified in
It should be apparent to one of ordinary skill in the art given the present disclosure that the process described may be recursive and multiplex; that is, cells may go through the workflow described in relation to
introducing into the cell:
introducing into the cell:
13. The method of claim 12, wherein each single-pack expression cassette of the plurality of single-pack expression cassettes further comprises a melting temperature booster sequence and a subpool primer binding sequence flanking the assembled gRNA and barcode.
a guide RNA (gRNA) or a nucleic acid molecule encoding the gRNA, wherein the gRNA comprises a first nucleotide sequence that is at least partially complementary to a coding sequence of the target nucleic acid and a second nucleotide sequence configured to interact with an inactive dMAD7 nuclease; and
an inactive dMAD7 nuclease polypeptide and/or a nucleotide sequence encoding an inactive MAD7® nuclease polypeptide, wherein the gRNA is configured to guide the inactive dMAD7 nuclease polypeptide to the target nucleic acid, and wherein binding of the inactive dMAD7 nuclease polypeptide to the target nucleic acid attenuates or prevents transcription of the target nucleic acid.
a plurality of guide RNAs (gRNAs) or one or more nucleic acid molecules encoding the plurality of gRNAs, wherein each gRNA comprising a first nucleotide sequence that is at least partially complementary to a coding sequence of a corresponding target nucleic acid of the plurality of target nucleic acids and a second nucleotide sequence configured to interact with an inactive dMAD7 nuclease; and
one or more inactive dMAD7 nuclease polypeptides and/or nucleotide sequences each encoding an inactive MAD7® nuclease polypeptide, wherein each gRNA of the plurality of gRNAs is configured to guide one of the one or more inactive dMAD7 nuclease polypeptides to the corresponding target nucleic acid of the plurality of target nucleic acids, and wherein binding of each inactive dMAD7 nuclease polypeptide to the corresponding target nucleic acid of the plurality of target nucleic acids attenuates or prevents transcription of the corresponding one of the plurality of target nucleic acids.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent or imply that the experiments below are all of or the only experiments performed. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific aspects without departing from the spirit or scope of the invention as broadly described. The present aspects are, therefore, to be considered in all respects as illustrative and not restrictive.
Custom 87bp single-stranded single-pack cassettes, each comprising from 5′ to 3′ an melting temperature booster sequence (T1), a gRNA repeat region (CR), a gRNA spacer region (SR), a barcode sequence (BC), and a subpool primer binding sequence (P2) as described with reference to
PCR-amplified single-pack cassettes were utilized as PCR templates to assemble multi-pack cassettes (e.g., two or more stitched single-pack cassettes). 50 μL reaction volumes for assembly PCR were formed by mixing the following reagents: 10 μL of 5× assembly PCR buffer (final concentration: 1×) (Watchmaker Genomics, Boulder, Colo.); 1 μL of 10 mM dNTP mix (final concentration 0.2 mM) (Watchmaker Genomics, Boulder, Colo.); 1.5 μL of 10 μM forward primers (final concentration: 0.3 μM); 1.5 μL of 10 μM reverse primers (final concentration: 0.3 μM); 10 ng of each single-pack cassettes; 1 μL of 50× DNA polymerase (final concentration: 1×); and up to 50 μL of PCR-grade water. Assembly PCR was facilitated by temperature cycling with the following parameters (in sequence):
Assembly efficiency was increased by adding forward and reverse primers after 5 cycles of denaturation, primer annealing, and extension. After performing the assembly PCR reaction to stitch the multi-pack cassettes, amplicons were run on a 1% agarose gel, and cassettes of the correct band size were identified and extracted for further processing.
gRNAs were designed using a custom software to automate gRNA design, which takes into account various criteria to systematically identify PAM sequences nearest to target sites (TS) of interest and design effective gRNAs in view thereof. gRNAs were designed to target and bind to specific coding and/or non-coding sequences on DNA template or non-template strands. For certain target genes, guide sequences of corresponding gRNAs were designed to target the beginning of the coding sequence (CDS) thereof and/or the upstream ribosome binding site (RBS) sequence to prevent transcription initiation or block transcription elongation.
Single-pack cassettes were prepared with gRNAs designed to target the lacZ gene on the template genomic DNA strand according to methods described herein. The single-pack cassettes were assembled with vector backbones into expression vectors via isothermal assembly, and co-transformed with engine vectors comprising one or more inactive dMAD7 nuclease variants into E. coli strain MG1655 cells via electroporation.
Electroporation of expression and engine vectors was performed per relevant electroporation protocols. See, e.g., Pigac J. Schrempf H., Appl Environ Microbiol. 1995;61W:352-356. Following transformation, the cells were plated on lysogeny broth (LB) agar supplemented with chloramphenicol (25 mg/ml) and carbenicillin (100 mg/mL), and the cultures were incubated at 30 ° C. overnight. Individual colonies were picked for plating on MacConkey agar to assess repression efficiency, with the results shown in
Additional experiments were performed to determine the effectiveness of dMAD7-mediated repression when targeting the template versus non-template genomic DNA strand, with gRNA design and results shown in
Experiments were also performed to determine the effect on repression efficiency of incubation temperatures of the E. coli strain MG1655 cells after transformation, with gRNA design and results shown in
As shown in
Multi-pack cassettes formed of two, three, four, or five 87 bp single-pack cassettes, each comprising a gRNA targeting a target gene on the template DNA strand and a barcode, were stitched according to methods described herein. The guide sequences of the gRNAs in each single-pack cassette were designed with sufficient homology to the beginning of the CDS region of a target gene selected from the group of lacZ, xylA, galK, GFP, and RFP, and/or the upstream RBS sequence. The multi-pack cassettes were assembled with vector backbones into expression vectors via isothermal assembly, and co-transformed with engine vectors comprising one or more inactive dMAD7 nuclease variants into electrocompetent E. coli strain K12 cells via electroporation. Electroporation was performed per relevant electroporation protocols. See, e.g., Piga.c J, Schrempf H. Appl Environ Microbiol. 1995; 61(1): 352-356. Following transformation, the cells were plated on lysogeny broth (LB) agar supplemented with chloramphenicol (25 mg/ml) and carbenicillin (100 mg/mL), and the cultures were incubated at 30 ° C. overnight. Individual colonies were picked for performance of flow cytometry or plating on MacConkey agar supplemented with lactose, xylose, or galactose to assess repression efficiency, with vector/cassette/gRNA design and results shown in
When stitched (e.g., assembled) in a multi-pack cassette, single-pack cassettes were linearly ordered in the following gRNA sequence, regardless of the number of single-pack cassettes utilized: lacZ-xylA-galK-GFP-RFP. That is, a two-pack cassette comprised the lacZ-targeting cassette in a first position (i.e., “register 1”) and the xylA-targeting cassette in a second position (i.e., “register 2”); a three-pack cassette comprised the lacZ-targeting cassette in the first position, the xylA-targeting cassette in the second position, and the galK-targeting cassette in a third position (i.e., “register 3”); a four-pack cassette comprised the lacZ-targeting cassette in the first position, the xylA-targeting cassette in the second position, the galK-targeting cassette in the third position, and the GFP-targeting cassette in a fourth position (i.e., “register 4”); a five-pack cassette comprised the lacZ-targeting cassette in the first position, the xylA-targeting cassette in the second position, the galK-targeting cassette in the third position, the GFP-targeting cassette in the fourth position, and the RFP-targeting cassette in a fifth position (i.e., “register 5”).
While this invention is satisfied by embodiments in many different forms, as described in detail in connection with preferred embodiments of the invention, it is understood that the present disclosure is to be considered as exemplary of the principles of the invention and is not intended to limit the invention to the specific embodiments illustrated and described herein. Numerous variations may be made by persons skilled in the art without departure from the spirit of the invention. The scope of the invention will be measured by the appended claims and their equivalents. The abstract and the title are not to be construed as limiting the scope of the present invention, as their purpose is to enable the appropriate authorities, as well as the general public, to quickly determine the general nature of the invention. In the claims that follow, unless the term “means” is used, none of the features or elements recited therein should be construed as means-plus-function limitations pursuant to 35 U.S.C. § 112, ¶6.
This application claims the benefit of U.S. Provisional Application No. 63/253,532, filed on Oct. 7, 2021, which is incorporated by reference herein in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63253532 | Oct 2021 | US |
