WO2020037057A1 - Improved detection of nuclease edited sequences in automated modules and instruments via bulk cell culture - Google Patents
Improved detection of nuclease edited sequences in automated modules and instruments via bulk cell culture Download PDFInfo
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Definitions
- This invention relates to automated modules and systems for culturing and editing live cells via bulk cell culture.
- nucleases have been identified that allow manipulation of gene sequence, and hence gene function.
- the nucleases include nucleic acid-guided nucleases, which enable researchers to generate permanent edits in live cells. Editing efficiencies in ceil populations can be high; however, in pooled or multiplex formats there tends to be selective enrichment of cells that have not been edited due to the lack of the double-strand DNA breaks that occur during the editing process. Double-strand DNA breaks dramatically negatively impact cell viability thereby leading to enhanced survival of unedited cells and making it difficult to identify edited cells in the background of unedited cells. In addition, cells with edits that confer growth advantages or disadvantages can lead to skewed representations for different edits in the population.
- the present disclosure provides methods, modules, instruments and systems for automated high-throughput and extremely sensitive enriching for cells edited by a nucleic acid-guided nuclease.
- the methods take advantage of isolation or singulation, where the term in this context refers to the process of separating cells and growing them into clonal colonies. Isolation (or singulation) overcomes growth bias from unedited cells, growth effects from differential editing rates, and growth bias resulting from fitness effects of different edits. Indeed, it has been determined that removing growth rate bias via isolation or substantial isolation and growing colonies from the isolated cells to saturation or terminal colony size (e.g., normalization of the colonies) improves the observed editing efficiency by up to 4x or more over conventional methods.
- One particularly facile method for isolation utilizes a bulk cell culture format, which is described in detail herein.
- a method for performing enrichment of cells edited by a nucleic acid-guided nuclease comprising: providing transformed cells at a dilution resulting in substantially cells in an appropriate liquid growth medium comprising 0.25%-6% alginate, wherein the cells comprise nucleic acid-guided nuclease editing components where the gRNA optionally is under the control of an inducible promoter; solidifying the alginate-containing medium with a divalent cation; allowing the isolated cells to grow for 2 to 50 doublings to establish cell colonies; optionally inducing transcription of the gRNA; allowing the cell colonies to grow to become normalized; and liquefying the alginate-containing medium with a divalent cation chelating agent.
- the nucleic acid-guided nuclease editing components are provided to the cells on two separate vectors and in some aspects, the nucleic acid-guided nuclease editing components are provided to the cells on a single vector, and in some aspects, the cells are bacterial cells, yeast cells, or mammalian cells.
- the percentage of alginate in the growth medium is l%-4%, and in some aspects, the percentage of alginate in the growth medium is 2%-3%.
- the inducible promoter driving the gRNA is a promoter that is activated upon an increase in temperature
- the inducible promoter is a pL promoter
- the cells are transformed with a coding sequence for the CI857 repressor
- transcription of the one or more nucleic acid-guided nuclease editing components is induced by raising temperature of the cells to 42°C.
- solidifying the alginate-containing medium is performed with divalent cations except Mg +2 , and in some embodiments, the divalent cation is Ca +2 .
- the divalent cation chelating agent e.g., liquefying agent
- the divalent cation chelating agent is citrate, ethylenediaminetetraacetic acid (EDTA), or hexametaphosphate.
- a module for performing automated enrichment of cells edited by a nucleic acid-guided nuclease editing comprising: means for providing cells transformed with one or more vectors comprising a coding sequence for a nuclease, a guide nucleic acid and a DNA donor sequence; means for diluting the transformed cells in a medium comprising 0.25% to 6% (w/v) alginate to a cell density appropriate to isolate the transformed cells in a vessel; means for solidifying the alginate-containing medium; means for providing a temperature to grow the cells; and means for re-liquefying the solidified alginate-containing medium with a chelating agent for a divalent cation.
- a multi-module cell editing instrument comprising the module for performing automated enrichment of cells edited by a nucleic acid-guided nuclease, where the multi-module cell editing instrument further comprises a liquid handling system for providing cells, diluting cells, dispensing a solidifying agent, and dispensing a liquefying agent. Also in some aspects of a multi-module cell editing instrument comprising the module for performing automated enrichment of cells edited by a nucleic acid-guided nuclease, there is further provided a transformation module, and /or a growth module, and/or a nucleic acid assembly module, and/or a reagent cartridge.
- the reagent cartridge comprises CaCb and Na 3 C 6 H 5 0 7 .
- a method for performing enrichment of cells edited by a nucleic acid-guided nuclease comprising providing transformed cells at a dilution in a vessel resulting in isolated cells in an appropriate liquid growth medium comprising a hydrogel, wherein the cells comprise a gRNA under the control of an inducible promoter; solidifying the hydrogel-containing medium with a solution of a divalent cation; allowing the isolated cells to grow for 2 and 50 doublings to establish cell colonies; inducing transcription of the gRNA; growing the cells for a period of time sufficient to allow the cell colonies to become normalized; and liquefying the hydrogel- containing medium with a chelating agent for the divalent cation.
- the hydrogel-containing medium may be liquefied by agitation of the gel by, e.g., beads.
- the inducible promoter is a temperature inducible promoter
- the means for providing a temperature to induce transcription of the nuclease or guide nucleic acid is a Peltier device.
- solidifying the alginate-containing medium is performed with divalent cations except Mg +2 , and in some embodiments, the divalent cation is Ca +2 .
- the divalent cation chelating agent e.g., liquefying agent
- EDTA ethylenediaminetetraacetic acid
- Figure 1A is a simplified flow chart of two exemplary methods that may be performed by an automated bulk cell culture module, either as a stand-alone instrument or as part of an automated multi-module cell processing instrument.
- Figure 1B is a plot of optical density vs. time showing the growth curves for edited cells (dotted line) and unedited cells (solid line).
- Figure 2A depicts a simplified graphic for a workflow for isolating, editing and normalizing cells after nucleic acid-guided nuclease genome editing in bulk cell culture, where reversible solidification of the bulk culture is utilized.
- Figure 2B depicts a simplified graphic for a workflow for isolating, editing and normalizing cells after nucleic acid-guided nuclease genome editing in bulk cell culture, where reversible solidification of the bulk culture is utilized, and editing is induced by inducing transcription of gRNA.
- Figure 2C is a photograph of E. coli cells expressing green fluorescent protein in 2.0% alginate and medium that has been solidified showing isolated colonies (left) and a photograph of E. coli cells expressing green fluorescent protein in 2.0% alginate and medium after the medium has been re-liquified.
- Figure 3A depicts an automated multi-module cell processing instrument.
- Figures 3B - 3D depict a reagent cartridge and a flow-through electroporation device that is configured to reside in the reagent cartridge.
- Figures 3E - 3L depict various components of one embodiment of a tangential flow filtration device which serves as a cell concentration and buffer exchange module in the automated multi-module cell processing instrument shown in Figure 3A.
- Figure 4A depicts one embodiment of a rotating growth vial for use with a cell growth module described herein.
- Figure 4B illustrates a perspective view of one embodiment of a cell growth module comprising a rotating growth vial housing.
- Figure 4C depicts a cut-away view of the cell growth module from Figure 4B.
- Figure 4D illustrates the cell growth module of Figure 4B coupled to LED, detector, and temperature regulating components.
- Figure 5 A is a simplified block diagram of an embodiment of an exemplary automated multi-module cell processing instrument comprising a bulk gel isolation/growth/editing/normalization module.
- Figure 5B is a simplified block diagram of an alternative embodiment of an exemplary automated multi-module cell processing instrument comprising a bulk gel isolation/growth/editing/normalization module.
- Figures 6A - 6C depict a process for determining whether normalization takes place when cells are cultured in bulk gel.
- Figure 7 is a photograph of a bulk gel cell culture workflow for automation in a bulk gel isolation/growth/editing/normalization module utilizing a rotating growth vial, such as that depicted in Figure 4A.
- Figures 8A, 8B, and 8C depict a graph, table, and two graphs, respectively, of the results obtained from editing experiments performed with liquid cell culture employing no isolation or normalization, but employing inducible editing; bulk cell gel culture, employing isolation, inducible editing, and normalization; solid agar plating (SPP) employing isolation, inducible editing, and normalization; solid agar plating (SPP- Cherry) employing isolation, inducible editing, and cherry picking; and solid agar plating (SPP) employing isolation, inducible editing, and normalization but without cherry picking and simply scraping the colonies from the plate and re -plating.
- SPP solid agar plating
- SPP- Cherry solid agar plating
- SPP solid agar plating
- Figure 9 depicts a recursive workflow using bulk gel cell culture with curing.
- Such conventional techniques include polymer array synthesis, hybridization and ligation of polynucleotides, and detection of hybridization using a label. Specific illustrations of suitable techniques can be had by reference to the examples herein. However, other equivalent conventional procedures can, of course, also be used. Such conventional techniques and descriptions can be found in standard laboratory manuals such as Green, et al., eds., Genome Analysis: A Laboratory Manual Series (Vols.
- CRISPR-specific techniques can be found in, e.g., Appasani and Church, Genome Editing and Engineering From TALENs and CRISPRs to Molecular Surgery (2016); and Lindgren and Charpentier, CRISPR: Methods and Protocols (2015); both of which are herein incorporated in their entirety by reference for all purposes.
- the terms “approximately,” “proximate,” “minor,” and similar terms generally refer to ranges that include the identified value within a margin of 20%, 10% or preferably 5% in certain embodiments, and any values therebetween.
- nucleic acid refers to Watson-Crick base pairing between nucleotides and specifically refers to nucleotides that are 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.
- a nucleic acid includes a nucleotide sequence described as having a "percent complementarity" or“percent homology” to a specified second nucleotide sequence.
- 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.
- 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'.
- 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.
- donor DNA or "donor nucleic acid” refers to nucleic acid that is designed to introduce a DNA sequence modification (insertion, deletion, substitution) into a locus by homologous recombination using nucleic acid- guided nucleases.
- the donor DNA For homology-directed repair, the donor DNA must have sufficient homology to the regions flanking the“cut site” or site to be edited in the genomic target sequence. The length of the homology arm(s) will depend on, e.g., the type and size of the modification being made.
- the donor DNA will have at least one region of sequence homology (e.g., one homology arm) to the genomic target locus.
- the donor DNA will have two regions of sequence homology (e.g., two homology arms) to the genomic target locus.
- an "insert" region or “DNA sequence modification” region the nucleic acid modification that one desires to be introduced into a genome target locus in a cell— will be located between two regions of homology.
- the DNA sequence modification may change one or more bases of the target genomic DNA sequence at one specific site or multiple specific sites. A change may include changing 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 or more base pairs of the target sequence.
- a deletion or insertion may be a deletion or insertion of 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 or more base pairs of the target sequence.
- the donor DNA optionally further includes an alteration to the target sequence, e.g., a PAM mutation that prevents binding of the nuclease at the PAM or spacer in the target sequence after editing has taken place.
- enrichment refers to enriching for edited cells by isolation or substantial isolation of cells, initial growth of cells into cell colonies (e.g., incubation), editing (optionally induced, particularly in bacterial systems), and growing the cell colonies into terminal-sized colonies (e.g., saturation or normalization of colony growth).
- 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.
- homologous region or“homology arm” refers to a region on the donor DNA with a certain degree of homology with the 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“isolation” or“isolate” mean to separate individual cells so that each cell (and the colonies formed from each cell) will be separate from other cells; for example, a single cell in a single microwell, or 100 single cells each in its own microwell.
- “Isolation” or“isolated cells” result in one embodiment, from a Poisson distribution in arraying cells.
- the terms“substantially isolated”,“largely isolated”, and “substantial isolation” mean cells are largely separated from one another, in small groups or batches. That is, when 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or up to 50— but preferably 10 or less cells— are delivered to a microwell.
- “Substantially isolated” or“largely isolated” result, in one embodiment, from a“substantial Poisson distribution” in arraying cells.
- cells be isolated via a Poisson distribution.
- operably linked refers to an arrangement of elements where the components so described are configured so as to perform their usual function.
- 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.
- 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.
- 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. In the methods described herein optionally the promoters driving transcription of the gRNAs is inducible.
- 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.
- Drug selectable markers such as ampilcillin/carbenicillin, kanamycin, chloramphenicol, erythromycin, tetracycline, gentamicin, bleomycin, streptomycin, puromycin, hygromycin, blasticidin, and G418 may be employed.
- selectable markers include, but are not limited to human nerve growth factor receptor (detected with a MAb, such as described in U.S. Pat. No.
- target genomic DNA sequence refers to any locus in vitro or in vivo, or in a nucleic acid (e.g., genome) of a cell or population of cells, in which a change of at least one nucleotide is desired using a nucleic acid-guided nuclease editing system.
- the target sequence can be a genomic locus or extrachromosomal locus.
- 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, YACs, BACs, mammalian synthetic chromosomes, and the like.
- the phrase“engine vector” comprises a coding sequence for a nuclease— optionally under the control of an inducible promoter— to be used in the nucleic acid-guided nuclease systems and methods of the present disclosure.
- the engine vector may also comprise, in a bacterial system, the l Red recombineering system or an equivalent thereto, as well as a selectable marker.
- the phrase“editing vector” comprises a donor nucleic acid, including an alteration to the target sequence which prevents nuclease binding at a PAM or spacer in the target sequence after editing has taken place, and a coding sequence for a gRNA optionally under the control of an inducible promoter (and preferably under the control of an inducible promoter in bacterial systems).
- the editing vector may also comprise a selectable marker and/or a barcode.
- the engine vector and editing vector may be combined; that is, the contents of the engine vector may be found on the editing vector.
- the present disclosure provides instruments, modules and methods for nucleic acid-guided nuclease genome editing that provide 1) enhanced observed editing efficiency of nucleic acid-guided nuclease editing methods, and 2) improvement in enriching for cells whose genomes have been properly edited, including high-throughput screening techniques.
- methods that take advantage of isolation (separating cells and growing them into clonal colonies) and normalization. Isolation or substantial isolation, incubation, followed by editing (optionally with a gRNA under the control of an inducible promoter) and normalization overcomes growth bias from unedited cells.
- the instruments, modules, and methods may be applied to all cell types including, archaeal, prokaryotic, and eukaryotic (e.g., yeast, fungal, plant and animal) cells.
- eukaryotic e.g., yeast, fungal, plant and animal
- Adherent cells may be grown on beads that are isolated in the bulk gel.
- Cell culture beads or scaffolds appropriate for this purpose typically have a diameter of 100-300 pm and have a density slightly greater than that of the culture medium (here, the liquefied 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.
- the culture medium here, the liquefied culture medium thus facilitating an easy separation of cells and medium for, e.g., medium exchange
- 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.
- Cytodex 1 (dextran-based, GE Healthcare), DE-52 (cellulose- based, Sigma-Aldrich Labware), DE-53 (cellulose-based, Sigma-Aldrich Labware), HLX 11-170 (poly styrene -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 HyQspheres P 102-4 (Thermo Scientific); or macroporous carriers based on gelatin (Cultisphere, Percell Biolytica) or cellulose (Cytopore, GE Healthcare).
- Cytodex 1 de-based, GE Healthcare
- DE-52 cellulose- based, Sigma-Aldrich Labware
- DE-53 cellulose-based, Sigma-Aldrich Labware
- HLX 11-170 poly styren
- the instruments, modules, and methods described herein employ editing cassettes comprising a guide RNA (gRNA) sequence covalently linked to a donor DNA sequence where, particularly in bacterial systems, the gRNA optionally is under the control of an inducible promoter (e.g., the editing cassettes are CREATE cassettes; see USPNs 9/982,278, issued 29 May 2019 and 10/240,167, issued 26 March 2019; 10/266,849, issued 23 April 2019; and US Pub. Nos. 15/948,785, filed 09 April 2018; 16/275,439, filed 14 February 2019: and 16/275,465, filed 14 February 2019, all of which are incorporated by reference in their entirety).
- the editing cassettes are CREATE cassettes; see USPNs 9/982,278, issued 29 May 2019 and 10/240,167, issued 26 March 2019; 10/266,849, issued 23 April 2019; and US Pub. Nos. 15/948,785, filed 09 April 2018; 16/275,439, filed 14 February 2019: and 16/275
- the disclosed methods allow' for cells to be transformed, substantially isolated, grown for several doublings (e.g., incubation), after which editing is allowed.
- the isolation process effectively negates the effect of unedited cells taking over the cell population.
- the combination of substantially isolating cells, then allowing for initial growth followed by optionally inducing transcription of the gRNA (and optionally the nuclease) and normalization of cell colonies leads to 2-250X, 10-225x, 25-200x, 40-175x, 50-!50x, 60-100x, or 50-100x gains in identifying edited cells over prior art methods and allows for generation of arrayed or pooled edited cells comprising cell libraries with edited genomes. Additionally, the methods may be leveraged to create iterative editing systems to generate combinatorial libraries of cells with two to many edits in each cellular genome.
- nucleic acid-guided nucleases e.g., RNA-guided nucleases
- a nucleic acid- guided nuclease complexed with an appropriate synthetic guide nucleic acid in a cell can cut the genome of the cell at a desired location.
- the guide nucleic acid helps the nucleic acid-guided nuclease recognize and cut the DNA at a specific target sequence.
- the nucleic acid-guided nuclease may be programmed to target any DNA sequence for cleavage as long as an appropriate protospacer adjacent motif (PAM) is nearby.
- the nucleic acid-guided nuclease editing 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).
- the guide nucleic acid may be a single guide nucleic acid that includes both the crRNA and tracrRNA sequences or a single guide nucleic acid that does not require a tracrRNA.
- a guide nucleic acid e.g., gRNA
- a guide nucleic acid complexes with a compatible nucleic acid-guided nuclease and can then hybridize with a target sequence, thereby directing the 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.
- a guide nucleic acid may comprise modified or non-naturally occurring nucleotides.
- the gRNA is encoded by a DNA sequence on a polynucleotide molecule such as a plasmid, linear construct, or resides within an editing cassette and is optionally— particularly in bacterial systems— under the control of an inducible promoter.
- 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 nucleic acid-guided 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.
- a guide sequence (the portion of the guide nucleic acid that hybridizes with the target 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.
- the guide nucleic acid is provided as a sequence to be expressed from a plasmid or vector and comprises both the guide sequence and the scaffold sequence as a single transcript.
- the guide nucleic acids may be transcribed from two separate sequences.
- the guide nucleic acid can be engineered to target a desired target DNA sequence by altering the guide sequence so that the guide sequence is complementary to the target DNA sequence, thereby allowing hybridization between the guide sequence and the target DNA sequence.
- the gRNA/nuclease complex binds to a target sequence as determined by the guide RNA, and the nuclease recognizes a protospacer adjacent motif (PAM) sequence adjacent to the target sequence.
- the target sequence can be any polynucleotide (either DNA or RNA) endogenous or exogenous to a prokaryotic or eukaryotic cell, or in vitro.
- the target sequence can be a polynucleotide residing in the nucleus of a eukaryotic cell.
- a target sequence can be a sequence encoding a gene product (e.g., a protein) and/or a non-coding sequence (e.g., a regulatory polynucleotide, an intron, a PAM, or“junk” DNA).
- a gene product e.g., a protein
- a non-coding sequence e.g., a regulatory polynucleotide, an intron, a PAM, or“junk” DNA.
- the guide nucleic acid may be part of an editing cassette that encodes the donor nucleic acid; that is, the editing cassette may be a CREATE cassette (see, e.g USPNs 9/982,278, issued 29 May 2019 and 10/240,167, issued 26 March 2019; 10/266,849, issued 23 April 2019; and US Pub. Nos. 15/948,785, filed 09 April 2018; 16/275,439, filed 14 February 2019; and 16/275,465, filed 14 February 2019, all of which are incorporated by reference in their entirety).
- the guide nucleic acid and the donor nucleic acid may be and typically are under the control of a single (optionally inducible) promoter.
- the guide nucleic acid may not be part of the editing cassette and instead may be encoded on the engine or editing vector backbone.
- a sequence coding for a guide nucleic acid can be assembled or inserted into a vector backbone first, followed by insertion of the donor nucleic acid.
- the donor nucleic acid can be inserted or assembled into a vector backbone first, followed by insertion of the sequence coding for the guide nucleic acid.
- the sequence encoding the guide nucleic acid and the donor nucleic acid are simultaneously but separately inserted or assembled into a vector.
- the sequence encoding the guide nucleic acid and the sequence encoding the donor nucleic acid are both included in the editing cassette.
- the target sequence is associated with a PAM, which is a short nucleotide sequence recognized by the gRNA/nuclease complex.
- PAM is a short nucleotide sequence recognized by the gRNA/nuclease complex.
- the precise 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 nuclease, can be 5' or 3' to the target sequence.
- Engineering of the PAM-interacting domain of a nucleic acid-guided nuclease may allow for alteration of PAM specificity, improve target site recognition fidelity, decrease target site recognition fidelity, and increase the versatility of a nucleic acid-guided nuclease.
- the genome editing of a target sequence both introduces a desired DNA change to a target sequence, e.g., the genomic DNA of a cell, and removes, mutates, or renders inactive a proto-spacer (PAM) region in the target sequence; that is, the donor DNA often includes an alteration to the target sequence that prevents binding of the nuclease at the PAM in the target sequence after editing has taken place. Rendering the PAM at the target sequence inactive precludes additional editing of the cell genome at that target sequence, e.g., upon subsequent exposure to a nucleic acid- guided nuclease complexed with a synthetic guide nucleic acid in later rounds of editing.
- a desired DNA change e.g., the genomic DNA of a cell
- PAM proto-spacer
- cells having the desired target sequence edit and an altered PAM can be selected using a nucleic acid-guided nuclease complexed with a synthetic guide nucleic acid complementary to the target sequence.
- Cells that did not undergo the first editing event will be cut rendering a double-stranded DNA break, and thus will not continue to be viable.
- the cells containing the desired target sequence edit and PAM alteration will not be cut, as these edited cells no longer contain the necessary PAM site and will continue to grow and propagate.
- nucleic acid-guided nucleases can recognize some PAMs very well (e.g., canonical PAMs), and other PAMs less well or poorly (e.g., non-canonical PAMs). Because the methods disclosed herein allow for identification of edited cells in a large background of unedited cells, the methods allow for identification of edited cells where the PAM is less than optimal; that is, the methods for identifying edited cells herein allow for identification of edited cells even if editing efficiency is very low. Additionally, the present methods expand the scope of target sequences that may be edited since edits are more readily identified, including cells where the genome edits are associated with less functional PAMs.
- the polynucleotide sequence encoding the nucleic acid-guided nuclease can be codon optimized for expression in particular cells, such as archaeal, prokaryotic or eukaryotic cells.
- Eukaryotic cells can be yeast, fungi, algae, plant, animal, or human cells.
- Eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human mammal including non-human primate.
- nucleic acid-guided nuclease The choice of nucleic acid-guided nuclease to be employed depends on many factors, such as what type of edit is to be made in the target sequence and whether an appropriate PAM is located close to the desired target sequence. Nucleases of use in the methods described herein include but are not limited to Cas 9, Cas l2/CpfI, MAD2, or MAD7 or other MADzymes.
- the nuclease may be encoded by a DNA sequence on a vector (e.g., the engine vector) and be under the control of a constitutive or an inducible promoter. Again, at least one of and preferably both of the nuclease and guide nucleic acid are under the control of an inducible promoter.
- the donor nucleic acid is another component of the nucleic acid-guided nuclease system.
- the donor nucleic acid is on the same polynucleotide (e.g., vector or editing (CREATE) cassette) as the guide nucleic acid.
- the donor nucleic acid is designed to serve as a template for homologous recombination with a target sequence nicked or cleaved by the nucleic acid-guided nuclease as a part of the gRNA/nuclease complex.
- a donor nucleic acid polynucleotide may be of any suitable length, such as about or more than about 30, 35, 40, 45, 50, 75, 100, 150, 200, 500, 1000, 2500, 5000 nucleotides or more in length.
- the donor nucleic acid can be provided as an oligonucleotide of between 40-300 nucleotides, more preferably between 50-250 nucleotides.
- the donor nucleic acid comprises a region that is complementary to a portion of the target sequence (e.g., a homology arm).
- the donor nucleic acid overlaps with (is complementary to) the target sequence by, e.g., about 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or more nucleotides.
- the donor nucleic acid comprises two homology arms (regions complementary to the target sequence) flanking the mutation or difference between the donor nucleic acid and the target template.
- the donor nucleic acid comprises at least one mutation or alteration compared to the target sequence, such as an insertion, deletion, modification, or any combination thereof compared to the target sequence.
- the donor nucleic acid is provided as an editing cassette, which is inserted into a vector backbone where the vector backbone may comprise a promoter driving transcription of the gRNA and the donor nucleic acid.
- the vector backbone may comprise a promoter driving transcription of the gRNA and the donor nucleic acid.
- the promoter driving transcription of the gRNA and the donor nucleic acid is optionally an inducible promoter (and in bacterial systems is preferably an inducible promoter) and the promoter driving transcription of the nuclease is optionally an inducible promoter as well.
- Inducible editing is advantageous in that substantially or largely isolated cells can be grown for several to many cell doublings before editing is initiated, which increases the likelihood that cells with edits will survive, as the double- strand cuts caused by active editing are largely toxic to the cells. This toxicity results both in cell death in the edited colonies, as well as a lag in growth for the edited cells that do survive but must repair and recover following editing. However, once the edited cells have a chance to recover, the size of the colonies of the edited cells will eventually catch up to the size of the colonies of unedited cells (e.g., the process of“normalization” or growing colonies to“terminal size”; see, e.g., FIG. 1B described infra).
- an editing cassette may comprise one or more primer sites.
- the primer sites can be used to amplify the editing cassette by using oligonucleotide primers; for example, if the primer sites flank one or more of the other components of the editing cassette.
- the donor nucleic acid may comprise— in addition to the at least one mutation relative to a target sequence— one or more PAM sequence alterations that mutate, delete or render inactive the PAM site in the target sequence.
- the PAM sequence alteration in the target sequence renders the PAM site“immune” to the nucleic acid-guided nuclease and protects the target sequence from further editing in subsequent rounds of editing if the same nuclease is used.
- the editing cassette also may comprise a barcode.
- a barcode is a unique DNA sequence that corresponds to the donor DNA sequence such that the barcode can identify the edit made to the corresponding target sequence.
- the barcode can comprise greater than four nucleotides.
- the editing cassettes comprise a collection of donor nucleic acids representing, e.g., gene-wide or genome-wide libraries of donor nucleic acids.
- the library of editing cassettes is cloned into vector backbones where, e.g., each different donor nucleic acid design is associated with a different barcode, or, alternatively, each different cassette molecule is associate with a different barcode.
- an expression vector or cassette encoding components of the nucleic acid-guided nuclease system further encodes a nucleic acid- guided 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.
- NLSs nuclear localization sequences
- the engineered nuclease comprises NLSs at or near the amino-terminus, NLSs at or near the carboxy-terminus, or a combination.
- the engine and editing vectors comprise control sequences operably linked to the component sequences to be transcribed.
- the promoters driving transcription of one or more components of the nucleic acid-guided nuclease editing system may be inducible.
- a number of gene regulation control systems have been developed for the controlled expression of genes in plant, microbe, and animal cells, including mammalian cells, including the pL promoter (induced by heat inactivation of the CI857 repressor), the pBAD promoter (induced by the addition of arabinose to the cell growth medium), and the rhamnose inducible promoter (induced by the addition of rhamnose to the cell growth medium).
- At least one of the nucleic acid-guided nuclease editing components is under the control of a promoter that is activated by a rise in temperature, as such a promoter allows for the promoter to be activated by an increase in temperature, and de-activated by a decrease in temperature, thereby“turning off’ the editing process.
- editing in the cell can be turned off without having to change media or addition of other genetic factors in, e.g., an additional genetic element on an existing plasmid, an another plasmid, or integrated into the genome; to remove, e.g., an inducing agent in the medium that is used to induce editing.
- Figure 1A shows simplified flow charts for two alternative exemplary methods lOOa and lOOb for isolating or substantially isolating cells and normalizing cell colony size where method lOOa does not employ induction of the editing machinery and method lOOb does employ an inducible promoter driving transcription of the gRNA.
- method lOOa begins by transforming cells 110 with the components necessary to perform nucleic acid-guided nuclease editing.
- the cells may be transformed simultaneously with separate engine and editing vectors; the cells may already be expressing the nuclease (e.g., the cells may have already been transformed with an engine vector or the coding sequence for the nuclease may be stably integrated into the cellular genome) such that only the editing vector needs to be transformed into the cells; or the cells may be transformed with a single vector comprising all components required to perform nucleic acid-guided nuclease genome editing.
- the cells may be transformed simultaneously with separate engine and editing vectors; the cells may already be expressing the nuclease (e.g., the cells may have already been transformed with an engine vector or the coding sequence for the nuclease may be stably integrated into the cellular genome) such that only the editing vector needs to be transformed into the cells; or the cells may be transformed with a single vector comprising all components required to perform nucleic acid-guided nuclease genome editing.
- a variety of delivery systems can be used to introduce (e.g., transform or transfect) nucleic acid-guided nuclease editing system components into a host cell 110.
- These delivery systems include the use of yeast systems, lipofection systems, microinjection systems, biolistic systems, virosomes, liposomes, immunoliposomes, polycations, lipid ucleic acid conjugates, virions, artificial virions, viral vectors, electroporation, cell permeable peptides, nanoparticles, nanowires, exosomes.
- molecular trojan horse liposomes may be used to deliver nucleic acid- guided nuclease components across the blood brain barrier.
- a multi-module cell editing instrument 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., USSNs 16/147,120, filed 28 September 2018; 16/147,353, filed 28 September 2018; 16/147,865, filed 30 September 2018; and 16/426,310, filed 30 May 2019; and USPN 10,323,258, issued 18 June 2019.
- the isolation/growth/editing/normalization module is one module in an automated multi-module cell editing instrument, the cells are likely transformed in an automated cell transformation module.
- the cells are isolated or substantially isolated 120; that is, the cells are diluted (if necessary) in a liquid culture medium so that cells are sequestered or separated from one another in the liquid. Isolation can then be performed by solidifying or“gelling” the liquid medium in which the cells are separated from one another in a three-dimensional gel; that is, e.g., the cells are suspended in liquid, then the liquid is solidified into a gel thereby fixing the isolated or singulated cells in three- dimensional space.
- the cells are actively editing, as the editing machinery is under the control of a constitutive promoter.
- the cells are editing, they are grown into colonies of terminal size 130; that is, the colonies arising from the isolated cells are grown into colonies to a point where cell growth has peaked and is normalized or saturated for both edited and unedited cells. Normalization occurs as the nutrients in the medium around a growing cell colony are depleted.
- the editing components are under the control of a constitutive promoter; thus, editing begins immediately (or almost immediately) upon transformation.
- At least the guide nucleic acid may be under the control of an inducible promoter, in which case editing may be induced after, e.g., a desired number of cell doublings.
- the terminal-size colonies are pooled 140 by liquefying the solidified medium and, e.g., vortexing the liquid to mix the cells from the normalized colonies.
- isolation/normalization alone enriches the total population of cells with cells that have been edited; that is, isolation combined with normalization (e.g., growing colonies to terminal size) allows for high-throughput enrichment of edited cells.
- the method lOOb shown in Figure 1A is similar to the method lOOa in that cells of interest are transformed 110 with the components necessary to perform nucleic acid- guided nuclease editing.
- the cells may be transformed simultaneously with both the engine and editing vectors, the cells may already be expressing the nuclease (e.g., the cells may have already been transformed with an engine vector or the coding sequence for the nuclease may be stably integrated into the cellular genome) such that only the editing vector needs to be transformed into the cells, or the cells may be transformed with a single vector comprising all components required to perform nucleic acid-guided nuclease genome editing.
- the isolation/growth/editing/normalization module is one module in an automated multi module cell editing instrument, cell transformation may be performed in an automated transformation module utilizing a flow-through electroporation module as described in relation to Figures 3A-3D below.
- the cells are isolated 120; that is, the cells are diluted (if necessary) in liquid medium so that cells are separated from one another. Once the cells are diluted properly in the liquid medium, the liquid medium is solidified or“gelled” fixing and sequestering the diluted cells in three-dimensional space.
- the cells are allowed to grow to, e.g., between 2 and 50, or between 5 and 40, or between 10 and 30 doublings, establishing clonal colonies 150.
- lOOb editing is induced 160 by, e.g., activating inducible promoters that control transcription of one or more of the components needed for nucleic acid-guided nuclease editing, such as, e.g., transcription of the gRNA, donor DNA, nuclease, or, in the case of bacteria, a recombineering system.
- Figure 1B is a plot of OD versus time for unedited cells (solid line) versus edited cells (dashed line). Note that the OD (e.g., growth) of the edited cells lags behind the unedited cells initially, but eventually catches up due, e.g., to unedited cells exhausting the nutrients in the medium and exiting log-phase growth. The colonies are allowed to grow long enough for the growth of the edited colonies to catch up with (approximate the size of, e.g., number of cells in) the unedited colonies.
- the methods described herein provide enhanced observed editing efficiency of nucleic acid-guided nuclease editing methods as the result of isolation/growth/editing/normalization.
- the combination of the isolation and normalization processes overcomes the growth bias in favor of unedited cells— and the fitness effects of editing (including differential editing rates)— thus allowing all cells “equal billing” with one another.
- the result of the methods described herein is that even in nucleic acid-guided nuclease systems where editing is not optimal (such as in systems where non-canonical PAMs are targeted), there is an increase in the observed editing efficiency; that is, edited cells can be identified even in a large background of unedited cells. Observed editing efficiency can be improved up to 80% or more.
- Figure 2A is an exemplary workflow 200 for optimizing the observed presence of edited cells after nucleic acid-guided nuclease genome editing that may be performed in an automated isolation/growth/editing/normalization module, and, optionally, as part of an automated multi-module cell editing instrument.
- transformed cells 204 are suspended at a pre-determined density in medium plus alginate (solidifying agent) in a vessel 202 containing, optionally, antibiotics or other selective compounds to allow only cells that have been transformed with both the engine vector and editing vector (if two vectors are used) or a combined engine/editing vector to grow.
- medium plus alginate solidifying agent
- two vectors, an engine vector and an editing vector are used in some embodiments a single vector comprising all necessary exogenous components for nucleic acid-guided nuclease editing is used.
- the medium used with depend, of course, on the type of cells being edited— e.g., bacterial, yeast or mammalian.
- medium for bacterial growth includes LB, SOC, M9 Minimal medium, and Magic medium
- medium for yeast cell growth includes TPD, YPG, YPAD, and synthetic minimal medium
- medium for mammalian cell growth includes MEM, DMEM, IMDM, RPMI, and Hanks.
- Natural polymers and proteins able to form hydrogels are alginate, chitosan, hyaluronan, dextran, collagen, and fibrin; synthetic examples of synthetic polymers and proteins able to form hydrogels include polyethylene glycol, poly(hydroxyethyl methacrylate, polyvinyl alcohol, and polycaprolactone.
- Alginate has been used as a preferred solidifying agent in the methods described herein due to a number of advantageous properties.
- Alginates are polysaccharides that consist of linear (unbranched) 1,4 linked residues of b-D-mannuronic acid and its C5-epimer a-D- guluronic acid.
- Alginates have a high affinity for alkaline earth metals and ionic hydrogels can be formed in the presence of divalent cations except Mg +2 . Chelation of the gel-forming ion occurs between two consecutive residues in the alginate chain, and an intermolecular gel network is formed as a result of a cooperative binding of consecutive residues in different alginate chains.
- ionically-gelled alginate can be dissolved by treatment with chelating agents for divalent cations such as citrate and ethylenediaminetetraacetic acid (EDTA) or hexametaphosphate.
- a 2% (w/v) alginate in medium was found to properly isolate cells; however appropriate ranges for the percentage of alginate in a growth medium include 0.25% to 6% (w/v) alginate, or 0.5% to 5% (w/v) alginate, or 1% to 4% (w/v) alginate, or 2% to 3% (w/v) alginate.
- neither of the processes of solidifying and of re-liquefying the alginate/medium impact cell viability.
- induction of editing by elevating the temperature of the bulk gel to 42°C (described in more detail below) does not impact the integrity of the solidified medium or the segregation of the isolated clonal cell colonies.
- the culture of mammalian cells using hydrogels has been performed to mimic the 3D cell environments found in tissue, allowing for more biologically-relevant cellular environments.
- tissue mimetics in the context of mammalian cell editing alginates may be chemically functionalized to alter physiochemical and biological characteristics and properties so as to better bind and promote the growth of mammalian cells once the cells have been isolated in the solidified alginate medium.
- cells do not have receptors that recognize alginate, proliferation and differentiation of some mammalian cells within an alginate hydrogen require signaling molecules and matrix interaction.
- cell attachment peptides especially the sequence RGD (arginine-glycine-aspartic acid), have been shown to improve cellular adaptability to matricies, as is the case with alginate.
- RGD arginine-glycine-aspartic acid
- alginate can be modified by covalently grafting peptide sequence to the alginate molecule.
- mammalian cells can be grown on beads where the beads are then suspended in the alginate medium.
- the alginate in the medium is solidified 201 by, e.g., addition of CaCb (described below in relation to Example 5). Note that some areas of the solidified alginate have no cells 206 and some areas have one cell 204. Next, the cells are allowed to grow 203 for a pre-determined approximate number of doublings. Because the cells are fixed in three-dimensional space, the resulting colonies 208 are fixed in three-dimensional space.
- the colonies are grown to terminal size 207 (that is, edited and non-edited cell colony growth is normalized), sodium citrate is added 209 to the medium such that the solidified medium/alginate re liquifies and the cells from the colonies 214, 216 (comprising to edited and unedited cells, respectively) are suspended in liquid medium once again. Once the medium is re liquified, the cells are recovered and subjected to analysis 211 or are used in a second round of editing 213.
- the combination of the processes of isolation and normalization overcomes growth bias from unedited cells or cells exhibiting fitness effects as the result of edits made
- the combination of the processes of isolation and normalization alone enriches the total population of cells with cells that have been edited; that is, isolation and normalization (e.g., growing colonies to terminal size) allows for high-throughput enrichment of edited cells.
- Figure 2B depicts a simplified graphic for a workflow 250 for isolating, editing and normalizing cells after nucleic acid-guided nuclease genome editing in bulk cell culture, where reversible solidification of the bulk culture is utilized, and editing is induced by inducing transcription of gRNA.
- First cells 204 are suspended in a vessel at an appropriate density, then at step 201 the alginate in the medium is solidified by, e.g., addition of CaCb (described below in relation to Example 5). Note that some areas of the solidified alginate have no cells 206 and some areas have one cell 204. Next, the cells are allowed to grow 203 for a pre-determined approximate number of doublings.
- the resulting colonies 208 are fixed in three-dimensional space. Editing is then induced 205 by inducing transcription of the gRNA. Once editing is induced, a number of cells in the edited colonies 212 die due to the toxicity of the double-stranded breaks as the result of editing. Growth of cells in colonies that have not been edited 210 are not affected by double-stranded breaks and continue to thrive.
- the colonies of cells— both edited and unedited— are grown to terminal size (that is, edited 212 and non-edited 210 cell colony growth is normalized), then sodium citrate is added 209 to the medium such that the solidified medium/alginate re-liquifies and the cells from the colonies 214, 216 (comprising to edited and unedited cells, respectively) are suspended in liquid medium once again. Once the medium is re-liquified, the cells are recovered and subjected to analysis 211 or are used in a second round of editing 213.
- the combination of the processes of isolation and normalization overcomes growth bias from unedited cells or cells exhibiting fitness effects as the result of edits made
- the combination of the processes of isolation and normalization alone enriches the total population of cells with cells that have been edited; that is, isolation and normalization (e.g., growing colonies to terminal size) allows for high-throughput enrichment of edited cells.
- Figure 2C is a photograph of E. coli cells expressing green fluorescent protein in 2.0% alginate and medium that has been solidified showing isolated colonies (left) and a photograph of E. coli cells expressing green fluorescent protein in 2.0% alginate and medium after the medium has been re-liquified.
- Figure 3A depicts an exemplary automated multi-module cell processing instrument 300 comprising a bulk cell culture isolation/growth/editing/normalization module 340 to, e.g., perform the exemplary workflows described above in relation to Figures 2A and 2B, as well as additional exemplary modules. Illustrated is a gantry 302, providing an automated mechanical motion system (actuator) (not shown) that supplies XYZ axis motion control to, e.g., modules of the automated multi-module cell processing instrument 300, including, e.g., an air displacement pipette 332.
- actuator automated mechanical motion system
- wash or reagent cartridge 304 comprising reservoirs 306.
- wash or reagent cartridge 304 may be configured to accommodate large tubes, for example, wash solutions, or solutions that are used often throughout an iterative process.
- wash or reagent cartridge 304 may be configured to remain in place when two or more reagent cartridges 310 are sequentially used and replaced.
- reagent cartridge 310 and wash or reagent cartridge 304 are shown in Figure 3A as separate cartridges, the contents of wash cartridge 304 may be incorporated into reagent cartridge 310.
- the exemplary automated multi-module cell processing instrument 300 of Figure 3A further comprises a cell growth module 334.
- the cell growth module 334 comprises two rotating growth vials (RGVs) 318, 320 (described in detail below with relation to Figure 3E) as well as a cell concentration module 322.
- RGVs rotating growth vials
- cell concentration processes for, e.g., medium exchange and cell concentration may be used to prepare (e.g., concentrate and render electrocompetent) the edited cells for another transformation for another round of editing.
- the cell concentration module 322 is part of the cell growth module 334; however, in some embodiments the cell concentration module 322 may be separate from cell growth module 334, e.g., in a separate, dedicated module.
- isolation/growth/editing/normalization module 340 separate from growth module 334, where the module 340 is served by, e.g., air displacement pipettor 332.
- the isolation/growth/editing/normalization module implementing the workflows described in Figures 2A and 2B and illustrated in Figure 3A may employ “off the shelf’ liquid handling instrumentation such as that sold by Opentrons (OT-2TM system, Brooklyn, NY); ThermoFisher Scientific (VersetteTM system, Carlsbad, CA); Labcyte (AccessTM system, San Jose, CA); Perkin Elmer (JanusTM system, San Jose, CA); Agilent Inc.
- FIG. 3A Also seen in Figure 3A is a waste repository 326, and a nucleic acid assembly/desalting module 314 comprising a reaction chamber or tube receptacle (not shown) and further comprising a magnet 316 to allow for purification of nucleic acids using, e.g., magnetic solid phase reversible immobilization (SPRI) beads (Applied Biological Materials Inc., Richmond, BC).
- SPRI magnetic solid phase reversible immobilization
- Figure 3B depicts an exemplary combination reagent cartridge and electroporation device 310 (“cartridge”) that may be used in an automated multi-module cell processing instrument along with the isolation/growth/editing/normalization module.
- the material used to fabricate the cartridge is thermally- conductive, as in certain embodiments the cartridge 310 contacts a thermal device (not shown), such as a Peltier device or thermoelectric cooler, that heats or cools reagents in the reagent receptacles or reservoirs 312.
- a thermal device such as a Peltier device or thermoelectric cooler
- Reagent receptacles or reservoirs 312 may be receptacles into which individual tubes of reagents are inserted as shown in Figure 3B, or the reagent receptacles may hold the reagents without inserted tubes. Additionally, the receptacles in a reagent cartridge may be configured for any combination of tubes, co joined tubes, and direct-fill of reagents.
- the reagent receptacles or reservoirs 312 of reagent cartridge 310 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.
- all receptacles 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 (not shown).
- the reagent reservoirs hold reagents without inserted tubes.
- 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.
- the reagents contained in the reagent cartridge will vary depending on workflow; that is, the reagents will vary depending on the processes to which the cells are subjected in the automated multi module cell processing instrument.
- Reagents such as cell samples, medium, CaCb, Na 3 C 6 H 5 0 7 (sodium citrate), enzymes, buffers, nucleic acid vectors, expression cassettes, proteins or peptides, reaction components (such as, e.g., MgCb, 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.
- the cartridge comprises a script (not shown) readable by a processor (not shown) for dispensing the reagents.
- the cartridge 310 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.
- the reagent cartridge is disposable and is pre-packaged with reagents tailored to performing specific cell processing protocols, e.g., 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 may match the reagents used and cell processes performed.
- reagent cartridges may be pre-packaged with reagents for genome editing and a script that specifies the process steps for performing single or recursive genome editing in an automated multi-module cell processing instrument.
- the reagent cartridge may comprise a script to pipette competent cells from a reservoir, transfer the cells to a transformation module (such as flow through electroporation device 330 in reagent cartridge 310), 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 isolation, editing, and growth module in the automated multi-module cell processing instrument.
- a transformation module such as flow through electroporation device 330 in reagent cartridge 310
- 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 isolation, editing, and growth module in the automated
- 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 editing oligonucleotide cassettes in a reservoir in the reagent cassette, and a nucleic acid assembly mix from another reservoir to the nucleic acid assembly /desalting module (314 of Figure 3A).
- the script may also specify process steps performed by other modules in the automated multi-module cell processing instrument.
- 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.
- the exemplary reagent cartridges 310 for use in the automated multi-module cell processing instruments may include one or more electroporation devices 330, preferably flow-through electroporation devices. Electroporation is a widely -used method for permeabilization of cell membranes that works by temporarily generating pores in the cell membranes with electrical stimulation. Applications of electroporation include the delivery of DNA, RNA, siRNA, peptides, proteins, antibodies, drugs or other substances to a variety of cells such as mammalian cells (including human cells), plant cells, archea, yeasts, other eukaryotic cells, bacteria, and other cell types.
- electroporation is a widely -used method for permeabilization of cell membranes that works by temporarily generating pores in the cell membranes with electrical stimulation.
- Applications of electroporation include the delivery of DNA, RNA, siRNA, peptides, proteins, antibodies, drugs or other substances to a variety of cells such as mammalian cells (including human cells), plant cells, archea, yeasts
- Electrical stimulation may also be used for cell fusion in the production of hybridomas or other fused cells.
- cells are suspended in a buffer or medium that is favorable for cell survival.
- low conductance mediums such as water, glycerol solutions and the like, are often used to reduce the heat production by transient high current.
- the cells and material to be electroporated into the cells are placed in a cuvette embedded with two flat electrodes for electrical discharge.
- Bio-Rad (Hercules, Calif.) makes the GENE PULSER XCELLTM line of products to electroporate cells in cuvettes.
- the reagent cartridges of the disclosure allow for particularly easy integration with robotic liquid handling instrumentation that is typically used in automated instruments and systems such as air displacement pipettors.
- automated instrumentation includes, but is not limited to, off-the-shelf automated liquid handling systems from Tecan (Mannedorf, Switzerland), Hamilton (Reno, NV), Beckman Coulter (Fort Collins, CO), etc. as described above.
- Figures 3C and 3D are top perspective and bottom perspective views, respectively, of an exemplary flow-through electroporation device 350 that may be part of reagent cartridge 300 in Figure 3B or may be contained in a separate module (e.g., a transformation/transfection module).
- Figure 3C depicts a flow-through electroporation unit 350.
- the flow-through electroporation unit 350 has wells that define cell sample inlets 352 and cell sample outlets 354.
- Figure 3D is a bottom perspective view of the flow-through electroporation device 350 of Figure 3C. An inlet well 352 and an outlet well 354 can be seen in this view.
- flow-through electroporation devices may comprise push-pull pneumatic means to allow multi-pass electroporation procedures; that is, cells to be 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 electroporation device toward the inlet end to pass between the electrodes again for another pass of electroporation.
- Exemplary flow-through electroporation devices of use in the automated multi-module cell processing instruments disclosed herein include those described in USSNs 16/147,120, filed 28 September 2018; 16/147,353, filed 28 September 2018; 16/147,865, filed 30 September 2018; and 16/426,310, filed 30 May 2019; and USPN 10,323,258, issued 18 June 2019, all of which are herein incorporated by reference in their entirety. Further, this process may be repeated one to many times. Moreover, other embodiments of the reagent cartridge may provide or accommodate electroporation devices that are not configured as flow-through devices, such as those described in USSN 16/109,156, filed 22 August 2018.
- Exemplary automated multi-module cell processing instrument 300 of Figure 3A also comprises a nucleic acid assembly module.
- the nucleic acid assembly module 314 is configured to perform, e.g., an isothermal nucleic acid assembly.
- An isothermal nucleic acid assembly joins multiple DNA fragments in a single, isothermal reaction, requiring few components and process manipulations.
- an isothermal nucleic acid assembly can combine simultaneously up to 20 or more nucleic acid fragments based on sequence identity.
- the assembly method requires that the nucleic acids to be assembled comprise at least a 15-base overlap with adjacent nucleic acid fragments.
- the fragments are mixed with a cocktail of three enzymes— an exonuclease, a polymerase, and a ligase— along with buffer components. Because in some embodiments the process is isothermal and can be performed in a l-step or 2-step method using a single reaction vessel, the isothermal nucleic acid assembly method is suited for use in an automated multi-module cell processing instrument.
- the 1 -step method allows for the assembly of up to five different fragments using a single step isothermal process.
- the fragments and the master mix of enzymes are combined and incubated at 50°C for up to one hour.
- the 2-step method For the creation of more complex constructs or for incorporating fragments from 100 bp up to lOkb, typically the 2-step method is used, where the 2-step reaction requires two separate additions of master mix; one for the exonuclease and annealing step and a second for the polymerase and ligation steps.
- aliquots of a vector, an oligonucleotide (e.g., a gene of interest or an editing sequence) to be inserted into the vector, and the nucleic acid assembly mix may be retrieved from three of the sixteen reagent reservoirs 312 disposed within reagent cartridge 310.
- the vector, oligonucleotide, and reaction mix are combined in a reaction chamber or tube located in a tube receptacle (not shown) in the nucleic acid assembly module, and the module is heated to 50°C.
- magnetic beads may be retrieved from one of the reagent reservoirs 312 disposed within reagent cartridge 310 and added to the nucleic acid assembly mix in the reaction chamber of the nucleic acid module 314.
- magnet 316 such as a solenoid magnet, is adjacent or proximal to the nucleic acid assembly module 314.
- the reaction solution (supernatant) in the nucleic acid assembly module 314 can be removed by air displacement pipettor 332, and a wash solution and/or ethanol may be pipetted from a reagent reservoir 312 in reagent cartridge 310, or from a wash solution reservoir 306 in wash cartridge 304 and used to wash the nucleic acids coupled to the beads.
- the magnet may be disengaged while the beads and coupled nucleic acids are being washed, then the magnet would be re-engaged to remove the wash solution from the nucleic acid assembly module. Alternatively, the magnet may not be disengaged while the beads and coupled nucleic acids are washed.
- the de-salted assembled vector + oligo may then be moved to, e.g., the flow-through electroporation device (transformation/transfection module) as described in relation to Figures 3B through 3D.
- FIG. 3E is a model of tangential flow filtration used in the TFF module described below.
- the TFF device is an integral module in the automated multi-module cell processing instrument.
- the TFF is used to concentrate and render electrocompetent cells after growth in the cell growth module.
- the cells may be cells that were loaded into a rotating growth vial for a first round of editing, or the cells may be cells that have been through one round of editing, recovered from liquefied alginate medium, re-grown in a rotating growth vial and are being prepared for a second round of editing.
- the TFF device was designed to take into account two primary design considerations. First, the geometry of the TFF device leads to filtering of the cell culture over a large surface area so as to minimize processing time.
- FIG. 3E is a general model 30 of tangential flow filtration.
- the TFF device operates using tangential flow filtration, also known as cross-flow filtration.
- FIG. 3E shows cells flowing over a membrane 34, where the feed flow of the cells 32 in medium or buffer is parallel to the membrane 34.
- TFF is different from dead-end filtration where both the feed flow and the pressure drop are perpendicular to a membrane or filter.
- FIGs. 3F - 7F depict an embodiment of a tangential flow filtration (TFF) device/module.
- FIG. 3F depicts a configuration of an retentate member 3022 (on left), a membrane or filter 3024 (middle), and a permeate member 3020 (on the right).
- retentate member 3022 comprises a tangential flow channel 3002, which has a serpentine configuration that initiates at one lower corner of retentate member 3022— specifically at retentate port 3028— traverses across and up then down and across retentate member 3022, ending in the other lower corner of retentate member 3022 at a second retentate port 3028.
- energy director 3091 which circumscribes the region where membrane or filter 3024 is seated.
- Energy director 3091 in this embodiment mates with and serves to facilitate ultrasonic wending or bonding of retentate member 3022 with permeate member 3020 via the energy director component on permeate member 3020.
- Membrane or filter 3024 has through-holes for retentate ports 3028 and is configured to seat within the circumference of energy directors 3091 between the retentate member 3022 and permeate member 3020.
- Permeate member 3020 comprises, in addition to energy director 3091, through-holes for retentate port 3028 at each bottom corner (which mate with the through-holes for retentate ports 3028 at the bottom corners of membrane 3024 and retentate ports 3028 in retentate member 3022), as well as a tangential flow channel 3002 and a single permeate port 3026 positioned at the top and center of permeate member 3020.
- the tangential flow channel 3002 structure in this embodiment has a serpentine configuration and an undulating geometry, although other geometries may be used. In some aspects, the length of the tangential flow channel is from 10 mm to 1000 mm, from 60 mm to 200 mm, or from 80 mm to 100 mm.
- the width of the channel structure is from 10 mm to 120 mm, from 40 mm to 70 mm, or from 50 mm to 60 mm.
- the cross section of the tangential flow channel 1202 is rectangular. In some aspects, the cross section of the tangential flow channel 1202 is 5 pm to 1000 pm wide and 5 pm to 1000 pm high, 300 pm to 700 pm wide and 300 pm to 700 pm high, or 400 pm to 600 pm wide and 400 pm to 600 pm high. In other aspects, the cross section of the tangential flow channel 1202 is circular, elliptical, trapezoidal, or oblong, and is 100 pm to 1000 pm in hydraulic radius, 300 pm to 700 pm in hydraulic radius, or 400 pm to 600 pm in hydraulic radius.
- FIG. 3G is a side perspective view of a reservoir assembly 3050.
- Reservoir assembly 3050 comprises retentate reservoirs 3052 on either side of a single permeate reservoir 3054.
- Retentate reservoirs 3052 are used to contain the cells and medium as the cells are transferred through the TFF device or module and into the retentate reservoirs during cell concentration.
- Permeate reservoir 3054 is used to collect the filtrate fluids removed from the cell culture during cell concentration, or old buffer or medium during cell growth.
- buffer or medium is supplied to the permeate member from a reagent reservoir separate from the device module. Additionally seen in FIG.
- 3G are grooves 3032 to accommodate pneumatic ports (not seen), permeate port 3026, and retentate port through-holes 3028.
- the retentate reservoirs are fluidically coupled to the retentate ports 3028, which in turn are fluidically coupled to the portion of the tangential flow channel disposed in the retentate member (not shown).
- the permeate reservoir is fluidically coupled to the permeate port 3026 which in turn are fluidically coupled to the portion of the tangential flow channel disposed in permeate member (not shown), where the portions of the tangential flow channels are bifurcated by membrane (not shown).
- up to 120 mL of cell culture can be grown and/or filtered, or up to 100 mL, 90 mL, 80 mL, 70 mL, 60 mL, 50 mL, 40 mL, 30 mL or 20 mL of cell culture can be grown and/or concentrated.
- FIG. 3H depicts a top-down view of the reservoir assembly 3050 shown in FIG. 3G
- FIG. 31 depicts a cover 3044 for reservoir assembly 3050 shown in FIG. 3G
- 3J depicts a gasket 3045 that in operation is disposed on cover 3044 of reservoir assembly 3050 shown in FIG. 3G.
- FIG. 3H is a top-down view of reservoir assembly 3050, showing two retentate reservoirs 3052, one on either side of permeate reservoir 3054.
- FIG. 31 depicts a cover 3044 that is configured to be disposed upon the top of reservoir assembly 3050. Cover 3044 has round cut-outs at the top of retentate reservoirs 3052 and permeate reservoir 3054.
- FIG. 3J depicts a gasket 3045 that is configured to be disposed upon the cover 3044 of reservoir assembly 3050. Seen are three fluid transfer ports 3042 for each retentate reservoir 3052 and for permeate reservoir 3054. Again, three pneumatic ports 3030, for each retentate reservoir 3052 and for permeate reservoir 3054, are shown.
- FIG. 3K depicts an exploded view of a TFF module 3000. Seen are components reservoir assembly 3050, a cover 3044 to be disposed on reservoir assembly 3050, a gasket 3045 to be disposed on cover 3044, retentate member 3022, membrane or filter 3024, and permeate member 3020. Also seen is permeate port 3026, which mates with permeate port 3026 on permeate reservoir 3054, as well as two retentate ports 3028, which mate with retentate ports 3028 on retentate reservoirs 3052 (where only one retentate reservoir 3052 can be seen clearly in this FIG. 3K). Also seen are through-holes for retentate ports 3028 in membrane 3024 and permeate member 3020.
- FIG 3L depicts an embodiment of assembled TFF module 3000.
- Retentate member 3022, membrane member 3024, and permeate member 3020 are coupled side-to- side with reservoir assembly 3050.
- tangential flow channel 3002 which is formed by the mating of retentate member 3022 and permeate member 3020, with membrane 3024 sandwiched between and bifurcating tangential flow channel 3002.
- FIG. 3L also shows the length, height, and width dimensions of the TFF module 3000.
- the assembled TFF device 3000 typically is from 50 to 175 mm in height, or from 75 to 150 mm in height, or from 90 to 120 mm in height; from 50 to 175 mm in length, or from 75 to 150 mm in length, or from 90 to 120 mm in length; and is from 30 to 90 mm in depth, or from 40 to 75 mm in depth, or from about 50 to 60 mm in depth.
- An exemplary TFF device is 110 mm in height, 120 mm in length, and 55 mm in depth.
- Figure 4A depicts one embodiment of a rotating growth vial (RGV) that may be used 1) to grow cells to an appropriate OD for transformation, and 2) as a vessel for the bulk cell culture procedures depicted in Figures 1A, 2 A and 2B.
- RGV rotating growth vial
- the RGV may constantly measure the optical density of a growing cell culture.
- One advantage of the cell growth module 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 or so on minutes.
- OD can be measured at specific time intervals early in the cell growth cycle, and continuously after the OD of the cell culture reaches a set point OD.
- the cell growth module is controlled by a processor, which can be programmed to measure OD constantly or at intervals as defined by a user.
- a script on, e.g., the reagent cartridge(s) may also specify the frequency for reading OD, as well as the target OD and target time.
- a user manually can set a target time at which the user desires the cell culture hit a target OD.
- the processor measures the OD of the growing cells, calculates the cell growth rate in real time, and predicts the time the target OD will be reached.
- the processor then automatically adjusts the temperature of the RGV (and the cell culture) as needed. Lower temperatures slow growth, and higher temperatures increase growth.
- the RGV 400 is a transparent container having an open end 424 for receiving liquid media and cells, a central vial region 406 that defines the primary container for growing cells, a tapered-to-constricted region 418 defining at least one light path 410, a closed end 416, and a drive engagement mechanism 412.
- the RGV has a central longitudinal axis 420 around which the vial rotates, and the light path 410 is generally perpendicular to the longitudinal axis of the vial.
- the first light path 410 is positioned in the lower constricted portion of the tapered- to-constricted region 408.
- some embodiments of the RGV 400 have a second light path 418 in the tapered region of the tapered-to-constricted region 408.
- Both light paths in this embodiment are positioned in a region of the RGV that is constantly filled with the cell culture (cells + growth media) and are not affected by the rotational speed of the RGV.
- the first light path 410 is shorter than the second light path 418 allowing for sensitive measurement of OD values when the OD values of the cell culture in the vial are at a high level (e.g., later in the cell growth process), whereas the second light path 418 allows for sensitive measurement of OD values when the OD values of the cell culture in the vial are at a lower level (e.g., earlier in the cell growth process).
- the drive engagement mechanism 412 engages with a motor (not shown) to rotate the vial.
- the RGV 400 may be reusable, or preferably, the RGV— like the reagent cartridge— is consumable.
- the RGV is consumable and is presented to a user pre-filled with growth medium, where the vial is sealed at the open end 424 with a foil seal.
- the growth module comprising the rotating growth vial depicted in Figures 4A - 4D may also be employed as the vessel for bulk cell culture, isolation, editing, and pooling (see, e.g., Figure 7).
- the rotating growth vial RGV When the rotating growth vial RGV) is used as an isolation module, cells in medium containing 0.25%-6% alginate are transferred into the rotating growth vial by, e.g., a liquid handling system, where first, the cells are at an appropriate dilution to allow each cell to be isolated or substantially isolated from other cells when the medium is gelled, and second, the cell colonies that grow from the isolated cells in the gelled or solidified medium are isolated from other cell colonies.
- solidification or gelling of the medium is triggered by slowing adding an appropriate amount of, e.g., CaCb dropwise to the RGV, preferably while the RGV is spinning at a low speed.
- the cells can be grown to colonies of terminal size (e.g., normalized) (see, e.g., FIG. 2A, where no induction of editing takes place) or the cells can be grown for, e.g., 2-50 doublings, editing is then induced by, e.g., raising the temperature of the RGV to 42°C for a period of time to induce a pL promoter driving transcription of the gRNA, then the temperature is lowered and the cells are allowed to grow to terminal size or a desired concentration of cells (see, e.g., FIG. 2B).
- terminal size e.g., normalized
- FIG. 2A where no induction of editing takes place
- editing is then induced by, e.g., raising the temperature of the RGV to 42°C for a period of time to induce a pL promoter driving transcription of the gRNA, then the temperature is lowered and the cells are allowed to grow to terminal size or a desired concentration of cells (see, e.g., FIG
- the gelled or solidified medium is liquefied by adding an appropriate amount of, e.g., sodium citrate to the solidified medium dropwise preferably while the RGV is spinning at a low speed.
- the cells and medium may then be removed from the RGV by the liquid handling system and filtered in, e.g., a filtration module such as the FTT device as described in relation to FIGs. 3F - 3L.
- FIG. 4B is a perspective view of one embodiment of a cell growth device 430.
- FIG. 4C depicts a cut-away view of the cell growth device 430 from FIG. 4B.
- the rotating growth vial 400 is seen positioned inside a main housing 436 with the extended lip 402 of the rotating growth vial 400 extending above the main housing 436.
- end housings 452, a lower housing 432 and flanges 434 are indicated in both figures.
- Flanges 434 are used to attach the cell growth device 430 to heating/cooling means or other structure (not shown).
- FIG. 4C depicts additional detail.
- upper bearing 442 and lower bearing 440 are shown positioned within main housing 636.
- Upper bearing 442 and lower bearing 440 support the vertical load of rotating growth vial 400.
- Lower housing 432 contains the drive motor 438.
- the cell growth device 430 of FIG. 4C comprises two light paths: a primary light path 444, and a secondary light path 450.
- Light path 444 corresponds to light path 410 positioned in the constricted portion of the tapered-to-constricted portion of the rotating growth vial 400
- light path 450 corresponds to light path 408 in the tapered portion of the tapered-to- constricted portion of the rotating growth vial 400.
- Light paths 410 and 408 are not shown in FIG. 4C but may be seen in FIG. 4A.
- the motor 438 engages with drive mechanism 412 and is used to rotate the rotating growth vial 400.
- motor 438 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.
- RPM revolution per minute
- other motor types such as a stepper, servo, brushed DC, and the like can be used.
- the motor 438 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 436, end housings 452 and lower housing 432 of the cell growth device 430 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 400 is envisioned in some embodiments to be reusable, but preferably is consumable, the other components of the cell growth device 430 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 430 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 430- 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.).
- a second spectrophotometer and vessel may be included in the cell growth device 430, where the second spectrophotometer is used to read a blank at designated intervals.
- FIG. 4D illustrates a cell growth device 430 as part of an assembly comprising the cell growth device 430 of FIG. 4B coupled to light source 490, detector 492, and thermal components 494.
- the rotating growth vial 400 is inserted into the cell growth device.
- Components of the light source 490 and detector 492 e.g., such as a photodiode with gain control to cover 5-log
- the lower housing 432 that houses the motor that rotates the rotating growth vial 400 is illustrated, as is one of the flanges 434 that secures the cell growth device 430 to the assembly.
- the thermal components 494 illustrated are a Peltier device or thermoelectric cooler.
- thermal control is accomplished by attachment and electrical integration of the cell growth device 430 to the thermal components 494 via the flange 434 on the base of the lower housing 432.
- Thermoelectric coolers are capable of“pumping” heat to either side of a junction, either cooling a surface or heating a surface depending on the direction of current flow.
- a thermistor is used to measure the temperature of the main housing and then, through a standard electronic proportional-integral-derivative (PID) controller loop, the rotating growth vial 400 is controlled to approximately +/- 0.5°C.
- PID proportional-integral-derivative
- 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 400 by piercing though the foil seal or film.
- the programmed software of the cell growth device 430 sets the control temperature for growth, typically 30 °C, then slowly starts the rotation of the rotating growth vial 400.
- the cell/growth media mixture slowly moves vertically up the wall due to centrifugal force allowing the rotating growth vial 400 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.
- 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 430 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 430 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.
- 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.
- the cell growth device 630 may include additional sensors for measuring, e.g., dissolved oxygen, carbon dioxide, pH, conductivity, and the like.
- FIG. 5A is a simplified block diagram of an embodiment of an exemplary automated multi-module cell processing instrument comprising an isolation/growth/editing/normalization module for enrichment for edited cells.
- the cell processing instrument 500 may include a housing 544, a reservoir of cells to be transformed or transfected 502, and a growth module (a cell growth device) 504.
- the cells to be transformed are transferred from a reservoir to the growth module to be cultured until the cells hit a target OD. Once the cells hit the target OD, the growth module may cool or freeze the cells for later processing, or the cells may be transferred to an optional filtration module 530 where the cells are rendered electrocompetent and concentrated to a volume optimal for cell transformation. Once concentrated, the cells are then transferred to the electroporation device 608 (e.g., transformation/transfection module).
- the electroporation device 608 e.g., transformation/transfection module
- the system 500 may include a reservoir for storing editing oligonucleotide cassettes 516 and a reservoir for storing an expression vector backbone 518. Both the editing oligonucleotide cassettes and the expression vector backbone are transferred from the reagent cartridge to a nucleic acid assembly module 520, where the editing oligonucleotide cassettes are inserted into the expression vector backbone.
- the assembled nucleic acids may be transferred into an optional purification module 522 for desalting and/or other purification and/or concentration procedures needed to prepare the assembled nucleic acids for transformation.
- pre-assembled nucleic acids may be stored within reservoir 516 or 518.
- the assembled nucleic acids are transferred to, e.g., an electroporation device 508, which already contains the cell culture grown to a target OD and rendered electrocompetent via filtration module 530.
- electroporation device 508 the assembled nucleic acids are introduced into the cells.
- the cells are transferred into a combined recovery/selection module 510.
- the cells are transferred to an isolation, editing, and growth module 540, where the cells are diluted and compartmentalized such that there is an average of one cell per compartment. Once isolated, the cells are allowed to grow to terminal size (e.g., the colonies are normalized). Once the colonies are grown to terminal size, the colonies are pooled. Again, isolation overcomes growth bias from unedited cells and growth bias resulting from fitness effects of different edits.
- the recovery, selection, isolation, editing and growth modules may all be separate, may be arranged and combined as shown in Figure 5A, or may be arranged or combined in other configurations. In certain embodiments, such as those described in relation to the rotating growth vial shown in Figures 4A, all of recovery, selection, isolation, editing, and normalization are performed in a single vessel/module (e.g., a rotating growth vial 400 in growth module 430 of Figure 4B).
- a single vessel/module e.g., a rotating growth vial 400 in growth module 430 of Figure 4B.
- the cells may be stored, e.g., in a storage module 512, where the cells can be kept at, e.g., 4°C until the cells are retrieved for further study. Alternatively, the cells may be used in another round of editing.
- the multi-module cell processing instrument is controlled by a processor 542 configured to operate the instrument based on user input, as directed by one or more scripts, or as a combination of user input or a script.
- the processor 542 may control the timing, duration, temperature, and operations of the various modules of the system 600 and the dispensing of reagents.
- the processor 542 may cool the cells post transformation until editing is desired, upon which time the temperature may be raised to a temperature conducive of genome editing and cell growth.
- the processor may be programmed with standard protocol parameters from which a user may select, a user may specify one or more parameters manually or one or more scripts associated with the reagent cartridge may specify one or more operations and/or reaction parameters.
- the processor may notify the user (e.g., via an application to a smart phone or other device) that the cells have reached the target OD as well as update the user as to the progress of the cells in the various modules in the multi-module system.
- the automated multi-module cell processing instrument 500 is a nuclease- directed genome editing system and can be used in single editing systems (e.g., introducing one or more edits to a cellular genome in a single editing process).
- the system of Figure 5B described below, is configured to perform sequential editing, e.g., using different nuclease-directed systems sequentially to provide two or more genome edits in a cell; and/or recursive editing, e.g. utilizing a single nuclease-directed system to introduce sequentially two or more genome edits in a cell.
- FIG. 5B illustrates another embodiment of a multi-module cell processing instrument.
- This embodiment depicts an exemplary system that performs recursive gene editing on a cell population.
- the cell processing instrument 550 may include a housing 544, a reservoir for storing cells to be transformed or transfected 502, and a cell growth module comprising a rotating growth vial 504. The cells to be transformed are transferred from a reservoir to the cell growth module to be cultured until the cells hit a target OD.
- the growth module may cool or freeze the cells for later processing or transfer the cells to an a TFF module 530 where the cells are subjected to buffer exchange and rendered electrocompetent, and the volume of the cells may be reduced substantially.
- the cells are transferred to electroporation device 508.
- the multi module cell processing instrument includes a reservoir for storing the vector pre assembled with editing oligonucleotide cassettes 506.
- the pre-assembled nucleic acid vectors are transferred to the electroporation device 508, which already contains the cell culture grown to a target OD.
- the nucleic acids are electroporated into the cells.
- the cells are transferred into an optional recovery module 556, where the cells are allowed to recover briefly post transformation.
- the cells may be transferred to a storage module 512, where the cells can be stored at, e.g., 4°C for later processing, or the cells may be transferred to a selection/isolation/editing/normalization module 558.
- the isolation/edit/growth module 558 the cells are diluted such that cells are isolated from one another in three- dimensional space.
- the arrayed cells are in selection medium to select for cells that have been transferred or transfected with the editing vectors. Once isolated, the liquid medium is solidified, and the cells continue to grow to form clonal colonies in three-dimensional space.
- editing is induced by providing conditions (e.g., temperature, addition of an inducing or repressing chemical) to induce editing.
- the cells are allowed to grow to terminal size or desired cell concentration or optical density (e.g., normalization of the colonies) and then are pooled and transferred to the storage unit 514 or can be transferred to a growth module 504 for another round of growth, transformation and editing.
- a growth module there may be one or more additional steps, such as medium exchange, cell concentration, etc., by, e.g., filtration via a TFF.
- the selection/isolation/growth/editing and normalization modules may be the same module, where all processes are performed in the same vessel such as the rotating growth vial of Figures 4A - 4D.
- the putatively-edited cells may be subjected to another round of editing, beginning with growth, cell concentration and treatment to render electrocompetent, and transformation by yet another donor nucleic acid in another editing cassette via the electroporation module 508.
- the cells from the first round of editing are transformed by a second set of editing oligos (or other type of oligos) and the cycle is repeated until the cells have been transformed and edited by a desired number of, e.g., editing cassettes.
- the multi-module cell processing instrument exemplified in Figure 5B is controlled by a processor 542 configured to operate the instrument based on user input or is controlled by one or more scripts including at least one script associated with the reagent cartridge.
- the processor 542 may control the timing, duration, and temperature of various processes, the dispensing of reagents, and other operations of the various modules of the system 550.
- a script or the processor may control the dispensing of cells, reagents, vectors, and editing oligonucleotides; which editing oligonucleotides are used for cell editing and in what order; the time, temperature and other conditions used in the recovery and expression module, the wavelength at which OD is read in the cell growth module, the target OD to which the cells are grown, and the target time at which the cells will reach the target OD.
- the processor may be programmed to notify a user (e.g., via an application) as to the progress of the cells in the automated multi-module cell processing instrument.
- r00116lEditing Cassette Preparation 5 nM oligonucleotides synthesized on a chip were amplified using Q5 polymerase in 50 pL volumes. The PCR conditions were 95°C for 1 minute; 8 rounds of 95°C for 30 seconds/60°C for 30 seconds/72°C for 2.5 minutes; with a final hold at 72°C for 5 minutes. Following amplification, the PCR products were subjected to SPRI bead cleanup, where 30pL SPRI mix was added to the 50 pL PCR reactions and incubated for 2 minutes. The tubes were subjected to a magnetic field for 2 minutes, the liquid was removed, and the beads were washed 2x with 80% ethanol, allowing 1 minute between washes.
- the beads were allowed to dry for 2 minutes, 50 pL 0.5x TE pH 8.0 was added to the tubes, and the beads were vortexed to mix. The slurry was incubated at room temperature for 2 minutes, then subjected to the magnetic field for 2 minutes. The eluate was removed and the DNA quantified.
- r00118lBackbone Preparation A lO-fold serial dilution series of purified backbone was performed, and each of the diluted backbone series was amplified under the following conditions: 95°C for 1 minute; then 30 rounds of 95°C for 30 seconds/60°C for 1.5 minutes/72°C for 2.5 minutes; with a final hold at 72°C for 5 minutes. After amplification, the amplified backbone was subjected to SPRI cleanup as described above in relation to the cassettes. The backbone was eluted into 100 pL ddPhO and quantified before nucleic acid assembly.
- r00119llsothermal Nucleic Acid Assembly 150 ng backbone DNA was combined with 100 ng cassette DNA. An equal volume of 2x Gibson Master Mix was added, and the reaction was incubated for 45 minutes at 50°C. After assembly, the assembled backbone and cassettes were subjected to SPRI cleanup, as described above.
- r00120lTransformation 1 pL of the engine vector DNA (comprising a coding sequence for MAD7 nuclease under the control of the pL inducible promoter, a chloramphenicol resistance gene, and the l Red recombineering system) was added to 50 pL EC1 strain E. coli cells. The transformed cells were plated on LB plates with 25 pg/mL chloramphenicol (chlor) and incubated overnight to accumulate clonal isolates. The next day, a colony was picked, grown overnight in LB + 25 pg/mL chlor, and glycerol stocks were prepared from the saturated overnight culture by adding 500 pL 50% glycerol to 1000 pL culture. The stocks of EC1 comprising the engine vector were frozen at -80°C.
- Example 4 Bulk Cell 3D Isolation, Colony Normalization, and Processing within a Rotating Growth Vial
- This protocol describes a standard bulk culture protocol using alginate as the solidifying agent.
- Alginate is solidified by addition of CaCl2 and liquefies upon the addition of a chelating agent, and both processes can take place at a temperature appropriate for enriching for nucleic acid-guided nuclease editing of bacterial and yeast cells by isolation, growth, editing, and normalization.
- This protocol was used to leverage the inducible system for both the nuclease and gRNAs to allow for a phenotypic difference in colonies.
- Alginate Alginate, Al l 12 Sigma- Aldrich (St. Louis, MO), Alginic acid sodium salt from brown algae, low viscosity).
- LB and DI fLO in desired quantities as listed in Table 1 were combined in a flask.
- a stir bar was added to the flask and the alginate was added slowly while the LB/alginate mixture was stirred on a stir plate.
- the LB/alginate mixture was then sterilized by autoclavation using standard conditions (e.g., 121 °C, 20 min, liquid cycle). After autoclavation, the solution was immediately cooled on ice. Before using the LB/ alginate solution, cells and desried antibiotics were added to the appropriate concentration.
- Table 2 LB Alginate, composition for arabinose induction (1% final cone) r version of LB Broth
- LB and DI H 2 0 in desired quantities as listed in Table 2 were combined in a bottle.
- a stir bar was added to the bottle and the alginate was added slowly while the LB/alginate mixture was stirred on a stir plate.
- the LB/alginate mixture was then sterilized by autoclavation using standard conditions (e.g., 121 °C, 20 min, liquid cycle). After autoclavation, the solution was immediately cooled on ice. Before using the LB/ alginate solution, cells and desired antibiotics were added to the appropriate concentration, and 1 ml of 20% arabinose also was added to 19 ml of the LB alginate solution to obtain a 1% arabinose final concentration.
- Editing was performed following the above protocols to make LB Alginate (25ml per sample) and LB Alginate +1% arabinose (25ml per sample). lOml of alginate + 1% arabinose solution was added to each 50ml conical tube. The conical tubes were kept at 30°C, to be ready for use after transformation protocol was complete. Transformation was performed using 500ng of the nucleic acid assembly (vector + editing cassette library) into ec83 (recombineering competent cells) using the Nepagene electroporator settings for E. coli. The cells were allowed to recover in 3000m1 of SOB in l5ml conical tubes while shaking at 30°C for 3 hours.
- the alginate tubes and the transformation tubes were removed from the 30°C incubator, and 250m1 of cells was added to each tube with the 25 ml of Alginate solution (1 : 10).
- the Alginate solution was solidified by slowly transferring 20 ml of the alginate + cells solution into 30 ml of 100 mM CaCb solution.
- the alginate slurry was then centrifuged for 10 min at 4000 x g. The supernatant was decanted, and the bulk gel was incubated at 30°C for 9 hours. After the 9-hour 30°C incubation, the temperature was shifted to 42°C for 2 hours for induction of editing.
- the cells were spun at 5,000 x g for 10 minutes. The supernatant was removed and the cells were resuspended in 500 m ⁇ of 0.8 NaCl.
- a ZyppyTM Plasmid Miniprep kit (Zymo Research, Bath, UK) was used to extract the plasmid DNA from the library, and the samples were prepped for PCR of the inserts, and for assaying the amplicons via next-gen sequencing.
- Figures 6 A - 6C is a depiction of an experiment performed to demonstrate that normalization is achieved in bulk culture, which compares the quantity of wildtype (inert) plasmid and editing plasmid (GalK) in bulk gel versus liquid cell culture (see Example 7 below).
- the wildtype plasmid was used to transform an E. coli cell line, and separately, an editing plasmid was used to transform the E. coli cell line.
- pools of the transformed cells were combined in the following ratios: 50:50, 10: 1, and 1: 10 (wildtype to editing cells, respectively) and dispensed between both bulk culture and liquid culture, where six replicates were prepared for each.
- Controls included 100% wildtype and 100% editing cells, and standard plating controls.
- the bulk and liquid cultures (experimental and controls) were grown at 30°C for 6 hours, 42°C for 2 hours, and at 30°C overnight.
- live cells were recovered from each culture (e.g., six experimental cultures and controls for each of the bulk and liquid cultures).
- Figure 6C depicts plasmid extraction and isolation of the cells recovered from the bulk gel cultures and from the liquid cultures (shown) as well as the controls (not shown).
- Phenotypic assessment was used to determine whether normalization takes place in the bulk gel culture. The phenotypic read out comprised red/white screening on MacConkey agar. The results obtained demonstrated cells edited in bulk gel match most closely the loaded ratio of the 50:50 mix of cells edited at 25% in alginate and 7% liquid and on a plate.
- Figure 7 depicts the workflow for bulk alginate isolation, growth, induction, editing, and normalization in a module comprising a rotating growth vial (as shown in Figure 4E and described above), which can be used in a multi-module cell editing system (as shown in Figure 3A and described above).
- a first step 10 ml LB medium comprising alginate was added to the rotating growth vial, which already contained the transformed cells to be edited.
- the medium also comprises antibiotics to select for the cells that have been properly transformed.
- the medium was then solidified by slowly adding 1.5 ml of 1M CaCL 2 to the LB alginate cell culture in the rotating growth vial.
- the cells were allowed to grow for 6 hours at 30°C to establish cell colonies, 2 hours at 42°C (which induces editing), then overnight at 30°C to normalize the edited and unedited cell colonies.
- the solidified LB alginate medium was liquified by adding 10 ml 1 M sodium citrate to the solidified medium, and the liquified normalized cell culture was filtered in a filtration module allowing for buffer exchange, cell concentration, and, if desired, rendering the cells electrocompetent for an additional round of editing. Liquification disperses all cells throughout the culture.
- the process described may be recursive; that is, cells may go through the workflow described in relation to Ligure 7, then the resulting edited culture may go through another (or several to many) rounds of additional editing (e.g., recursive editing) with different editing vectors.
- the cells from round 1 of editing may be diluted and an aliquot of the edited cells edited by editing vector A may be combined with editing vector B, an aliquot of the edited cells edited by editing vector A may be combined with editing vector C, an aliquot of the edited cells edited by editing vector A may be combined with editing vector D, and so on for a second round of editing.
- an aliquot of each of the double-edited cells may be subjected to a third round of editing, where, e.g., aliquots of each of the AB-, AC-, AD-edited cells are combined with additional editing vectors, such as editing vectors X, Y, and Z.
- double- edited cells AB may be combined with and edited by vectors X, Y, and Z to produce triple-edited edited cells ABX, ABY, and ABZ
- double-edited cells AC may be combined with and edited by vectors X, Y, and Z to produce triple-edited cells ACX, ACY, and ACZ
- double-edited cells AD may be combined with and edited by vectors X, Y, and Z to produce triple-edited cells ADX, ADY, and ADZ, and so on.
- many permutations and combinations of edits can be executed, leading to very diverse cell populations and cell libraries.
- “Curing” is a process in which one or more vectors used in the prior round of editing is eliminated from the transformed cells. Curing can be accomplished by, e.g., cleaving the vector(s) using a curing plasmid thereby rendering the editing and/or engine vector (or single, combined vector) nonfunctional; diluting the vector(s) in the cell population via cell growth (that is, the more growth cycles the cells go through, the fewer daughter cells will retain the editing or engine vector(s)), or by, e.g., utilizing a heat- sensitive origin of replication on the editing or engine vector (or combined vector).
- the conditions for curing will depend on the mechanism used for curing; that is, in this example, how the curing plasmid cleaves the editing and/or engine plasmid. Curing in the context of the isolation, growth, editing, and normalization reactions described herein is described in Example 8.
- This protocol describes a standard plating protocol for enriching for nucleic acid- guided nuclease editing of bacterial cells by isolation, growth, editing, and normalization. This protocol was used to leverage the inducible system for both the nuclease and gRNAs to allow for a phenotypic difference in colonies. From the resulting agar plates, it is possible to select edited cells with a high degree (-80%) of confidence. Though clearly this protocol can be employed for enriching for edited cells, in the experiments described herein this“standard plating protocol” of “SPP” was used to compare efficiencies of isolation, editing, and normalization with the bulk cell culture. The protocols for liquid cell culture described in Example 7 were used for the same purpose.
- Protocol Inputs for this protocol are frozen electrocompetent cells and purified nucleic acid assembly product. Immediately after electroporation, the cell/DNA mixture was transferred to a culture tube containing 2.7mL of SOB medium. Preparing 2.7mL aliquots in l4mL culture tubes prior to electroporation allowed for a faster recovery of cells from the electroporation cuvette; the final volume of the recovery was 3mL. All culture tubes were placed into a shaking incubator set to 250RPM and 30°C for three hours. While the cultures were recovering, the necessary number of LB agar plates with chloramphenicol and carbenicillin + 1% arabinose were removed from the refrigerator and warmed to room temperature. Multiple dilutions were used for each plating so as to have countable and isolated colonies on the plates. Plating suggestions:
- Additional or fewer dilutions may be used based on library/competent cell knowledge.
- the cultures were evenly spread across the agar using sterile, plating beads. The beads were then removed from the plate and the plates were allowed to dry uncovered in the flow hood. While the plates were drying, an incubator was programmed according to the following settings: 30°C for 9 hours -> 42°C for 2 hours -> 30°C for 9 hours.
- the agar plates were placed in the pre-set incubator, and after the temperature cycling was complete ( ⁇ 2l hours), the agar plates were removed from the incubator. If induction of editing has been successful, size differences in the colonies will be visible.
- r00134lLiquid culture process for control The editing cassette libraries were transformed via electroporation into specific strains of E. coli expressing Mad7 (nuclease) and Lambda Red (recombination) proteins. Transformation of process control vectors— alongside the editing cassette libraries— is essential to calculate the transformation efficiency and editing efficiency (sgRNA efficiency). Immediately post-transformation, the electroporated cells were transferred to medium for recovery.
- Figures 8 A, 8B, and 8C show the results of the editing rates and clonality resulting from editing experiments performed with liquid cell culture employing no isolation or normalization, but employing inducible editing; bulk cell gel culture, employing isolation, inducible editing, and normalization; solid agar plating (SPP) employing isolation, inducible editing, and normalization; solid agar plating (SPP- Cherry) employing isolation, inducible editing, and cherry picking; and solid agar plating (SPP) employing only isolation and inducible editing and simply scraping the colonies from the plate and re -plating.
- SPP solid agar plating
- SPP- Cherry solid agar plating
- SPP solid agar plating
- “cherry” refers to cherry-picking of small colonies, which, when cells are plated and grown into colonies, it has been determined that small colonies are likely to be colonies of edited cells, where large, fast-growing colonies typically are non-edited cells (e.g., escapees). See, e.g., USPNs 10,253,316, filed 30 June 2018; 10,329,559, filed 07 February 2019; and 10,323,242, filed 07 February 2019; and USSNs 16/412,175, filed 14 May 2019; 16/412,195, filed 14 May 2019; 16/454,865, filed 06 June 2019; and 16/423,289, filed 28 May 2019, all of which are herein incorporated by reference in their entirety.
- Figure 8A shows that liquid culture results in a very low rate of observed editing, at about 1-2%; the standard plating procedure (SPP) results in an approximate 75% rate of observed editing in the cell population; the bulk alginate cell culture protocol results in an approximate 50% rate of observed editing; the standard plating procedure plus cherry picking (SPP-cherry) (e.g., manual picking of only small colonies from the plated cells, where the presumption is that small colonies represent colonies of cells that have been edited) protocol results in an approximate 95% rate of observed editing; and the standard plating procedure (SPP) without normalization or cherry picking results in an approximate 8% rate of observed editing.
- SPP + cherry picking produces the highest rate of observed editing but requires manual intervention for picking colonies.
- SPP without cherry picking, but including isolation, induced editing, and normalization results in a high (75%) rate of observed editing, and the easily- automatable bulk gel cell culture process resulted in an approximate 50% rate of observed editing.
- Figure 8B provides the observed clonality for the standard plating procedure (SPP), the standard plating procedure + cherry picking (SPP cherry), the standard plating procedure + scraping the plate comprising the colonies where editing has been induced (but also comprising unedited cells), and for the bulk procedure.
- the first column gives the fraction of colonies examined with more than half the reads being called edits.
- the second column gives the fraction of colonies that have more than 90% of the reads being called edit reads. The higher fraction here shows how complete the edits are if there are some colonies examined between the 50% and 90% cut offs that demonstrate that not all of the cells in the colony that is being picked are edited.
- the third column provides the number of unique edits for the colonies in the >50% clonal colonies. Note that SPP-cherry provides the highest clonality and number of unique edits, but that the bulk gel cell culture provides good clonality (44/95 at >50%) and a high proportion of the clonal colonies consist of unique edits (42/44).
- Figure 8C provides a graph of the data in Figure 8B. This graph indicates the extent of incomplete editing.
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