Dial-out cloning methods and systems

Abstract

This application describes methods, compositions, and kits for enriching cells based on DNA sequences. The method may include one or more of cloning nucleic acids into a population of cells, culturing the cells, obtaining nucleic acids from a portion of the cultured cells, sequencing the obtained nucleic acids, identifying a set of one or more barcode sequences that distinguish a population of cultured cells that have a desired library sequence, and enriching one or more cultured cells that comprise the identified set of one or more barcode sequences.

Description

DIAL-OUT CLONING METHODS AND SYSTEMSCROSS-REFERENCE TO RELATED APPLICATIONS[1] This application claims the benefit of U.S. Provisional Application No. 63 / 525,258 filed on July 6, 2023, the disclosure of which is incorporated herein by reference in its entirety.BACKGROUND OF THE INVENTION[2] In the creation of long DNA constructs with high accuracy, one challenge is finding ways to link sequence curation data with individual molecules in high volume. Traditional methods allow sequence validation of an individual clone, but to take full advantage of the vast increases in sequencing throughput that have occurred in the past 20 years, methods to allow simultaneous sequence verification of millions of individual molecules are needed. One method that has been proposed is the use of dial-out PCR. In this method, adapters are connected to molecules that contain a barcode or UMI in the primer region, allowing amplification of molecules that are identical-by-descent with another molecule that was sequenced in another process. However, as molecules become long, this process encounters a fundamental issue: PCR introduces sequence variation. Once a molecule is longer than a few thousand bases in length, the probability that two molecules descended from the same progenitor molecule retain correct sequence drops to a small value and at 100,000 bases it becomes a statistical impossibility to retain a correct sequence.[3] Cloning methods can preserve sequence identity far better than PCR amplification. This invention is directed to methods for achieving dial -out with clones in a manner which is analogous or partly analogous with dial -out PCR.BRIEF SUMMARY OF THE INVENTION[4] Aspects of the subject application include a method of enriching cells based on DNA sequences. The method may include one or more of: cloning nucleic acids into a population of cells, wherein the nucleic acids comprise library sequences and barcode sequences; culturing the cells; obtaining nucleic acids from a portion of the cultured cells, wherein the nucleic acids comprise copies of the library sequences and barcode sequences of nucleic acids that were cloned into different cells of the population of cells; sequencing the obtained nucleic acids;identifying, based on the sequencing, a set of one or more barcode sequences that distinguish a population of cultured cells that have a desired library sequence; and enriching one or more cultured cells that comprise the identified set of one or more barcode sequences.[5] Additional aspects of the method are described further hereien. Kits and compositions comprising one or more reagents or elements of the methods described herein are also within the scope of this application.DETAILED DESCRIPTION OF THE INVENTION[6] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments can be utilized, and other changes can be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein and made part of the disclosure herein.[7] Unless defined otherwise, technical, and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. See, e.g., Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al, Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory, New York (2001). For purposes of the present disclosure, the following terms are defined below.[8] As used herein, the singular forms “a,” “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an antigen” includes mixtures of antigens; reference to “a pharmaceutically acceptable carrier” includes mixtures of two or more such carriers, and the like. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.[9] Furthermore, “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” asused in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A (alone)”, and “B (alone)”.I. Overview of Techniques and Molecular Tools Useful for Dial-Out Cloning

[0010] In the most basic form, long DNA constructs are transfected into cells using a vector that contains barcodes (e.g., markers), optionally adjacent or proximal (e.g., within 200 bases, or within 100 bases) to a sequence of interest, that allow for unique (or nearly unique) identification of cells that are carrying a particular payload.Exemplary Markers for Enrichment of Desired Cells

[0011] One method for doing this is to use a series of positive and negative selection markers.

[0012] Consider 8 commonly used resistance markers:

[0013] 1. Ampicillin resistance gene (amp): Confers resistance to the antibiotic ampicillin.

[0014] 2. Kanamycin resistance gene (kan): Confers resistance to the antibiotic kanamycin.

[0015] 3. Neomycin resistance gene (neo): Confers resistance to the antibiotic neomycin.

[0016] 4. Streptomycin resistance gene (str): Confers resistance to the antibiotic streptomycin.

[0017] 5. Chloramphenicol resistance gene (cat): Confers resistance to the antibiotic chloramphenicol.

[0018] 6. Tetracycline resistance gene (tet): Confers resistance to the antibiotic tetracycline.

[0019] 7. Gentamicin resistance gene (gent): Confers resistance to the antibiotic gentamicin.

[0020] 8. Spectinomycin resistance gene (spec): Confers resistance to the antibiotic spectinomycin.

[0021] Just by themselves, a 4-bit UMI can be constructed that will allow for selection of 1 of 16 elements, or correspondingly a 16-fold enrichment of the desired construct against the background of undesired constructs. This would be done by pairing them (for example, 1-4 paired with 5-8), so that bit 1 would have the value “0” if maker 1 is present but 5 is absent, and “1” if 1 is absent but 5 is present. This pattern would be repeated for 2,3, and 4. Then at selection time, if the 11thchannel is desired for amplification, this would be decoded into binary (1011), and correspondingly the 8 marker selectors would be chosen as follows:

[0022] Working through the survival conditions, it will be seen that only those cells carrying the correct “code” will survive, and selection will have taken place.

[0023] This can be extended to include a combination of solution / survival markers beyond just resistance genes. For example, nutritional markers can be used in the same way, and in many cases at the same time so as to increase the size of the code-space.

[0024] In another embodiment of the invention, fluorescent markers are used for FACS sorting of cells that contain a particular spectral code. An example set of fluorophores is give here, including fluorescent proteins that may be expressed in a cell and chemical dyes that may be associated with a cell through an affinity reagent (e g., antibody) intermediate:

[0025] GFP (green fluorescent protein)

[0026] mCherry (red fluorescent protein)

[0027] BFP (blue fluorescent protein)

[0028] Cerulean (cyan fluorescent protein)

[0029] mCitrine (yellow fluorescent protein)

[0030] TagBFP2 (bright blue fluorescent protein)

[0031] TagGFP2 (green fluorescent protein)

[0032] TagRFP (red fluorescent protein)

[0033] mPlum (far-red fluorescent protein)

[0034] mOrange (orange fluorescent protein)

[0035] EYFP (yellow fluorescent protein)

[0036] mTurquoise (turquoise fluorescent protein)

[0037] Cy7 (far-red fluorescent dye)

[0038] Cy5 (red fluorescent dye)

[0039] Alexa Fluor 647 (far-red fluorescent dye)

[0040] Alexa Fluor 594 (orange-red fluorescent dye)

[0041] Alexa Fluor 568 (red fluorescent dye)

[0042] Alexa Fluor 555 (red fluorescent dye)

[0043] Alexa Fluor 488 (green fluorescent dye)

[0044] Pacific Blue (blue fluorescent dye)

[0045] Pacific Orange (orange fluorescent dye)

[0046] DyLight 680 (near-infrared fluorescent dye)

[0047] DyLight 750 (near-infrared fluorescent dye)

[0048] DyLight 650 (near-infrared fluorescent dye)

[0049] ATTO 565 (red fluorescent dye)

[0050] ATTO 590 (orange-red fluorescent dye)

[0051] ATTO 647N (far-red fluorescent dye)

[0052] ATTO 647 (far-red fluorescent dye)

[0053] ATTO 488 (green fluorescent dye)

[0054] ATTO 390 (blue fluorescent dye)

[0055] Through combinations of copy number, or transcription factor efficiency, several different intensities can be coded, and by simultaneous use of modulating excitation spectrum and measuring emission spectrum, up to 10 or more of these could be introduced simultaneously leading to a code space that could have 32 bits of information or more. This would allow for selection of one from a code space of billions. This size of code space would likely require multiple passes through existing FACS hardware, or development of new hardware specifically designed to allow for serial application of different wavelengths of illumination. This would allow differentiation of over 4 billion different codes.

[0056] Another approach is to use sequence-codable transcription factors (TF), such as TALEs, that allow for modular design of transcription factors. A codable TF can be placed in front of the gene for another transcription factor, promoter or repressor. That promoter can be positioned in front of any of the other selection factors, allowing the code space to be multiplied by the number of biologically distinguishable TALE codes.Exemplary Vector Design and Mechanisms of Addressability

[0057] Another method of achieving sequence-coded selection would be through the use of CAS9 targeted degradation of a promoter or TSS for a gene coding a repressor for an antibiotic resistance gene or fluorescence marker. In this way a single fluorescence marker orantimicrobial resistance marker could be used in conjunction with a large CAS9 code space to allow dial-out from large populations. With current technology, the addressable code-space for CAS9 is estimated to be over lxlOA12 different codes(https: / / www.nature.com / articles / naturel6526). This is sufficient to take advantage of any commercially available sequence throughput today.

[0058] Another aspect of the invention is allowing for convenient access to the codes during the sequencing phase. Some of the methods mentioned above will produce a DNA construct that occupies a portion of the available read-length of the DNA sequencer that will have a negative effect on either the performance or the cost of applying this method. In these cases, a shortened version of the code can be “copied” into the space immediately adjacent to the location of any potential construct. For example, in one embodiment, the transfection target library can be pre- loaded with diverse di al -out-codes (e.g., a combination of barcodes) as well as UMI codes (e.g., random er barcodes) that are adjacent or proximal to the insert site. A prior sequencing run can be conducted to associate the UMIs with the dial -out codes. Though this step produces an added cost, this library can be cultured, and re-used many times over, thus reducing the effective cost of this added library sequencing step.

[0059] In another method of providing the code adjacent to the DNA sequence insert location, LoxP sites can be introduced into the region where the dial -out codes are located, and positioned in such a way as to cause the bulk of the length of the segment to be excised, while leaving behind key nucleotides that will allow identification of which element was present. This would be done only on the library to be sequenced, leaving an aliquot of the library unaltered for the dial-out step. The Flp recombinase and PhiC31 integrase could be used in a similar manner.

[0060] Another aspect of the invention is providing methods for processing DNA inserts longer than the read-length of the sequencing technology used. There would be a strong commercial motivation to allow the constructs to grow beyond the read-length of the sequencing technology. Given that assembly methods are well-known in the art for producing high-quality reconstructions of long sequences of DNA, we require only a way to link the identifier at the end of the construct to each sub-section of the insert that we wish to sequence.

[0061] Methods for single-cell sequencing are known in the art, and can be applied with modification to make this possible. First, a small clonal population of each cell type may be grown, using methods known in the art. In one method, a small gelatin bead will contain a singlecell and will prevent replication progeny from diffusing away from the bead. This bead can then be treated as a single cell for the purposes of existing single-cell sequencing systems, in which an additional cell label UMI is introduced for the purpose of labeling the individual fragments for later assembly. Emulsion methods for containing replicates can also be used. Then, standard single-cell sequencing is applied followed by assembly of the insert. Note that even if sequencing yield can reach 100%, more than one replicate of the insert is still required to allow sequence overall for assembly. One method for this is to use a cloning vector known to produce multiple copies inside a single cell.

[0062] Another method is to use retrotransposon insertion to place UMI codes in the interior of the insert. This would be done only in the aliquot to be sequenced, as the desired fragment should not be disturbed for the final output of the system. This retrotansposition could be done randomly, allowing a process analogous to haplotype resolution to link the UMI at the flank with the sequence of the insert. However, because of the likely very large number of similar sequences, this process would require extra sequencing coverage and a dense network of insertion sites. Another related approach is to use enzymatic or other mutagenesis to “damage” the insert sequence in a way that would allow for unique assembly later.

[0063] Another method would be to have the sequence inserts reflect the code involved. This could be done by changing the retrotransposon (rt) sequence to reflect the UMI or to be linked with the UMI in a prior sequencing step. Then the RT could be biologically induced and the cells would self-label the insert with RT sites.

[0064] Another method would be to re-close DNA loops after degradation to link the UMI at the edge with various parts of the interior of a circular molecule.

[0065] These methods can be combined with each other to increase the code space, if needed. In some cases this could be simultaneous — such as with nutritional and antibiotic markers, and in other cases it may be serial, such as with fluorescence selection and antibiotic resistance.

[0066] In one preferred embodiment, a repressor of an antibiotic resistance gene can be targeted with the CAS9 cutter to release a specific library member and allow it to replicate.

[0067] These can be applied in multiplex to a mixture if there are specific signals in the ends of the inserts to allow for programmed linkage between the ends. For example, a type Ils enzyme target can be included in the insert that will create a sticky overhang. In this way, several fragment can be selected at the same time and combined in vitro without the need for furtherisolation. With appropriate selection of the end sequences, this could be done more than once in a hierarchical assembly process.

[0068] It should be noted that due to the extreme difference in the cost of sequencing compared to synthesis, strategies can be employed that do not rely on perfect specificity, or that do not rely on specificity at all. For example, if 4 fragments are being assembled, even with no specificity whatsoever, there is a finite number of ways to ligate these fragments together while still being approximately the correct length. A later sequencing step can simply select for the one that was correctly assembled.

[0069] In certain embodiments, the enrichment of step e) may comprise selecting a set of guide RNAs that match barcodes of the identified set of barcodes, transfecting the cultured cells with from a library of guide RNAs.

[0070] Additional embodiments are described further herein, and may be combined with any of the above described embodiments.Gene Assembly and Cloning

[0071] A first step is designing a library of sequences for insertion into a cell. As described further herein, library sequences can be sequences of interest for a particular study, different variations of a gene, a sequence that is to be amplified through cell culture and later purified, a cassette of one or more genes for engineering a cell in a cell therapy, or other genetic elements. The sequences may be DNA sequences. Each sequence may be flanked by homology arms that match the sequence at the desired insertion site in the genome, such as for homology based repair approached discussed further herein.

[0072] Once the library is designed, it is synthesized before being cloned into a suitable vector. Longer library sequences, such as gene sequences or cassettes that comprise multiple genes and promoters, may be assembled from shorter synthesized sequences, such as through Gibson assembly or polymerase cycle assembly. After assembly, errors may be reduced through homology based error correction, such as through exposure to one or more DNA repair enzymes. Homology based error correction may comprise mismatch excision, gap filling, and / or ligation. In certain aspects, error correction does not include nick translation and does not include extension with a strand displacing polymerase. In certain aspects, the library element may be a short sequence (e.g., less than 100 bases in length) that is cloned into a vector that comprises oneor more genes and / or promoter, such as to modify the vector or to correct an error in the vector sequence.[731 The vector could be a plasmid, viral vector, or other delivery mechanism. The vector is then introduced into the cells. This could be done by a variety of methods such as electroporation, chemical transfection, or viral transduction. The choice of method will depend on the cell type and the specific requirements of the experiment. In certain aspects, library sequences are cloned alongside barcode sequences.Crispr-associated endonuclease cloning

[0074] The vector may further contain a sequence encoding a Crispr-associated endonuclease (Cas protein), such as Cas9, and one or more guide RNAs (gRNAs) that target the endonuclease to the correct location in a cell’s genome. While Cas9 and Crispr-Cas9 is used in embodiment herein, it will be understood that any gRNA mediated gene editing, such as another Crispr-Cas protein, may be used in place of Cas9. The vector may further comprise one or more library sequences and / or barcode sequences as described elsewhere herein, or may be separate from a vector comprising one or more library sequences and / or barcode sequences. In certain aspects, a cell line may have been engineered to express a Crispr-associated endonuclease.

[0075] The Cas9 enzyme is an RNA-guided DNA endonuclease, derived from bacterial adaptive immune systems. Once inside the cell, the Cas9 protein binds to the guide RNA (gRNA) sequence that is between 10 and 30 base pairs in length, such as a 20 base pair length sequence. This complex then searches the cell's DNA for a sequence matching the gRNA's target sequence. The target sequence is complementary to the gRNA, and may be followed by a protospacer adjacent motif (PAM) the Cas protein binds to, which for Cas9 from Streptococcus pyogenes (the most commonly used variant) is 'NGG'. In certain aspects, the target sequence may be a barcode as described elsewhere herein. When Cas9, bound to its gRNA, encounters this target sequence, it cleaves the DNA, introducing a double-strand break (DSB). The generation of DSBs is an essential step for CRISPR-Cas9 mediated gene editing. The specificity of CRISPR-Cas9 mediated cleavage is highly dependent on the design of the gRNA.

[0076] After the DSB is introduced, the cell mobilizes its DNA repair machinery to mend the break. There are two major pathways the cell can use to repair DSBs: non-homologous end joining (NHEJ) and homology directed repair (HDR). NHEJ is an error-prone repair pathway that can result in insertions or deletions (indels) at the break site. It is fast and efficient but lacksprecision. In contrast, HDR is a slower but far more accurate process that uses a DNA template to repair the break and restore the original sequence. A DNA repair template, provided along with the Cas9 and gRNA, which contains the sequence to be inserted, may flanked by homology arms that match the sequences up and downstream of the break site. When the DSB is repaired through the HDR pathway, the cell's repair machinery uses this template to incorporate the desired sequence into the genome at the break site. The balance between NHEJ and HDR can vary greatly depending on the cell type, cell cycle stage, and the specific conditions of the experiment. The repair may incorporate the library sequences described herein. For example, in HDR, if homology arms on the library sequence match the sequences on either side of the break, the cell's repair machinery will incorporate the sequence from the library into the genome. In certain aspects, the library sequence may be incorporated alongside one or more barcodes sequences, such as when library sequence and a barcode sequence are on the same nucleic acid that is incorporated into a locus, or when library sequences and barcode sequences may be incorporated at separate loci.

[0077] Once the cells have had time to repair the DNA break and incorporate the library sequence and any barcode sequences, cells can be analyzed to confirm successful integration. This is typically done by sequencing, but other methods such as PCR or fluorescence microscopy (if the sequence codes for a fluorescent protein) may also be used. gRNA Targeted Cell Enrichment

[0078] Guide RNA (gRNA) mediated expression control can be used to specifically express, or suppress, one or more genes. The genes may be for resistance (e.g., resistance to an antibiotic), metabolism (e.g., surviving withdrawal of a nutrient such as an amino acid), or expressing one or more fluorescent proteins or protein that may be labeled (e.g., for cell sorting). Enrichment, such as antibiotic treatment, nutrient withdrawal, or cell sorting, can then be applied to the cell culture to specifically obtain desired cells. Methods of gRNA-mediated expression control include CRISPR interference, CRISPR activation, and Crispr-Cas editing of a promoter.

[0079] In some cases, rather than cutting the DNA, the Cas9 enzyme is deactivated (dCas9) so it can still bind to the target sequence but can no longer cut the DNA. This technique is known as CRISPR interference (CRISPRi) when used for gene repression, and CRISPR activation (CRISPRa) when used for gene activation. In CRISPRi, dCas9 is fused to a transcriptional repressor protein. The gRNA directs the dCas9-repressor complex to bind to the promoterregion, blocking transcription and effectively silencing the gene. Examples of repressor proteins include KRAB and SID4X. For CRISPRa, dCas9 is fused to a transcriptional activator protein. When directed to a promoter by the gRNA, the dCas9-activator complex enhances transcription of the downstream gene, increasing its expression. Examples of activator proteins include VP64, p65, and Rta.

[0080] CRISPR / Cas9 can be used to delete a promoter (e.g., or part of a promoter), thus reducing the expression of a gene associated with the promoter. The process may inludie designing two gRNAs to target sequences flanking the promoter. When Cas9 introduces cuts at these two locations, the region in between is excised and the remaining ends are ligated back together by the cell's repair machinery. Alternatively, a strong promoter sequence can be inserted adjacent to the gene of interest to increase its expression. In this case, a DNA repair template containing the promoter sequence (flanked by homology arms matching the sequences up and downstream of the cut site) is supplied along with the Cas9 and gRNA. When Cas9 introduces a cut at the target location, the cell's repair machinery incorporates the promoter sequence into the genome at the break site. Alternatively, or in addition, CRISPR / Cas9 can be used to delete or insert a sequence within a gene, either to correct the gene sequence or to disrupt the gene sequence.II. Overview of Methods and Systems for Dial-Out Cloning

[0081] In a first and general aspect, a method of enriching cells based on DNA sequences may include one or more of the following steps:

[0082] a) cloning nucleic acids into a population of cells, wherein the nucleic acids include library sequences and barcode sequences;

[0083] b) culturing the cells;

[0084] c) obtaining nucleic acids from a portion of the cultured cells, wherein the nucleic acids include copies of the library sequences and barcode sequences of nucleic acids that were cloned into different cells of the population of cells;

[0085] d) sequencing the obtained nucleic acids;

[0086] e) identifying, based on the sequencing, a set of one or more barcode sequences that distinguish a population of cultured cells that have a desired library sequence; and

[0087] f) enriching one or more cultured cells that include the identified set of one or more barcode sequences.

[0088] Additional aspects of the method are described further hereien. For example, the above method can be modified with appropriate cell types, library sequences, sequenceing approaches, culturing approaches, barcodes and related enrichment schemes as well as applications for enriched cells as described in the exemplary aspects below.Cell type

[0089] The method of aspect 1, wherein the cloned cells include bacteria.

[0090] The method of aspect 1 or 2, Wherein the nucleic acids include plasmids having the library sequences and the barcode sequences.

[0091] The method of aspect 1, wherein the cloned cells include eukaryotic cells.

[0092] The method of aspect 4, wherein the cloned cells include yeast cells.

[0093] The method of aspect 4, wherein the cloned cells include mammalian cells.

[0094] The method of aspect 6, wherein the cloned cells include human cells.

[0095] The method of aspect 7, wherein the human cells are immune cells.

[0096] The method of any one of aspects 1 to 8, wherein step a) of cloning includes a transfection in which the nucleic acids are inserted into the cells.

[0097] The method of aspect 9, wherein the transfection is by electroporation, chemical transfection, or mechanical disruption of the cell membrane.

[0098] The method of aspect 9 or 10, wherein the nucleic acids are in a viral vector.Library Production and Size

[0099] The method of any one of aspects 1 to 11, wherein the library sequences are assembled DNA synthesis products.

[0100] The method of aspect 12, wherein the majority of assembled DNA synthesis products are at least 500 bases in length.

[0101] The method of aspect 12 or 13, wherein the majority of library sequences of step a) are at least 90% identical.

[0102] The method of any one of aspects 1 to 14, wherein the desired library sequence is at least 1 kb in length.

[0103] The method of aspect 15, wherein the desired library sequence is at least 10 kb in length.

[0104] The method of aspect 15 or 16, wherein the desired library sequence is at least 1 kb in length, or at least 10 kb in length, and wherein sequencing include long-read sequencing and wherein individual reads include an entire library sequence and one or more barcode sequences.Sequencing Approaches

[0105] The method of any one of aspects 1 to 17, wherein step d) of sequencing includes obtaining individual sequencing reads that include both a library sequence and one or more barcode sequences from an individual nucleic acid of the nucleic acids of step c).

[0106] The method of any one of aspects 1 to 18, wherein step d) of sequencing is single molecule real time sequencing.

[0107] The method of aspect 19, wherein step d) of sequencing is nanopore sequencing.

[0108] The method of aspect 19, wherein step d) of sequencing is in optical confinement volumes.

[0109] The method of any one of aspects 1 to 21, wherein step d) of sequencing includes concatenating nucleic acids from different cells and sequencing the concatenated nucleic acids.[HO] The method of any one of aspects 1 to 22, wherein step d) of sequencing includes obtaining individual sequencing reads from multiple passes of a library sequence.[Hl] The method of aspect 23, wherein the multiple passes are by circular consensus sequencing.

[0112] The method of any one of aspects 1 to 24, wherein step d) of sequencing includes obtaining individual sequencing reads for which the majority of base calls are at an accuracy of Q40 or higher.

[0113] The method of aspect 25, wherein step d) of sequencing includes obtaining individual sequencing reads for which the majority of base calls are at an accuracy of Q50 or higher.

[0114] The method of any one of aspects 1 to 26, wherein the desired library sequence encodes a plurality of genes.

[0115] The method of aspect 27, wherein expression of each of the genes is controlled by a different promoter.

[0116] The method of any one of aspects 1 to 28, wherein library sequence encodes one or more genes encoding micro RNA.

[0117] The method of any one of aspects 1 to 29, wherein library sequence encodes one or more genes encoding a protein.

[0118] The method of any one of aspects 1 to 30, wherein the desired library sequence has over 90% homology to the majority of library sequences of the cells cultured in step b).

[0119] The method of any one of aspects 1 to 31, wherein the desired library sequence is 20 bases or more in length and replaced a sequence of over 90% homology in step a).

[0120] The method of any one of aspects 1 to 32, wherein individual library sequences of step a) each include a set of barcode sequences, wherein at least one of the library sequences of step a) includes the identified set of one or more barcode sequences of steps e) and f).Library Sequence Composition

[0121] The method of any one of aspects 1 to 33, wherein step e) includes identifying, based on the sequencing, a set of one or more barcode sequences that distinguish cultured cells that have a combination of separate desired library sequences.

[0122] The method of aspect 34, wherein different library sequences of the combination of separate desired library sequences is identified by a different barcode.

[0123] The method of any one of aspects 1 to 33, wherein step e) includes identifying, based on the sequencing, a set of one or more barcode sequences that distinguish cultured cells that have a library sequence including a combination of genes.

[0124] The method of aspect 36, wherein different genes of the combination of genes is identified by a different barcode.

[0125] The method of any one of aspects 1 to 33, wherein each library sequence of the combination of separate desired library sequences includes a unique barcode.

[0126] The method of any one of aspects 1 to 33, wherein each library sequence of the combination of separate desired library sequences includes a unique combination of barcodes, wherein different library sequences share one or more barcode sequences.General barcode and enrichment schemes

[0127] The method of any one of aspects 1 to 39, wherein cells cloned in step a) include more than 1,000 distinguishable sets of one or more barcode sequences.

[0128] The method of aspect 41, wherein cells cloned in step a) include more than 1,000,000 distinguishable sets of one or more barcode sequences.

[0129] The method of any one of aspects 1 to 41, wherein the set of one or more barcode sequences includes a unique combination of a plurality of barcode sequences that is distinct from the combination of barcode sequences of all cells that do not have the desired barcode sequence.

[0130] The method of any one of aspects 1 to 41, wherein the set of one or more barcode sequences includes a unique barcode that is distinct from the barcode sequences of all cells that do not have the desired barcode sequence.

[0131] The method of any one of aspects 1 to 43, wherein the barcode sequences encode markers that are used in step f) of enriching to sort cultured cells that have the desired library sequence.

[0132] The method of any one of aspects 1 to 43, wherein the barcode sequences are used to modify the expression of one or more genes that encode markers, and wherein the markers are used in step f) of enriching to sort cultured cells that have the desired library sequence, optionally wherein the modification of the expression of the genes is mediated by guide RNA complementary to the barcodes sequences or by sequence-codable transcription factors (TF) that are specific for the barcode sequences.Barcodes and markers for cell capture, sorting or depletion

[0133] The method of aspect 44 or 45, wherein the markers are fluorescent proteins or are surface markers stained by fluorescently labeled affinity reagents, and wherein step f) includes sorting the cultured cells that have the desired library sequence by fluorescence-activated cell sorting.

[0134] The method of aspect 44 or 45, wherein the markers are surface markers and wherein step f) includes capturing cells expressing the surface markers on a solid support.

[0135] The method of aspect 47, wherein the solid support includes a temporal series of beads that each include an affinity reagent to a different surface marker.

[0136] The method of any one of aspects 45 to 48, wherein the surface markers are each less than 100 amino acids in length.

[0137] The method of aspect 49, wherein the surface markers include two or more of 6xHis, myc, HA, AU1 and AU5.

[0138] The method of aspect 46, wherein the surface markers are each less than 100 amino acids in length, and wherein different cells express the same surface markers at different combinations of expression levels.

[0139] The method of aspect 51, wherein the expression level of each surface marker is determined by the number of copies of genes, or segments of a gene, encoding the surface marker.

[0140] The method of aspect 45, wherein enriching includes binding surface markers that are not expressed by any barcode sequences of the set of one or more barcode sequences, thereby depleting cells that are not the cultured cells that have the desired library sequence.

[0141] The method of aspect 53, wherein the solid support includes a mixture of beads that include an affinity reagents to different surface markers.

[0142] The method of aspect 47, wherein enriching includes serial binding and release of each of the surface markers that distinguish cultured cells that have the desired library sequence.

[0143] The method of any one of aspects 1 to 42, wherein the set of one or more barcode sequences provides a unique combination of expression levels of a plurality of barcodes.

[0144] The method of any one of aspects 1 to 42, wherein the set of one or more barcode sequences includes a unique combination of barcode sequences that each express a different marker and wherein each of the barcode sequences includes an associated promoter.Combinatorial barcodes

[0145] The method of any one of aspects 1 to 57, wherein each barcode of the identified set of one or more barcode sequences includes a gene expressing a different marker.

[0146] The method of any one of aspects 1 to 57, wherein each barcode of the identified set of one or more barcode sequences is within 50 bases of a promoter to a gene expressing a different marker.

[0147] The method of aspect 58 or 59, each different gene is a marker used in the enrichment of step f).

[0148] The method of any one of aspects 58 to 60, wherein the barcode sequences are random er sequences.

[0149] The method of any one of aspects 1 to 60, wherein nucleic acids of step a) include different nucleic acid molecules having different combinations of barcode sequences of a population of barcode sequences.

[0150] The method of aspect 62, wherein the population of barcode sequences includes 1000 or less barcode sequences.

[0151] The method of aspect 63, wherein the population of barcode sequences includes 100 or less barcode sequences.

[0152] The method of any one of aspects 62 to 64, wherein the number of barcode sequences on a nucleic acid is between 2 and 10, and wherein the set of barcode sequences is a combination of 2 and 10 barcode sequences.

[0153] The method of any one of aspects 62 to 65, wherein the different combinations of barcode sequences include 100 or more combinations of barcode sequences.

[0154] The method of aspect 66, wherein the different combinations of barcode sequences include 1000 or more combinations of barcode sequences.Multiplexed enrichment

[0155] The method of any one of aspects 1 to 67, wherein the cloned cells of step a) each include a set of a plurality of barcode sequences.

[0156] The method of any one of aspects 1 to 68, wherein the nucleic acids of step a) each have a library sequence and a set of one or more barcode sequence of the barcode sequences.

[0157] The method of aspect 1 or 69, wherein step e) of identifying further includes identifying additional sets of one or more barcode sequences that each distinguish cultured cells that have a desired library sequence, and wherein step f) of enriching further includes enriching cultured cells that include the identified additional sets of one or more barcode sequences.Culturing in partitions

[0158] The method of any one of aspects 1 to 70, wherein the cells are cultured in a vessel in step b).

[0159] The method of any one of aspects 1 to 71, wherein the cells are cultured in more than 10 partitions, optionally wherein the partitions are beads or droplets, optionally wherein the majority of partitions include monoclonal cultures.

[0160] The method of step 72, wherein enrichment includes sorting beads including monoclonal cultures of cells having a desired library sequence.

[0161] The method of aspect 73, wherein the particles include a plurality of fluorescent labels that are matched to the set of one or more barcodes distinguishing cells in or on the particle.

[0162] The method of aspect 74, wherein sequencing further includes sequencing particle specific barcodes and associating them with sets of one or more barcodes that distinguish cells in or on the particles.

[0163] The method of aspect 76, wherein the particle specific barcodes and associated fluorescent labels are associated with the particles in a split pool workflow, optionally wherein the particle specific barcodes include lipid anchors that embed in the surface of the cultured cells.

[0164] The method of any one of aspects 1 to 71, wherein cells from step a) are cultured in different aliquots in step b), optionally wherein the different aliquots are more than 10 aliquots.

[0165] The method of step 77, wherein step f) of enriching includes aliquot selection.

[0166] The method of step 78, wherein aliquot selection includes culturing the enriched cells in separate aliquots in step b) of culturing, obtaining nucleic acids from a portion of the cultured enriched cells in each aliquot and adding an aliquot specific barcode to each nucleic acids.

[0167] The method of aspect 79, wherein sequencing of step d) further includes sequencing aliquot specific barcodes in addition to the sets of one or more barcodes.

[0168] The method of aspect 80, wherein enriching of step f) includes separately enriching cells of each aliquot.

[0169] The method of aspect 81, wherein cells in different aliquots that have the desired library sequence are identified by a different set of one or more barcodes.Combinatorial enrichment schemes

[0170] The method of any one of aspects 1 to 82, wherein the cells enriched in step f) were cultured from a plurality of different cells cloned with different instances of the same desired library sequence and different instances of the same set of one or more barcodes in step a).

[0171] The method of any one of aspects 1 to 82, wherein the cells enriched in step f) were cultured from a plurality of different cells cloned with different instances of the same desired library sequence and different sets of one or more barcodes in step a).

[0172] The method of aspect 83 or 84, wherein another set of one or more barcodes distinguish cells that have both desired and undesired library sequences are not enriched in step f).

[0173] The method of any one of aspects 1 to 85, wherein enrichment of step f) includes a temporal series of selections, optionally wherein the selections include cell sorting, cell capture, nutrient withdrawal, and / or resistance selection.

[0174] The method of any one of aspects 1 to 85, wherein step f) of enriching includes two independent events that are both necessary to enrich cells.

[0175] The method of aspect 87, wherein the events include modification of sequences at two or more loci, modification of expression of two or more genes, modification of two or more culture conditions, and / or sorting cells based on expression of two or more markers.

[0176] The method of aspect 88, wherein the events include correction of two or more loci in one or more promoter sequences, transcriptional start sites, and / or gene sequences in the enriched cells.

[0177] The method of aspect 88, wherein the events include disruption of two or more loci in one or more promoter sequences, transcription start sites, and / or gene sequences in the enriched cells.

[0178] The method of aspect 88, wherein the two or more events include modulating expression of two or more genes, optionall wherein the expression is modulated by Cas9 or by sequence- codable transcription factors (TF).

[0179] The method of aspect 91, wherein expression of the two or more genes is increased.

[0180] The method of aspect 91, wherein expression of the two or more genes is decreased.

[0181] The method of any one of aspects 87 to 93, wherein the events further include the modification of culture conditions over the conditions in which cells were cultured in step b).

[0182] The method of aspect 94, wherein each modification includes removal of one or more nutrients.

[0183] The method of aspect 95, further including modifying cells with the set of one or more barcodes are modified to metabolize the one or more removed nutrients.

[0184] The method of any one of aspects 95 to 96, wherein the one or more removed nutrients include an amino acid.

[0185] Wherein the modification includes addition of one or more component that induce or suppress expression of one or more genes in cells with the set of one or more barcodes.

[0186] The method of any one of aspects 1 to 86, wherein the set of one or more barcode sequences includes, or controlles expression of, a unique combination of markers.

[0187] The method of aspect 99, wherein the markers are a combination of surface markers or fluorescent proteins.

[0188] The method of aspect 99, wherein the unique combination of barcodes is specifically targeted in step f) of enriching to express a combination of surface markers or fluorescentproteins, optionally wherein the barcodes are targeted by guide RNA complementary to each of the barcodes.[1891 The method of aspect 100 or 101, wherein step f) of enriching is by fluoresecence activated cell sorting of cells expressing the combination of surface markers or fluorescent proteins.

[0190] The method of aspect 100 or 101, further including sorting cells that do not express the combination of surface markers or fluorescent proteins prior to inducing expression of the surface markers or fluorescent proteins, optionally wherein the expression is induced by guide RNA complementary to the barcode sequences.

[0191] The method of any one of aspects 100 to 103, wherein the combination of surface markers are short peptide sequences that are on the same surface protein, optionally wherein the surface markers include two or more of 6xHis, myc, HA, AU1 and AU5.

[0192] The method of aspect 100 or 101, wherein step f) of enriching is by bead-based capture of the combination of surface markers.

[0193] The method of aspect 99, wherein the combinatorial barcodes includes a combination of resistances, or is the basis for modifying cells to have the combination of resistances.

[0194] The method of aspect 106, wherein step f) of enriching further includes exposing the cultured cells to a combination of compounds that only cells including the combination of markers provide resistant to.

[0195] The method of aspect 99, wherein the combinatorial barcode includes a combination of amino acid synthesis, or is the basis for modifying cells to have the combination of amino acid synthesis, and wherein step f) of enriching includes growing the cells in the absence of the combination of amino acids.

[0196] The method of aspect 99, wherein enriching includes removing cultured cells that do not include the combinatorial barcode.

[0197] Wherein the removal is by exposure of the cultured cells to a combination of antibiotics or by withdrawal of a combination of nutrients.

[0198] The method of aspect 99, wherein step f) of enriching includes transfection of the cultured cells to express a combination of markers in cells having the set of one or more barcode sequences, optionally wherein the transfection is mediated by guide RNA complementary to two or more barcode sequences of the set of one or more barcode sequences.

[0199] The method of aspect 111, wherein step f) of enriching further includes selection of transfected cultured cells having combination of markers.

[0200] The method of aspect 99, wherein step f) of enriching includes correcting or disrupting the sequence of two or more genes or their associated promoters or transcription start sites, wherein the two or more genes express the combination of markers.

[0201] The method of aspect 99, The method of wherein modifying further includes expression of each marker of the combination of markers by exposure of the cultured cells to supplements that induce expression of different markers.

[0202] The method of aspect 114, wherein the one or more supplements include an RNA switch.

[0203] The method of aspect 115, wherein the RNA switch is selected from theophylline and caffeine.

[0204] The method of any one of aspects 114 to 116, wherein two or more supplements are applied to the cells it different times.

[0205] The method of any one of aspects 99 to 117, wherein enrichment of step f) includes a temporal series of selections, optionally wherein the selections include cell sorting, cell capture, nutrient withdrawal, and / or resistance selection that specifically enriches cells having the unique combination of markers.

[0206] The method of any one of aspects 1 to 118, wherein step f) of enriching includes enriching cells by two or more different schemes.

[0207] The method of aspect 119, wherein the different schemes include two or more of nutrient withdrawal, resistance selection, temporal barcoding, cell sorting, and aliquot selection.

[0208] The method of aspect 120, wherein the different schemes include cell sorting and aliquot selection.

[0209] Avoiding evolutionary escape

[0210] The method of any one of aspects 1 to 121, wherein enrichment includes modifying two or more loci in a promoter, transcriptional start sequence (TSS), or gene that is selected on in step f) of enrichment.

[0211] The method of aspect 122, wherein modifying includes correcting a sequence.

[0212] The method of aspect 122 or 123, wherein modifying includes disrupting a sequence.

[0213] The method of aspect 122, wherein the two or more loci are within the same promoter or gene sequences.

[0214] The method of aspect 122, wherein the two or more loci are in different promoter, TSS and / or gene sequences.

[0215] The method of aspect 122, wherein the plurality of loci are each proximal to a gene necessary for cell survival.

[0216] The method of aspect 122, wherein the proximity is less than 500 bases, optionally wherein the proximity is less than 100 bases.

[0217] The method of aspect 122, wherein each of the loci are in a promoter, TSS or gene for synthesis of a nutrient withdrawn from culture in step f) of enrichment and / or for resistance to a compound that is introduced into culture in step f) of enrichment.

[0218] The method of aspect 122, wherein each of the loci are in a promoter, TSS or gene for synthesis of a nutrient withdrawn from culture in step f).

[0219] The method of aspect 131, wherein the nutrient is an amino acid.

[0220] The method of aspect 122, wherein each of the loci are in a promoter, TSS or gene for resistance to a compound that is introduced into culture in step f) of enrichment.

[0221] The method of aspect 132, wherein the compound is an antibiotic, optionally wherein the antibiotic is selected from ampicillin, kanamycin, neomycin, streptomycin, chloramphenicol, gentamicin, and spectinomycin.Modifying genes based on barcodes

[0222] The method of any one of aspects 1 to 133, wherein step f) of enriching includes modifying at least one gene, or promoter or transcription start site for the gene, wherein the gene expressing a marker that cells are enriched for in step f).

[0223] The method of aspect 132, wherein modifying includes increasing expression of the gene, decreasing expression of the gene, correcting a sequence of the gene, or disrupting a sequence of the gene.

[0224] The method of aspect 134 or 135, further including modifying of at least two genes.

[0225] The method of aspect 136, wherein the set of barcode sequences include two or more barcode sequences that are each proximal to a different promoter or transcription start sequence (TSS) of a different gene of the at least two genes.

[0226] The method of aspect 137, wherein each of the barcode sequences are within 50 nucleotides of their respective promoter or TSS.Guide RNA mediated enrichment

[0227] The method of any one of aspects 1 to 138, wherein step f) of enriching includes gRNA- mediated modulation of an expression level of a gene and further includes enriching cells with the modulated expression level.

[0228] The method of any one of aspects 1 to 139, wherein enriching includes synthesizing a guide RNA (gRNA) sequence, and wherein step f) of enriching includes gRNA-mediated modulation of expression level and further includes enriching cells with the modulated expression level.

[0229] The method of any one of aspects 1 to 140, wherein step f) of enriching includes CRISPR-Cas directed change to expression of one or more genes, wherein a Cas protein is guided by one or more guide RNAs complementary the identified set of one or more barcode sequences or a subsequence thereof, optionally wherein the Cas protein is Cas9.

[0230] The method of aspect 141, wherein the barcode sequences are random er sequences.

[0231] The method of aspect 142, wherein step f) of enriching includes synthesizing a guide RNA sequence that is complementary to one of the set of one or more barcode sequences.

[0232] The method of aspect 141, wherein Cas9 is guided by a plurality of guide RNAs each complementary to a different barcode of the identified set of one or more barcode sequences.

[0233] The method of any one of aspects 139 to 144, wherein the change in expression includes an increase in expression of a plurality of genes.

[0234] The method of aspect 145, wherein the change in expression of each of the plurality of genes is targeted by a different guide RNA.

[0235] The method of any one of aspects 139 to 144, wherein the gene express a surface markers or fluorescence protein.

[0236] The method of any one of aspects 145 to 147, wherein enriching of step e) further includes sorting cells that express the gene, optionally wherein sorting is by fluorescence activated cell sorting.

[0237] The method of aspect 145 or 146, wherein the plurality of genes each express a surface markers or fluorescence protein.

[0238] The method of aspect 149, wherein enriching of step e) further includes sorting cells that express the plurality of genes, optionally wherein sorting is by fluorescence activated cell sorting.

[0239] The method of aspect 147, wherein the fluorescence protein is selected from green fluorescent protein, red fluorescent protein, and yellow fluorescent protein.

[0240] The method of any one of aspects 145 to 151, further including removing cultured cells that express one of the plurality of genes prior to guide RNA modulation of expression level.

[0241] The method of any one of aspects 145 to 151, further including removing cultured cells that express all of the plurality of genes prior to guide RNA modulation of expression level.

[0242] The method of any one of aspects 1 to 153, wherein guide RNA modulation of expression level is by CRISPR-Cas directed change to expression is by CRISPR Interference (CRISPRi) that reduces an expression of one or more genes.

[0243] The method of any one of aspects 1 to 153, wherein guide RNA modulation of expression level is by CRISPR-Cas directed change to expression is by CRISPR Activation (CRISPRa) that increases an expression of one or more genes.

[0244] The method of any one of aspects 1 to 153, wherein enriching includes CRISPR-Cas directed of one or more loci in a promoter, transcriptional start site (TSS), and / or gene.

[0245] The method of any one of aspects 1 to 156, wherein the enriching includes CRISPR-Cas editing of two or more loci in a promoter, transcriptional start site (TSS), and / or gene.

[0246] The method of aspect 156 or 157, wherein enriching includes CRISPR-Cas directed editing of a promoter or transcriptional start site (TSS), wherein Cas9 is directed by guide RNA specific for at least one barcode sequence of the set of one or more barcode sequences.

[0247] The method of aspect 158, wherein the set of one or more barcode sequences includes a randomer sequence adjacent to a promoter or TSS, and wherein the CRISPR-Cas editing is directed based a guide RNA complementary to the randomer sequence.

[0248] The method of aspect 159, further including synthesizing the guide RNA after step d) of sequencing.

[0249] The method of any one of aspects 156 to 160, wherein the promoter or TSS is replaced or corrected by CRISPR-Cas editing.

[0250] The method of any one of aspects 156 to 160, wherein the promoter sequence or TSS is deleted or disrupted by the CRISPR-Cas directed editing.

[0251] The method of aspect 162, wherein the promoter or TSS is for a gene coding a repressor for an antibiotic resistance gene or a marker.

[0252] The method of any one aspects 139 to 163, wherein the gene encodes a resistance, wherein step f) of enriching includes exposing cultured cells to a compound that only cells expressing the gene are resistant to, optionally wherein the compound is an antibiotic, optionally wherein antibiotic is selected from ampicillin, kanamycin, neomycin, streptomycin, chloramphenicol, gentamicin, and spectinomycin.

[0253] The method of any one aspects 139 to 163, wherein the gene encodes a synthesis of a nutrient, wherein the nutrient is an amino acid that is withdrawn from a culture of the cells in step f) of enriching.

[0254] The method of any one of aspects 139 to 165, wherein the cell population of step a) expresses a Crispr-Cas complex.

[0255] The method of any one of aspects 139 to 165, wherein the nucleic acids of step a) further include a Cas9 gene and / or a guide RNA sequence.

[0256] The method of any one of aspects 139 to 167, wherein step f) of enriching includes treating the culture with a guide RNA complementary the one or more barcodes to modulate expression of the gene.

[0257] The method of any one of aspects 139 to 168, further including gRNA-mediated modulation of a second gene.Applications of Enriched Cells

[0258] The method of any one of aspects 139 to 169, further including g) isolating copies of the library sequence, or a portion thereof, from the enriched cells.

[0259] The method of any one of aspects 139 to 169, further including re-cloning the isolated copies into a population of cells and performing a second round of enrichment.

[0260] The method of aspect 171, further including transfecting the cells enriched in step f) to modify a sequence within the library sequence.

[0261] The method of aspect 172, wherein the modification of the sequence corrects the library sequence.

[0262] The method of any one of aspects 139 to 169, further including separating the enriched cells into aliquots, wherein each aliquot has more than one cell but less than 1000 cells, culturing cells in the separated aliquots, sequencing nucleic acids from cultured cells of the separated aliquots, and combining aliquots for which the cultured cells have the desired nucleic acids but do not have one or more undesired nucleic acids present in aliquots that are not combined.

[0263] The method of any one of aspects 139 to 169, wherein the cell is an immune cell, and wherein the desired library sequence includes a plurality of genes that together generate a immunotherapeutic response to a tumor.

[0264] The method of aspect 175, further including applying enriched cells, or clonally expanded enriched cells, to a subject having the tumor, thereby generating an immune response to the tumor.

[0265] The method of any one of aspects 139 to 169, wherein the cloned cells are human immune cells and further including expansion of the enriched cultured cells.

[0266] The method of aspect 177, wherein the expansion is a clonal expansion.

[0267] The method of aspect 177 or 178, further including administering the expanded cells to a subject.

[0268] The method of aspect 179, wherein the cloned cells of step a) were derived from the subject prior to step a).

[0269] The method of aspect 179, wherein the cloned cells of step a) are allogenic to the human subject.Kits and Compositions

[0270] Kits and compositions comprising one or more nucleic acid, cells, and / or reagents for performing the methods described herein are also within the scope of this application.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method of enriching cells based on DNA sequences, comprising: a) cloning nucleic acids into a population of cells, wherein the nucleic acids comprise library sequences and barcode sequences; b) culturing the cells; c) obtaining nucleic acids from a portion of the cultured cells, wherein the nucleic acids comprise copies of the library sequences and barcode sequences of nucleic acids that were cloned into different cells of the population of cells; d) sequencing the obtained nucleic acids; e) identifying, based on the sequencing, a set of one or more barcode sequences that distinguish a population of cultured cells that have a desired library sequence; and f) enriching one or more cultured cells that comprise the identified set of one or more barcode sequences.

2. The method of claim 1, wherein the cloned cells comprise bacteria.

3. The method of claim 1 or 2, Wherein the nucleic acids comprise plasmids having the library sequences and the barcode sequences.

4. The method of claim 1, wherein the cloned cells comprise eukaryotic cells.

5. The method of claim 4, wherein the cloned cells comprise yeast cells.

6. The method of claim 4, wherein the cloned cells comprise mammalian cells.

7. The method of claim 6, wherein the cloned cells comprise human cells.

8. The method of claim 7, wherein the human cells are immune cells.

9. The method of any one of claims 1 to 8, wherein step a) of cloning comprises a transfection in which the nucleic acids are inserted into the cells.

10. The method of claim 9, wherein the transfection is by electroporation, chemical transfection, or mechanical disruption of the cell membrane.

11. The method of claim 9 or 10, wherein the nucleic acids are in a viral vector.

12. The method of any one of claims 1 to 11, wherein the library sequences are assembled DNA synthesis products.

13. The method of claim 12, wherein the majority of assembled DNA synthesis products are at least 500 bases in length.

14. The method of claim 12 or 13, wherein the majority of library sequences of step a) are at least 90% identical.

15. The method of any one of claims 1 to 14, wherein the desired library sequence is at least 1 kb in length.

16. The method of claim 15, wherein the desired library sequence is at least 10 kb in length.

17. The method of claim 15 or 16, wherein the desired library sequence is at least 1 kb in length, or at least 10 kb in length, and wherein sequencing comprise long-read sequencing and wherein individual reads comprise an entire library sequence and one or more barcode sequences.

18. The method of any one of claims 1 to 17, wherein step d) of sequencing comprises obtaining individual sequencing reads that comprise both a library sequence and one or more barcode sequences from an individual nucleic acid of the nucleic acids of step c).

19. The method of any one of claims 1 to 18, wherein step d) of sequencing is single molecule real time sequencing.

20. The method of claim 19, wherein step d) of sequencing is nanopore sequencing.

21. The method of claim 19, wherein step d) of sequencing is in optical confinement volumes.

22. The method of any one of claims 1 to 21, wherein step d) of sequencing comprises concatenating nucleic acids from different cells and sequencing the concatenated nucleic acids.

23. The method of any one of claims 1 to 22, wherein step d) of sequencing comprises obtaining individual sequencing reads from multiple passes of a library sequence.

24. The method of claim 23, wherein the multiple passes are by circular consensus sequencing.

25. The method of any one of claims 1 to 24, wherein step d) of sequencing comprises obtaining individual sequencing reads for which the majority of base calls are at an accuracy of Q40 or higher.

26. The method of claim 25, wherein step d) of sequencing comprises obtaining individual sequencing reads for which the majority of base calls are at an accuracy of Q50 or higher.

27. The method of any one of claims 1 to 26, wherein the desired library sequence encodes a plurality of genes.

28. The method of claim 27, wherein expression of each of the genes is controlled by a different promoter.

29. The method of any one of claims 1 to 28, wherein library sequence encodes one or more genes encoding micro RNA.

30. The method of any one of claims 1 to 29, wherein library sequence encodes one or more genes encoding a protein.

31. The method of any one of claims 1 to 30, wherein the desired library sequence has over 90% homology to the majority of library sequences of the cells cultured in step b).

32. The method of any one of claims 1 to 31, wherein the desired library sequence is 20 bases or more in length and replaced a sequence of over 90% homology in step a).

33. The method of any one of claims 1 to 32, wherein individual library sequences of step a) each comprise a set of barcode sequences, wherein at least one of the library sequences of step a) comprises the identified set of one or more barcode sequences of steps e) and f).

34. The method of any one of claims 1 to 33, wherein step e) comprises identifying, based on the sequencing, a set of one or more barcode sequences that distinguish cultured cells that have a combination of separate desired library sequences.

35. The method of claim 34, wherein different library sequences of the combination of separate desired library sequences is identified by a different barcode.

36. The method of any one of claims 1 to 33, wherein step e) comprises identifying, based on the sequencing, a set of one or more barcode sequences that distinguish cultured cells that have a library sequence comprising a combination of genes.

37. The method of claim 36, wherein different genes of the combination of genes is identified by a different barcode.

38. The method of any one of claims 1 to 33, wherein each library sequence of the combination of separate desired library sequences comprises a unique barcode.

39. The method of any one of claims 1 to 33, wherein each library sequence of the combination of separate desired library sequences comprises a unique combination of barcodes, wherein different library sequences share one or more barcode sequences.

40. The method of any one of claims 1 to 39, wherein cells cloned in step a) comprise more than 1,000 distinguishable sets of one or more barcode sequences.

41. The method of claim 41, wherein cells cloned in step a) comprise more than 1,000,000 distinguishable sets of one or more barcode sequences.

42. The method of any one of claims 1 to 41, wherein the set of one or more barcode sequences comprises a unique combination of a plurality of barcode sequences that is distinct from the combination of barcode sequences of all cells that do not have the desired barcode sequence.

43. The method of any one of claims 1 to 41, wherein the set of one or more barcode sequences comprises a unique barcode that is distinct from the barcode sequences of all cells that do not have the desired barcode sequence.

44. The method of any one of claims 1 to 43, wherein the barcode sequences encode markers that are used in step f) of enriching to sort cultured cells that have the desired library sequence.

45. The method of any one of claims 1 to 43, wherein the barcode sequences are used to modify the expression of one or more genes that encode markers, and wherein the markers are used in step f) of enriching to sort cultured cells that have the desired library sequence, optionally wherein the modification of the expression of the genes is mediated by guide RNA complementary to the barcodes sequences or by sequence-codable transcription factors (TF) that are specific for the barcode sequences.

46. The method of claim 44 or 45, wherein the markers are fluorescent proteins or are surface markers stained by fluorescently labeled affinity reagents, and wherein step f) comprises sorting the cultured cells that have the desired library sequence by fluorescence-activated cell sorting.

47. The method of claim 44 or 45, wherein the markers are surface markers and wherein step f) comprises capturing cells expressing the surface markers on a solid support.

48. The method of claim 47, wherein the solid support comprises a temporal series of beads that each comprise an affinity reagent to a different surface marker.

49. The method of any one of claims 45 to 48, wherein the surface markers are each less than 100 amino acids in length.

50. The method of claim 49, wherein the surface markers comprise two or more of 6xHis, myc, HA, AU1 and AU5.

51. The method of claim 46, wherein the surface markers are each less than 100 amino acids in length, and wherein different cells express the same surface markers at different combinations of expression levels.

52. The method of claim 51, wherein the expression level of each surface marker is determined by the number of copies of genes, or segments of a gene, encoding the surface marker.

53. The method of claim 45, wherein enriching comprises binding surface markers that are not expressed by any barcode sequences of the set of one or more barcode sequences, thereby depleting cells that are not the cultured cells that have the desired library sequence.

54. The method of claim 53, wherein the solid support comprises a mixture of beads that comprise an affinity reagents to different surface markers.

55. The method of claim 47, wherein enriching comprises serial binding and release of each of the surface markers that distinguish cultured cells that have the desired library sequence.

56. The method of any one of claims 1 to 42, wherein the set of one or more barcode sequences provides a unique combination of expression levels of a plurality of barcodes.

57. The method of any one of claims 1 to 42, wherein the set of one or more barcode sequences comprises a unique combination of barcode sequences that each express a different marker and wherein each of the barcode sequences comprises an associated promoter.

58. The method of any one of claims 1 to 57, wherein each barcode of the identified set of one or more barcode sequences comprises a gene expressing a different marker.

59. The method of any one of claims 1 to 57, wherein each barcode of the identified set of one or more barcode sequences is within 50 bases of a promoter to a gene expressing a different marker.

60. The method of claim 58 or 59, each different gene is a marker used in the enrichment of step f).61 . The method of any one of claims 58 to 60, wherein the barcode sequences are randomer sequences.

62. The method of any one of claims 1 to 60, wherein nucleic acids of step a) comprise different nucleic acid molecules having different combinations of barcode sequences of a population of barcode sequences.

63. The method of claim 62, wherein the population of barcode sequences comprises 1000 or less barcode sequences.

64. The method of claim 63, wherein the population of barcode sequences comprises 100 or less barcode sequences.

65. The method of any one of claims 62 to 64, wherein the number of barcode sequences on a nucleic acid is between 2 and 10, and wherein the set of barcode sequences is a combination of 2 and 10 barcode sequences.

66. The method of any one of claims 62 to 65, wherein the different combinations of barcode sequences comprise 100 or more combinations of barcode sequences.

67. The method of claim 66, wherein the different combinations of barcode sequences comprise 1000 or more combinations of barcode sequences.

68. The method of any one of claims 1 to 67, wherein the cloned cells of step a) each comprise a set of a plurality of barcode sequences.

69. The method of any one of claims 1 to 68, wherein the nucleic acids of step a) each have a library sequence and a set of one or more barcode sequence of the barcode sequences.

70. The method of claim 1 or 69, wherein step e) of identifying further comprises identifying additional sets of one or more barcode sequences that each distinguish cultured cells that have a desired library sequence, and wherein step f) of enriching further comprises enriching cultured cells that comprise the identified additional sets of one or more barcode sequences.

71. The method of any one of claims 1 to 70, wherein the cells are cultured in a vessel in step b).

72. The method of any one of claims 1 to 71, wherein the cells are cultured in more than 10 partitions, optionally wherein the partitions are beads or droplets, optionally wherein the majority of partitions comprise monoclonal cultures.

73. The method of step 72, wherein enrichment comprises sorting beads comprising monoclonal cultures of cells having a desired library sequence.

74. The method of claim 73, wherein the particles comprise a plurality of fluorescent labels that are matched to the set of one or more barcodes distinguishing cells in or on the particle.

75. The method of claim 74, wherein sequencing further comprises sequencing particle specific barcodes and associating them with sets of one or more barcodes that distinguish cells in or on the particles.

76. The method of claim 76, wherein the particle specific barcodes and associated fluorescent labels are associated with the particles in a split pool workflow, optionally wherein the particle specific barcodes comprise lipid anchors that embed in the surface of the cultured cells.

77. The method of any one of claims 1 to 71, wherein cells from step a) are cultured in different aliquots in step b), optionally wherein the different aliquots are more than 10 aliquots.

78. The method of step 77, wherein step f) of enriching comprises aliquot selection.

79. The method of step 78, wherein aliquot selection comprises culturing the enriched cells in separate aliquots in step b) of culturing, obtaining nucleic acids from a portion of the cultured enriched cells in each aliquot and adding an aliquot specific barcode to each nucleic acids.

80. The method of claim 79, wherein sequencing of step d) further comprises sequencing aliquot specific barcodes in addition to the sets of one or more barcodes.

81. The method of claim 80, wherein enriching of step f) comprises separately enriching cells of each aliquot.

82. The method of claim 81, wherein cells in different aliquots that have the desired library sequence are identified by a different set of one or more barcodes.

83. The method of any one of claims 1 to 82, wherein the cells enriched in step f) were cultured from a plurality of different cells cloned with different instances of the same desired library sequence and different instances of the same set of one or more barcodes in step a).

84. The method of any one of claims 1 to 82, wherein the cells enriched in step f) were cultured from a plurality of different cells cloned with different instances of the same desired library sequence and different sets of one or more barcodes in step a).

85. The method of claim 83 or 84, wherein another set of one or more barcodes distinguish cells that have both desired and undesired library sequences are not enriched in step f).

86. The method of any one of claims 1 to 85, wherein enrichment of step f) comprises a temporal series of selections, optionally wherein the selections comprise cell sorting, cell capture, nutrient withdrawal, and / or resistance selection.

87. The method of any one of claims 1 to 85, wherein step f) of enriching comprises two independent events that are both necessary to enrich cells.

88. The method of claim 87, wherein the events comprise modification of sequences at two or more loci, modification of expression of two or more genes, modification of two or more culture conditions, and / or sorting cells based on expression of two or more markers.

89. The method of claim 88, wherein the events comprise correction of two or more loci in one or more promoter sequences, transcriptional start sites, and / or gene sequences in the enriched cells.

90. The method of claim 88, wherein the events comprise disruption of two or more loci in one or more promoter sequences, transcription start sites, and / or gene sequences in the enriched cells.

91. The method of claim 88, wherein the two or more events comprise modulating expression of two or more genes, optionall wherein the expression is modulated by Cas9 or by sequence-codable transcription factors (TF).

92. The method of claim 91, wherein expression of the two or more genes is increased.

93. The method of claim 91, wherein expression of the two or more genes is decreased.

94. The method of any one of claims 87 to 93, wherein the events further include the modification of culture conditions over the conditions in which cells were cultured in step b).

95. The method of claim 94, wherein each modification comprises removal of one or more nutrients.

96. The method of claim 95, further comprising modifying cells with the set of one or more barcodes are modified to metabolize the one or more removed nutrients.

97. The method of any one of claims 95 to 96, wherein the one or more removed nutrients comprise an amino acid.

98. Wherein the modification comprises addition of one or more component that induce or suppress expression of one or more genes in cells with the set of one or more barcodes.

99. The method of any one of claims 1 to 86, wherein the set of one or more barcode sequences comprises, or controlles expression of, a unique combination of markers.

100. The method of claim 99, wherein the markers are a combination of surface markers or fluorescent proteins.

101. The method of claim 99, wherein the unique combination of barcodes is specifically targeted in step f) of enriching to express a combination of surface markers or fluorescent proteins, optionally wherein the barcodes are targeted by guide RNA complementary to each of the barcodes.

102. The method of claim 100 or 101, wherein step f) of enriching is by fluoresecence activated cell sorting of cells expressing the combination of surface markers or fluorescent proteins.

103. The method of claim 100 or 101, further comprising sorting cells that do not express the combination of surface markers or fluorescent proteins prior to inducing expression of the surface markers or fluorescent proteins, optionally wherein the expression is induced by guide RNA complementary to the barcode sequences.

104. The method of any one of claims 100 to 103, wherein the combination of surface markers are short peptide sequences that are on the same surface protein, optionally wherein the surface markers comprise two or more of 6xHis, myc, HA, AU1 and AU5.

105. The method of claim 100 or 101, wherein step f) of enriching is by bead-based capture of the combination of surface markers.

106. The method of claim 99, wherein the combinatorial barcodes comprises a combination of resistances, or is the basis for modifying cells to have the combination of resistances.

107. The method of claim 106, wherein step f) of enriching further comprises exposing the cultured cells to a combination of compounds that only cells comprising the combination of markers provide resistant to.

108. The method of claim 99, wherein the combinatorial barcode comprises a combination of amino acid synthesis, or is the basis for modifying cells to have the combination of amino acid synthesis, and wherein step f) of enriching comprises growing the cells in the absence of the combination of amino acids.

109. The method of claim 99, wherein enriching comprises removing cultured cells that do not comprise the combinatorial barcode.

110. Wherein the removal is by exposure of the cultured cells to a combination of antibiotics or by withdrawal of a combination of nutrients.

111. The method of claim 99, wherein step f) of enriching comprises transfection of the cultured cells to express a combination of markers in cells having the set of one or more barcode sequences, optionally wherein the transfection is mediated by guide RNA complementary to two or more barcode sequences of the set of one or more barcode sequences.

112. The method of claim 111, wherein step f) of enriching further comprises selection of transfected cultured cells having combination of markers.

113. The method of claim 99, wherein step f) of enriching comprises correcting or disrupting the sequence of two or more genes or their associated promoters or transcription start sites, wherein the two or more genes express the combination of markers.

114. The method of claim 99, The method of wherein modifying further comprises expression of each marker of the combination of markers by exposure of the cultured cells to supplements that induce expression of different markers.

115. The method of claim 114, wherein the one or more supplements comprise an RNA switch.

116. The method of claim 115, wherein the RNA switch is selected from theophylline and caffeine.

117. The method of any one of claims 114 to 116, wherein two or more supplements are applied to the cells it different times.

118. The method of any one of claims 99 to 117, wherein enrichment of step f) comprises a temporal series of selections, optionally wherein the selections comprise cellsorting, cell capture, nutrient withdrawal, and / or resistance selection that specifically enriches cells having the unique combination of markers.

119. The method of any one of claims 1 to 118, wherein step f) of enriching comprises enriching cells by two or more different schemes.

120. The method of claim 119, wherein the different schemes include two or more of nutrient withdrawal, resistance selection, temporal barcoding, cell sorting, and aliquot selection.

121. The method of claim 120, wherein the different schemes include cell sorting and aliquot selection.

122. The method of any one of claims 1 to 121, wherein enrichment comprises modifying two or more loci in a promoter, transcriptional start sequence (TSS), or gene that is selected on in step f) of enrichment.

123. The method of claim 122, wherein modifying comprises correcting a sequence.

124. The method of claim 122 or 123, wherein modifying comprises disrupting a sequence.

125. The method of claim 122, wherein the two or more loci are within the same promoter or gene sequences.

126. The method of claim 122, wherein the two or more loci are in different promoter, TSS and / or gene sequences.

127. The method of claim 122, wherein the plurality of loci are each proximal to a gene necessary for cell survival.

128. The method of claim 122, wherein the proximity is less than 500 bases, optionally wherein the proximity is less than 100 bases.

129. The method of claim 122, wherein each of the loci are in a promoter, TSS or gene for synthesis of a nutrient withdrawn from culture in step f) of enrichment and / or for resistance to a compound that is introduced into culture in step f) of enrichment.

130. The method of claim 122, wherein each of the loci are in a promoter, TSS or gene for synthesis of a nutrient withdrawn from culture in step f).

131. The method of claim 131, wherein the nutrient is an amino acid.

132. The method of claim 122, wherein each of the loci are in a promoter, TSS or gene for resistance to a compound that is introduced into culture in step f) of enrichment.

133. The method of claim 132, wherein the compound is an antibiotic, optionally wherein the antibiotic is selected from ampicillin, kanamycin, neomycin, streptomycin, chloramphenicol, gentamicin, and spectinomycin.

134. The method of any one of claims 1 to 133, wherein step f) of enriching comprises modifying at least one gene, or promoter or transcription start site for the gene, wherein the gene expressing a marker that cells are enriched for in step f).

135. The method of claim 132, wherein modifying comprises increasing expression of the gene, decreasing expression of the gene, correcting a sequence of the gene, or disrupting a sequence of the gene.

136. The method of claim 134 or 135, further comprising modifying of at least two genes.

137. The method of claim 136, wherein the set of barcode sequences comprise two or more barcode sequences that are each proximal to a different promoter or transcription start sequence (TSS) of a different gene of the at least two genes.

138. The method of claim 137, wherein each of the barcode sequences are within 50 nucleotides of their respective promoter or TSS.

139. The method of any one of claims 1 to 138, wherein step f) of enriching comprises gRNA-mediated modulation of an expression level of a gene and further comprises enriching cells with the modulated expression level.

140. The method of any one of claims 1 to 139, wherein enriching comprises synthesizing a guide RNA (gRNA) sequence, and wherein step f) of enriching comprises gRNA-mediated modulation of expression level and further comprises enriching cells with the modulated expression level.

141. The method of any one of claims 1 to 140, wherein step f) of enriching comprises CRISPR-Cas directed change to expression of one or more genes, wherein a Cas protein is guided by one or more guide RNAs complementary the identified set of one or more barcode sequences or a subsequence thereof, optionally wherein the Cas protein is Cas9.

142. The method of claim 141, wherein the barcode sequences are randomer sequences.

143. The method of claim 142, wherein step f) of enriching comprises synthesizing a guide RNA sequence that is complementary to one of the set of one or more barcode sequences.

144. The method of claim 141, wherein Cas9 is guided by a plurality of guide RNAs each complementary to a different barcode of the identified set of one or more barcode sequences.

145. The method of any one of claims 139 to 144, wherein the change in expression comprises an increase in expression of a plurality of genes.

146. The method of claim 145, wherein the change in expression of each of the plurality of genes is targeted by a different guide RNA.

147. The method of any one of claims 139 to 144, wherein the gene express a surface markers or fluorescence protein.

148. The method of any one of claims 145 to 147, wherein enriching of step e) further comprises sorting cells that express the gene, optionally wherein sorting is by fluorescence activated cell sorting.

149. The method of claim 145 or 146, wherein the plurality of genes each express a surface markers or fluorescence protein.

150. The method of claim 149, wherein enriching of step e) further comprises sorting cells that express the plurality of genes, optionally wherein sorting is by fluorescence activated cell sorting.

151. The method of claim 147, wherein the fluorescence protein is selected from green fluorescent protein, red fluorescent protein, and yellow fluorescent protein.

152. The method of any one of claims 145 to 151, further comprising removing cultured cells that express one of the plurality of genes prior to guide RNA modulation of expression level.

153. The method of any one of claims 145 to 151, further comprising removing cultured cells that express all of the plurality of genes prior to guide RNA modulation of expression level.

154. The method of any one of claims 1 to 153, wherein guide RNA modulation of expression level is by CRISPR-Cas directed change to expression is by CRISPR Interference (CRISPRi) that reduces an expression of one or more genes.

155. The method of any one of claims 1 to 153, wherein guide RNA modulation of expression level is by CRISPR-Cas directed change to expression is by CRISPR Activation (CRISPRa) that increases an expression of one or more genes.

156. The method of any one of claims 1 to 153, wherein enriching comprises CRISPR- Cas directed of one or more loci in a promoter, transcriptional start site (TSS), and / or gene.

157. The method of any one of claims 1 to 156, wherein the enriching comprises CRISPR-Cas editing of two or more loci in a promoter, transcriptional start site (TSS), and / or gene.

158. The method of claim 156 or 157, wherein enriching comprises CRISPR-Cas directed editing of a promoter or transcriptional start site (TSS), wherein Cas9 is directed by guide RNA specific for at least one barcode sequence of the set of one or more barcode sequences.

159. The method of claim 158, wherein the set of one or more barcode sequences comprises a randomer sequence adjacent to a promoter or TSS, and wherein the CRISPR- Cas editing is directed based a guide RNA complementary to the randomer sequence.

160. The method of claim 159, further comprising synthesizing the guide RNA after step d) of sequencing.

161. The method of any one of claims 156 to 160, wherein the promoter or TSS is replaced or corrected by CRISPR-Cas editing.

162. The method of any one of claims 156 to 160, wherein the promoter sequence or TSS is deleted or disrupted by the CRISPR-Cas directed editing.

163. The method of claim 162, wherein the promoter or TSS is for a gene coding a repressor for an antibiotic resistance gene or a marker.

164. The method of any one claims 139 to 163, wherein the gene encodes a resistance, wherein step f) of enriching comprises exposing cultured cells to a compound that only cells expressing the gene are resistant to, optionally wherein the compound is an antibiotic, optionally wherein antibiotic is selected from ampicillin, kanamycin, neomycin, streptomycin, chloramphenicol, gentamicin, and spectinomycin.

165. The method of any one claims 139 to 163, wherein the gene encodes a synthesis of a nutrient, wherein the nutrient is an amino acid that is withdrawn from a culture of the cells in step f) of enriching.

166. The method of any one of claims 139 to 165, wherein the cell population of step a) expresses a Crispr-Cas complex.

167. The method of any one of claims 139 to 165, wherein the nucleic acids of step a) further comprise a Cas9 gene and / or a guide RNA sequence.

168. The method of any one of claims 139 to 167, wherein step f) of enriching comprises treating the culture with a guide RNA complementary the one or more barcodes to modulate expression of the gene.

169. The method of any one of claims 139 to 168, further comprising gRNA-mediated modulation of a second gene.

170. The method of any one of claims 139 to 169, further comprising g) isolating copies of the library sequence, or a portion thereof, from the enriched cells.

171. The method of any one of claims 139 to 169, further comprising re-cloning the isolated copies into a population of cells and performing a second round of enrichment.

172. The method of claim 171, further comprising transfecting the cells enriched in step f) to modify a sequence within the library sequence.

173. The method of claim 172, wherein the modification of the sequence corrects the library sequence.

174. The method of any one of claims 139 to 169, further comprising separating the enriched cells into aliquots, wherein each aliquot has more than one cell but less than 1000 cells, culturing cells in the separated aliquots, sequencing nucleic acids from cultured cells of the separated aliquots, and combining aliquots for which the cultured cells have the desired nucleic acids but do not have one or more undesired nucleic acids present in aliquots that are not combined.

175. The method of any one of claims 139 to 169, wherein the cell is an immune cell, and wherein the desired library sequence comprises a plurality of genes that together generate a immunotherapeutic response to a tumor.

176. The method of claim 175, further comprising applying enriched cells, or clonally expanded enriched cells, to a subject having the tumor, thereby generating an immune response to the tumor.

177. The method of any one of claims 139 to 169, wherein the cloned cells are human immune cells and further comprising expansion of the enriched cultured cells.

178. The method of claim 177, wherein the expansion is a clonal expansion.

179. The method of claim 177 or 178, further comprising administering the expanded cells to a subject.

180. The method of claim 179, wherein the cloned cells of step a) were derived from the subject prior to step a).

181. The method of claim 179, wherein the cloned cells of step a) are allogenic to the human subject.

Images