Gramc: genome-scale reporter assay method for cis-regulatory modules

Abstract

To provide a GRAMC: genome-scale reporter assay method for cis-regulatory modules.SOLUTION: Disclosed herein are libraries of reporter nucleic acids for functional regulatory elements as well as methods and kits for constructing and using such libraries. Exemplary libraries, methods, and kits can be used for high-throughput detection, identification, and / or quantitation of functional regulatory elements. In some examples, the nucleic acids are genomic DNA obtained from a cell or population of cells of interest. The genomic DNA can be from any organism of interest, including, but not limited to animals (for example, mammals), plants, bacteria, fungi, or archaea.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 753,608, filed October 31, 2018, which is incorporated by reference in its entirety.

[0002] Field The present application provides libraries of reporter nucleic acids, eg, functional regulatory elements, as well as methods and kits for constructing and using such libraries. [Background technology]

[0003] Cis-regulatory modules (CRMs), such as enhancers, promoters, and repressors, are functional elements of the genome. It has been estimated that hundreds of thousands of CRMs are scattered throughout the human genome (Niu, et al. Nucleic acids research 46.11 (2018): 5395-5409; Visel, et al. Nature 461.7261 (2009):199;ENCODE Project Consortium. Nature 489.7414 (2012):57). CRMs determine when, where, and how genes are expressed. CRMs are involved in nearly every biological process because they regulate how many of the genes are expressed at the level of the transcription factor. Individual CRMs interact directly with multiple transcription factors, and multiple CRMs function together to mediate gene regulatory activity (Davidson. The Regulatory Genome, Elsevier (2006); Levine, et al. Cell 157.1 (2014): 13-25;De Laat, et al. Nature 502.7472 (2013): 499). Comprehensive experimental identification of such elements is difficult.

[0004] The standard reporter assay for identifying CRMs is to clone a candidate CRM upstream of a basal promoter and a reporter gene and examine its ability to drive expression of the reporter gene (Rosenthal, Methods in enzymology 152 (1987): 704-720; Arnone, et al. Methods in cell biology 74. (2004): 621-652; Banerji, et al. Cell 27.2 (1981): 299-308). The same reporter constructs allow the detection of CRMs in response to genetic perturbations (Nam, et al. PLoS One 7.4 (2012): e35934.) , and for mutations in transcription binding sites (Damle, et al. Developmental biology 357.2 (2011): 505-517;de-Leon, et al. PNAS USA 107.22 (2010): 10103-10108;Cui, et al. Cell reports 19.2 (2017): 364-374;Emison, et al. Nature 434.7035 (2005): 857; Guerreiro, et al. PNAS USA 110.26 (2013): 10682-10686), it can be monitored how the reporter responds. However, such traditional one-by-one reporter assays are not suitable for analyzing (e.g., high-throughput analysis) the millions of potential CRMs contained in a genome. Several high-throughput assays have been attempted, but bias can be an issue. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Rosenthal, Methods in enzymology (1987) 152:704~720 [Non-Patent Document 2] Arnone et al., Methods in cell biology (2004) 74:621~652 [Non-Patent Document 3] Banerji et al., Cell (1981) 27.2:299–308 Summary of the Invention [Means for solving the problem]

[0006] Disclosed herein are methods for constructing a nucleic acid molecule reporter library, and nucleic acid molecule reporter libraries produced using the methods disclosed herein. The genome-scale reporter assay method disclosed herein is effective for both enhancers and promoters, as is the case with standard reporter assays. The assay also accommodates long DNA inserts, allowing for screening of complete CRMs rather than partial CRMs. Excessive genome coverage and DNA barcodes increase experimental costs, while insufficient genome coverage and DNA barcodes result in unreliable data. However, with the libraries and methods disclosed herein, genome coverage and the number of DNA barcodes in the library are adjustable. Finally, the assay produces reproducible data with comparable or less input material than currently available methods.

[0007] In some embodiments, a method of constructing a nucleic acid molecule reporter library includes the steps of isolating a plurality of nucleic acid molecules (e.g., genomic DNA or synthetic DNA) of a selected size range (e.g., a size range of 100-3000 base pairs long, such as about 750-850 base pairs long); ligating the plurality of isolated nucleic acid molecules to at least one linear adapter sequence (such as an adapter comprising at least two consecutive ribonucleotides flanked by at least one deoxyribonucleotide at the 3' end and at least one deoxyribonucleotide at the 5' end) to form a plurality of circular nucleic acid molecules comprising inserts (isolated nucleic acid molecules) and adapters; contacting the plurality of circular nucleic acid molecules with an enzyme under conditions sufficient to produce a plurality of linear nucleic acid molecules; and fusing the plurality of linear nucleic acid molecules with at least one reporter nucleic acid to produce a plurality of reporter constructs, forming a nucleic acid molecule reporter library.

[0008] Any nucleic acid molecule can be used, including genomic DNA (such as genomic DNA fragments) or synthetic DNA. In some examples, the nucleic acid is genomic DNA obtained from a cell of interest or a population of cells of interest. The genomic DNA may be from any organism of interest, including, but not limited to, an animal (e.g., a mammal), a plant, a bacteria, a fungus, or an archaea. In some examples, the method includes using gel electrophoresis or bead-based size selection to select a size range of isolated nucleic acid molecules. In some examples, the method includes ligating a plurality of isolated nucleic acid molecules to at least one linear adaptor sequence using a ligase. In some examples, the ligase includes a DNA ligase, such as T4 DNA ligase. The linear adaptor sequence may include at least two consecutive ribonucleotides (e.g., nucleic acids of SEQ ID NO: 1 and / or SEQ ID NO: 2) flanked by at least one deoxyribonucleotide at the 3' end and at least one deoxyribonucleotide at the 5' end. Thus, the ligation produces a plurality of circular nucleic acid molecules that include the insert and the adaptor.

[0009] In some examples, the method further comprises contacting the plurality of circular nucleic acid molecules with an exonuclease (e.g., exonuclease I, exonuclease III, and / or lambda exonuclease) under conditions sufficient to remove linear nucleic acid molecules from the plurality of circular nucleic acid molecules prior to linearizing the circular nucleic acid. In some examples, the method then comprises contacting the plurality of circular nucleic acid molecules with an endoribonuclease (e.g., an endoribonuclease specific for ribonucleotides in a DNA duplex, such as RNase HII or uracil-DNA glycosylase) under conditions sufficient to produce a plurality of linear nucleic acid molecules, each of which comprises at least one deoxyribonucleotide at the 3' end and at least one deoxyribonucleotide at the 5' end, flanked by inserts. In some examples, the method comprises fusing the plurality of linear nucleic acid molecules with at least one reporter nucleic acid (e.g., a nucleic acid encoding a fluorescent protein and / or a nucleic acid comprising a barcode) to produce a plurality of reporter constructs.

[0010] In some examples, the method further comprises determining the genome coverage of the plurality of linear nucleic acid molecules. For example, determining the genome coverage may comprise selecting at least one genome region of interest, amplifying the plurality of linear nucleic acid molecules, and determining whether the selected genome region is present in the plurality of linear nucleic acid molecules, the copy number of the selected genome region in the plurality of linear nucleic acid molecules, and / or the genome coverage. In some examples, the genome coverage is determined by selecting one or more single copy targets for analysis. Exemplary single copy targets include ACTA1, ADM, ADAM12, AXL, CFB, DLX5, Kiss1, NCOA6, Notch2, RPP30, and TOP1. Depending on the source of the starting material of the library, additional or alternative single copy targets can be selected.

[0011] In some examples, the method includes fusing a plurality of nucleic acid molecules with a linear vector nucleic acid (e.g., a linear vector nucleic acid that includes a basal promoter). Thus, the method can be used to produce a plurality of linear vectors that include the nucleic acid molecules.

[0012] In some examples, the at least one reporter nucleic acid comprises a nucleic acid encoding a fluorescent protein, and fusing the plurality of linear nucleic acid molecules with the at least one reporter nucleic acid comprises fusing a plurality of linear vectors with a fluorescent reporter nucleic acid. Thus, the method can be used to produce a plurality of fluorescent reporter constructs. In another example, the at least one reporter nucleic acid comprises a nucleic acid encoding a barcode, and fusing the plurality of linear nucleic acid molecules with the at least one reporter nucleic acid comprises fusing a plurality of reporter linear vectors with the barcode nucleic acid. Thus, the method can be used to produce a plurality of barcode reporter constructs. In some examples, the at least one reporter nucleic acid comprises a nucleic acid encoding a barcode and a nucleic acid encoding a fluorescent protein, and fusing the plurality of linear vectors with the at least one reporter nucleic acid comprises fusing a plurality of reporter constructs with the barcode nucleic acid and the nucleic acid encoding a fluorescent protein. Thus, the method can be used to produce a plurality of fluorescent and barcode reporter constructs.

[0013] In some examples, the method further comprises contacting each of the plurality of linear vectors with a primer nucleic acid comprising a barcode reporter construct. In some examples, the method then comprises performing a polymerase chain reaction (PCR). Thus, the method herein can be used to produce a plurality of amplified vectors comprising a barcode reporter construct. In some examples, the method then comprises self-ligating the amplified vectors comprising the barcode reporter construct to produce a circular vector. Thus, the method herein can be used to produce a barcode reporter construct. In some examples, the method herein further comprises contacting the plurality of circular vectors comprising the barcode reporter construct with an exonuclease (e.g., exonuclease I, exonuclease III, and / or lambda exonuclease) under conditions sufficient to remove linear nucleic acid molecules from the plurality of circular vectors comprising the barcode reporter construct.

[0014] In a particular example of a method for constructing a nucleic acid molecule reporter library, the method includes the steps of isolating a plurality of nucleic acid molecules of a selected size range; ligating the plurality of isolated nucleic acid molecules to at least one linear adaptor sequence using a ligase, the linear adaptor sequence comprising at least two consecutive ribonucleotides flanked by at least one deoxyribonucleotide at the 3' end and at least one deoxyribonucleotide at the 5' end, thereby producing a plurality of circular nucleic acid molecules comprising an insert and an adaptor; contacting the plurality of circular nucleic acid molecules with an exonuclease under conditions sufficient to remove the linear nucleic acid molecule from the plurality of circular nucleic acid molecules; ligating the plurality of circular nucleic acid molecules to at least one linear adaptor sequence, each flanked by an insert, with an exonuclease under conditions sufficient to remove the linear nucleic acid molecule from the plurality of circular nucleic acid molecules; and fusing the plurality of linear nucleic acid molecules with at least one reporter nucleic acid to produce a plurality of reporter constructs, such as by (a) fusing the plurality of nucleic acid molecules with the linear vector nucleic acid, thereby producing a plurality of linear vectors comprising the nucleic acid molecules, (b) contacting each of the plurality of linear vectors comprising the nucleic acid molecules with a primer comprising a barcode nucleic acid, and (c) performing a polymerase chain reaction (PCR) and a ligation reaction to produce a plurality of circular vectors comprising the barcode reporter constructs; and contacting the plurality of circular vectors comprising the barcode reporter constructs with an exonuclease under conditions sufficient to remove the linear nucleic acid molecule from the plurality of circular vectors comprising the barcode reporter constructs. In some examples, the method further comprises determining the genome coverage of the insert before fusing the plurality of linear nucleic acid molecules with the at least one reporter nucleic acid.

[0015] Further disclosed herein is a method (e.g., a high-throughput method) for detecting functional nucleic acid regulatory elements. In some examples, the method includes transfecting or transforming any of the libraries disclosed herein into at least one cell of interest. Exemplary cells include animal (e.g., mammalian) cells, bacterial cells, plant cells, fungal cells, and archaeal cells. For example, mammalian cells can include cardiomyocytes, neurons, hepatocytes, endothelial cells, embryonic stem cells, organoid-derived cells, organoid-derived cells, and induced stem cells. In some examples, the method includes collecting at least one cell of interest from at least two subjects, the at least two subjects including at least one subject with a disease or condition and at least one subject without a disease or condition. In some examples, the method includes collecting at least one cell of interest from at least one subject, from which a plurality of cells are collected under different conditions.

[0016] Also, in some examples, the method includes measuring at least one reporter. For example, some methods may include identifying and / or quantifying at least one reporter. In some examples, the method includes isolating RNA from a cell of interest to produce an isolated RNA. In some examples, identifying the reporter includes reverse transcribing the isolated RNA to produce cDNA, such as using recombinant Moloney murine leukemia virus (rMoMuLV) reverse transcriptase or avian myeloblastosis virus (AMV) reverse transcriptase. Also, in certain examples, the isolated RNA can be reverse transcribed using RNA-dependent and DNA-dependent DNA polymerases.

[0017] In some examples, the method then comprises detecting the cDNA. In some examples, the detection comprises amplifying the cDNA. For example, when the at least one reporter is at least one unique barcode nucleic acid, amplifying the cDNA may comprise selecting a primer specific to the nucleotide comprising at least one unique nucleic acid barcode, contacting the primer with the cDNA, and carrying out PCR using the primer and the cDNA to produce amplified DNA.

[0018] In some examples, the method further comprises identifying at least one unique nucleic acid barcode. In some examples, the at least one unique nucleic acid barcode is identified by sequencing the amplified DNA. Also, in some examples, the method comprises quantifying the at least one unique nucleic acid barcode.

[0019] In some examples of the methods herein, the plurality of nucleic acid molecules, e.g., the plurality of nucleic acid molecules in a library produced using the methods described herein, comprises at least 80% of a selected genome of interest. In some examples of the methods herein, the plurality of nucleic acid molecules comprises at least 80% of the cis-regulatory elements in the selected genome of interest.

[0020] Also disclosed herein is a kit for constructing a nucleic acid molecule reporter library. In some examples, the kit comprises at least one of any of the reporter nucleic acids described herein. In some examples, the reporter nucleic acid comprises the linear adaptor sequence of SEQ ID NO:1 and / or SEQ ID NO:2. Exemplary kits may also comprise at least one ligase, exonuclease, endoribonuclease, and / or polymerase.

[0021] Further disclosed herein is a kit for high-throughput identification and / or quantification of functional nucleic acid regulatory elements.In some examples, the kit comprises any of the libraries disclosed herein, such as a library that covers at least 80% of the genome of interest.An additional example of the kit comprises at least one reverse transcriptase and / or PCR primer and high fidelity DNA polymerase.

[0022] The above and other features of the present disclosure will become more apparent from the following detailed description taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0023] [Figure 1-1]1A-1D are diagrams illustrating GRAMc library construction. FIG. 1A shows an exemplary method for controlling the genome coverage of the library. Size-selected and end-repaired random genomic DNA fragments were circularized by ligation with fusion adapters. Linear DNA was removed by exonuclease treatment, followed by digestion with RNaseHII to linearize the ligation products and diced adapter-concatamers. The adapter-ligated products were then serially diluted to determine the genome coverage of each dilution by QPCR. Dilutions of intended coverage were assembled using GIBSON ASSEMBLY® with the SCP-GFP cassette and vector backbone to form a barcode-free linear construct. FIG. 1B is a schematic diagram illustrating an exemplary method for controlling the number of barcodes in the library. Random 25 bp (N25) barcodes and core polyadenylation signals were added to the library of linear constructs by PCR. The barcoded constructs were allowed to self-ligate and linear DNA was removed with exonuclease I / III. A small portion of the ligates was transformed to determine the scale of transformation. To avoid an increase in colony counts due to cell division, transformants for colony counts should be plated immediately without rescue. The desired amount of ligates was transformed to produce a GRAMc library with the intended number of barcodes. Plasmids extracted from the liquid culture were used for library characterization and reporter assays. Inserts and associated barcodes were identified by Illumina paired-end sequencing. Figure 1C shows the size distribution of inserts in the human GRAMc library. Figure 1D shows the cumulative distribution of the number of barcodes per insert in the human GRAMc library. [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 1-4] Same as above.

[0024] [Figure 2-1]Figures 2A-2E show the reproducibility and accuracy of GRAMc. Figure 2A shows the reproducibility of GRAMc results. The human GRAMc library was tested in two batches of 200M HepG2 cells. CRM activity was double normalized to the copy number of the input plasmid and background activity (bg). Inserts that drove reporter expression ≥ 5 × bg in one batch and ≥ 4.5 × bg in another batch were considered CRMs ("active"), and CRM calls were 80% reproducible. Inserts that did not meet the cutoff but were still ≥ 3 × bg in one batch and ≥ 2.7 × bg in another batch were considered marginally active. Reproducibility was lower at 62%. Figure 2B shows validation of GRAMc results by individual reporter assays. A set of 11 CRMs ("active"), 5 marginally active inserts, and 4 inactive inserts were tested in 4 batches of individual reporter assays by QPCR. The average activity (solid bars) of four batches of individual reporter assays was compared to the GRAMc data (R2=0.83). Figure 2C shows the correlated genomic distribution of CRMs (top row) and expressed genes (middle row) on chromosome 1. The genomic distribution of the input library is shown in the bottom row. Inserts originating from centromeres were removed. Figure 2D shows the enrichment of CRMs in 2 kb windows with up to 100 kb of flanking regions of expressed (black dots) and non-expressed (grey dots) genes. The genomic average is shown by the dashed line. The gene region is at position 0 and includes both exons and introns. The upstream area of ​​the gene is shown in the left half and the downstream area in the right half. Figure 2E shows the relative enrichment of ENCODE chromatin annotations of CRMs (G5, greater than 5xbg) versus inactive inserts (L1, lower than 1xbg). ENCODE annotations are ranked based on their relative enrichment. [Figure 2-2] Same as above. [Figure 2-3] Same as above. [Figure 2-4] Same as above. [Figure 2-5] Same as above.

[0025] [Figure 3-1] Figures 3A-3G show that cis-regulatory activity and TFBS motif enrichment in ChromHMM predicted strong enhancers. Figure 3A shows predicted enhancer enrichment of CRMs (black bars) versus CRM activity measured by GRAMc (grey bars). Inserts were sorted by their average activity in two batches of GRAMc data: G5, greater than 5xbg; G3L5, equal to or greater than 3xbg and less than 5xbg; G2L3, equal to or greater than 2xbg and less than 3xbg; G1L2, equal to or greater than 1xbg and less than 2xbg; and L1, less than 1xbg. Figures 3B-3G show relative motif enrichment (log2 scale) of predicted enhancers with progressively weaker activity versus GRAMc-identified CRMs (G5). Each dot represents a TFBS motif and the lines indicate the two-fold difference between the two data sets. The upper left box of each plot indicates the percentage of predicted enhancers in each bin. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Diagram 3-4] Same as above. [Figure 3-5] Same as above. [Diagram 3-6] Same as above. [Diagram 3-7] Same as above.

[0026] [Figure 4-1]Figures 4A-4E show CRM-driven prediction of gene regulatory programs. Figure 4A shows the abundance and enrichment of TFBS motifs in CRMs. Abundance is the proportion of CRMs (G5 set) or inactive set (L1 set) that contain a given TFBS motif, and relative enrichment is the ratio of motif enrichment between the G5 set and the L1 set. Vertical lines indicate the border of relative enrichment of motifs. Several highly enriched and abundant motifs are labeled. Figure 4B shows a comparison of enrichment of predicted TFBS motifs and ENCODE ChIP-seq annotations in the G5 set. Figure 4C shows two alternative hypotheses regarding the role of PITX2 or IKZF1 on HepG2-CRM in other cells (cell X). Figures 4D-4E show the test of the hypothesis on enriched TFBS motifs of non-expressed transcription factors in HepG2 by ectopic expression of human pitx2 (Figure 4D) vs. CMV::gfp control and human ikzf1 (Figure 4E) vs. CMV::gfp control. Inserts belonging to the G5 set are indicated by red dots (motif+) or black dots (motif-). The two black diagonal lines indicate the 2-fold difference between the perturbed and control sets. The inset box plots show the difference between motif+ and motif-inserts with P-values ​​using a two-sample t-test. [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 4-4] Same as above. [Figure 4-5] Same as above.

[0027] [Figure 5-1] Figures 5A-5B show the enrichment of repetitive elements in the GRAMc data. Inserts were sorted by their average activity in two batches of GRAMc data as in Figures 3A-3G. Figure 5A shows a representative family of repetitive elements in the GRAMc data. The enrichment of repetitive elements in genomic regions with different activities is shown. The G5 set of genomic regions was considered as CRMs. Figure 5B shows the enrichment of the three major subfamilies of Alu elements in the GRAMc data. [Figure 5-2] Same as above.

[0028] [Figure 6-1] 6A-6B are diagrams illustrating the generation of fusion adapters and adapter-ligated inserts. FIG. 6A shows a fusion adapter. A fusion adapter is prepared by annealing two 5' phosphorylated oligomers (top row, SEQ ID NO:1; bottom row, SEQ ID NO:2). The fusion adapter contains two primer sites, P1 (yellow arrow) and P2 (crimson arrow), for amplifying the adapter-ligated genomic insert. The boxes indicate two ribonucleotides for RNase HII cleavage. FIG. 6B shows an exemplary method for preparing a pure population of adapter-ligated inserts. Ligation of the insert with the fusion adapter generated circular DNA that was resistant to exonuclease treatment. All undesired linear DNA was removed with exonuclease I / III. Because circular DNA is difficult to amplify using PCR, the circular ligation product was linearized with RNase HII. At this point, the linearized adapter-ligated insert was ready for PCR amplification with P1 and P2 primers. [Figure 6-2] Same as above.

[0029] [Figure 7]FIG. 7 is a schematic diagram showing an exemplary method for preparing the GRAMc vector for GIBSON ASSEMBLY®. The GRAMc vector is linearized by digestion with AflII and HindIII to increase the efficiency and reduce the cycles required for amplification. After digestion, the vector is amplified into two pieces, one containing the SCP-GFP cassette and the other containing the vector backbone. Primers NJ96 and NJ95 add P1 and P2 sites to the vector backbone cassette and the SCP-GFP cassette, respectively, for subsequent GIBSON ASSEMBLY® with adaptor-ligated inserts. Primers NJ146 and NJ145 contain a sequence of six phosphorothioated nucleotides (designated S6) at the 5' end to protect the terminal primer sites from degradation during GIBSON ASSEMBLY® and to allow efficient amplification of pre-barcoded libraries.

[0030] [Figure 8]FIG. 8 shows an exemplary method for constructing paired-end sequencing libraries for Illumina NextSeq500. PCR of the GRAMc library was performed with two pairs of primers (P2 / nP3 and P1 / P4) to the insert and adapter sequences flanked by the N25 barcode, followed by self-ligation. This generates two sub-libraries with N25 mated to either the 5' end of the insert (Hs800_14) or the 3' end of the insert (Hs800_23). Exonuclease treatment ensures that only mated circular ligates remain during the subsequent second round of amplification of the insert::N25 cassette with an alternative set of primers (P1 / P4 for Hs800_23 and P2 / nP3 for Hs800_14), generating the two sequencing libraries Hs800_2314 and Hs800_1423. PCR adds PE1 and PE2 sites for Illumina paired-end sequencing. To compensate for the lack of diversity in the flanking adapter sequences, seven out of phase primers were used per sequencing library to add the PE1 sites. Phased primers incorporate random sequences of 0N, 2N, 4N, 6N, 8N, 10N, and 12N between the PE1 site and the respective nP3 or P4 site. The 14 phased libraries were sequenced on an Illumina NextSeq500 platform.

[0031] [Figure 9]FIG. 9 shows an exemplary schematic for preparing a GRAMc sequencing library from total RNA. During the first QC step (QC1), the removal of contaminating DNA in the RNA sample is monitored by measuring GFP DNA by QPCR. If the Ct value of GFP DNA remains ≦30 after 12 hours of DNase treatment, DNA digestion is continued. The Ct value is observed every 6 hours, and this process is repeated until the Ct value is >30. As a quality control (QC) standard for reverse transcription (RT), 1000 ng of DNaseI / ExoI / ExoIII digested total RNA was used in a standard RT reaction. During the second QC (QC2) step, the genome-scale RT reaction is monitored, and additional reagents are replenished as necessary until the Ct value of GFP cDNA is within one cycle of the Ct value of the QC standard.

[0032] [Figure 10-1] 10A-10F show the densities of CRMs, expressed genes, and inputs across the human genome 38. FIGS. 10A-10B show GRAMc CRM densities across the human genome 38. FIGS. 10C-10D show expressed gene densities across the human genome 38. FIGS. 10E-10F show GRAMc input densities across the human genome 38. [Figure 10-2] Same as above. [Figure 10-3] Same as above. [Figure 10-4] Same as above. [Figure 10-5] Same as above. [Figure 10-6] Same as above.

[0033] [Figure 11] Figure 11 shows Western blot confirmation of ectopic transcription factor expression. Samples of cells co-transfected with 80K constructs from the GRAMc library and either Flag-tagged EGFP (control) or Flag-tagged transcription factors PITX2 or IKZF1 were subjected to anti-Flag detection of protein expression. Equivalent sample loading was confirmed with anti-GAPDH control blot.

[0034] [Figure 12] FIG. 12 shows an exemplary schematic of GRAMc, including library construction and characterization, and use of the library in reporter assays, and data deconvolution.

[0035] [Figure 13]FIG. 13 shows an exemplary stepwise synthesis of long random DNA sequences from short random oligomers. De novo synthesis of a large number of long random DNA sequences remains challenging, and therefore a simple method for generating a pool of long random DNA sequences from commercially available short random single-stranded DNA (ssDNA) is presented. First, 2 μg of ssDNA is phosphorylated using polynucleotide kinase, followed by conversion to double-stranded DNA (dsDNA) with random hexamers, dNTPs, and Klenow enzyme. In parallel, 1 μg of unphosphorylated ssDNA is converted to dsDNA using random hexamers, dNTPs, and Klenow enzyme. Second, a reaction tube is prepared with 200 ng of unphosphorylated dsDNA and T4 DNA ligase in 1×T4 DNA ligase buffer. The unphosphorylated dsDNA is ligated to the phosphorylated dsDNA. Third, to initiate ligation, 50 ng of phosphorylated dsDNA (or a portion of the unphosphorylated DNA, such as about 1 / 4) is added to the ligation reaction tube. Since there is an excess of unphosphorylated DNA in the reaction, most of the phosphorylated DNA will ligate with the unphosphorylated DNA. Each molecule of unphosphorylated DNA can accept up to two molecules of phosphorylated DNA (one molecule at each end). The ligation product contains an unphosphorylated 5' end. The ligation process is repeated for at least one cycle (e.g., at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 25, 30, 45, 50, 60, 75, 90, or 100 cycles, or about 1-5, 1-10, 1-15, 1-20, 5-20, 10-25, 25-50, or 50-100 cycles, or about 16 cycles). The number of cycles (X) is expected to be ≧2×L / I, where L and I are the desired length of the random DNA and the length of the starting oligomer, respectively. For example, to synthesize a pool of approximately 800 bp long DNA molecules with 100 bp long oligomers, X should be about ≧16.Fourth, nicks in the ligation products were repaired with DNA repair enzymes (NEB PreCR Repair Mix, Cat. No. M0309S). Fifth, DNA molecules of desired length were enriched by gel-based or bead-based size selection. The eluted DNA was then enriched for DNA molecules of desired length with inserts of at least about 50, 100, 200, 300, 400, 500, 750, 800, 900, 1000, 1200, 1500, 2000, 2500, or 3000 base pairs in length, such as about 50-3000 or 100-3000 base pairs in length, about 50-200, 100-200, 100-300, 300-500, 100-1500, 500-1200, 700-1000, or 750-850 base pairs in length, or about 800 base pairs in length. A library construction (e.g., CRM library) can be prepared, such as a library having at least about 10, 25, 50, 100, 250, 500, 103, 104, 105, 106, 107, 108, or 109 reporter constructs (e.g., having inserts), such as 10-100, 100-103, 103-104, 104-106, 106-107, 107-108, 108-109, or 106-109 reporter constructs, or about 107 reporter constructs. Stepwise synthesis of long random DNA sequences can be used in other applications.

[0036] [Figure 14] Figure 14 shows the reproducibility of perturbation experiments. Two independent batches of 80,000 randomly selected reporter constructs were compared in each perturbation experiment. All three experiments were highly reproducible (Pearson's r > 0.97). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] Sequence Listing The nucleic acid and amino acid sequences listed in the attached sequence listing are shown using standard letter abbreviations for nucleotide bases and three-letter code for amino acids as specified in U.S. Patent § 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the strand displayed. The sequence listing has been submitted as a 30kb ASCII text file created on October 30, 2019, and is incorporated herein by reference. In the attached sequence listing:

[0038] SEQ ID NOs:1 and 2 are exemplary linear adaptor nucleic acid sequences.

[0039] SEQ ID NOs: 3 to 116 are exemplary primer sequences.

[0040] SEQ ID NOs:117-124 are exemplary trimming adaptor sequences.

[0041] Unless otherwise indicated, technical terms are used according to conventional usage. Definitions of common terms in molecular biology can be found in the following references: Benjamin Lewin, Genes VII, published by Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN 0471186341); and and George P. Redei, Encyclopedic Dictionary of Genetics, Genomics, and Proteomics, 2nd Edition, 2003 (ISBN: 0-471-26821-6).

[0042] The singular forms "a," "an," and "the" refer to one or more, unless the context clearly dictates otherwise. The term "or" refers to a single element or a combination of two or more of the listed alternative elements, unless the context clearly dictates otherwise. As used herein, "comprise" means "include." Thus, "comprising A or B" means "including A, B, or A and B," and does not exclude additional elements.

[0043] Furthermore, it should be understood that all base or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and are provided for illustration purposes. Methods and materials similar or equivalent to those described herein can be used to practice or test the present disclosure, but the following describes suitable methods and materials. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety, as are GenBank® accession numbers (for sequences existing on October 31, 2018). In case of conflict, the present specification, including explanations of terms, shall control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0044] In order to facilitate review of the various embodiments of the disclosure, explanations of specific terms are provided below.

[0045] Adapter (or adapter sequence or linker): A single-stranded or double-stranded nucleic acid (e.g., DNA, RNA, or a combination of both) that can be ligated to the ends of other nucleic acid molecules (e.g., DNA and / or RNA). Double-stranded adapters can be synthesized with blunt ends, sticky ends, or sticky and blunt ends. In particular examples, the adapter sequence may comprise, for example, at least one deoxyribonucleotide at the 3' end and at least one deoxyribonucleotide at the 5' end (e.g., at least about 1, 2, 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 100, 250, 500, or 1000 deoxyribonucleotides at the 3' end and / or 5' end, or about 5-45, 10-40, 15-35, 20-30, 1-50, 1-100 , 1-250, 1-500, or 1-1000 deoxyribonucleotides, or about 21, 28, or 29, or about 15-35 or 20-30 deoxyribonucleotides) or at least two consecutive ribonucleotides (e.g., at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, or 100 ribonucleotides, such as about 2-5, 2-10, 2-25, 25-50, or 50-100 ribonucleotides, or about 2 ribonucleotides). Specific non-limiting examples of adapter sequences include SEQ ID NOs: 1 and 2.

[0046] Barcode: any nucleic acid or genetic marker. Barcodes may be random (e.g., for reporter applications, such as high throughput applications), semi-random, or non-random (e.g., for taxonomic applications, such as unique barcodes specific to taxonomic groups for such identification). In certain examples, the barcode is a random barcode. In some examples, the barcode is a random barcode with a random number of about 10-100, 100-10 3 , 10 3 ~10 4 , 10 4 ~106 , 10 6 ~10 7 , 10 7 ~10 8 , 10 8 ~10 9 , or 10 6 ~10 9 pcs barcodes, or approximately 10 7 ~2×10 7 pcs barcodes, or approximately 2 x 10 7 Libraries of at least 10, 25, 50, 100, 250, 500, 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9 The barcodes are derived from a library of barcodes (e.g., a pre-existing or algorithmically generated barcode library), such as a library of barcodes. In a particular example, the barcodes are about 2×10 7 In some examples, the barcodes are short barcodes, e.g., at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 250, 500, 1000, 2000, 3000, or 5000 nucleotides in length, or about 5-10, 10-20, 15-40, 20-30, 10-50, 10-75, 10-100, 100-250, 250-500, 500-1000, 1000-3000, or 1000-5000 nucleotides in length, or about 20, 25, 30, 15-40, or 20-30 nucleotides in length.

[0047] Complementary: A nucleic acid molecule is said to be complementary to another nucleic acid molecule if the two molecules share a sufficient number of complementary nucleotides (e.g., AT, AU, or GC) such that the strands bind (hybridize) to one another, for example by forming Watson-Crick, Hoogsteen, or reverse Hoogsteen base pairs, forming a stable duplex or triplex. Stable or specific binding occurs when a nucleic acid molecule remains detectably associated with another nucleic acid as a result of base pairing between complementary nucleotides of the nucleic acid molecule under the required conditions.

[0048] Conditions sufficient for: Any environment that allows for a desired activity, e.g., allows for specific binding between two molecules (such as between a nucleic acid and a protein or between two nucleic acids) or allows for an enzymatic activity (such as a ligase activity or a nuclease activity).

[0049] Contacting: Placing in direct physical association. Includes both solid and liquid forms. For example, contacting may occur in vitro or in a cell with nucleic acids, proteins, and / or enzymes (e.g., ligases or nucleases).

[0050] Detection: To determine whether an agent (such as a nucleic acid molecule and / or a reporter molecule) is present or absent. In some cases, detection may further include identification and / or quantification. For example, in certain cases, the method and detection probes of the present disclosure can be used to determine the presence, amount, and / or identity of a nucleic acid or reporter molecule (such as a reporter nucleic acid).

[0051] Hybridization: The ability of complementary single-stranded DNA, RNA, or DNA / RNA hybrids to form a double-stranded molecule (also called a hybridization complex).

[0052] Ligate: To join two nucleic acid molecules together by a phosphodiester bond between the 3' hydroxyl group of one nucleic acid molecule and the 5' phosphate group of a second nucleic acid molecule. Enzymes that catalyze the formation of a phosphodiester bond between juxtaposed 5' phosphate and 3' hydroxyl ends of nucleic acids are called ligases. Exemplary ligases include DNA ligases (including T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, Taq DNA ligase (e.g., Taq DNA ligase, or high fidelity Taq DNA ligase such as HiFi Taq DNA ligase)), thermostable DNA ligases (e.g., thermostable ligases that catalyze the formation of phosphodiester bonds between the 5' phosphate and 3' hydroxyl of two adjacent DNA strands that hybridize to complementary DNA strands without gaps and are precisely paired, such as 9°N® DNA ligase), and ligases that ligate adjacent single-stranded DNA splinted with a complementary RNA strand (e.g., SPLINTR® ligase). In some examples, the ligase is sufficient to ligate the blunt ends of double-stranded nucleic acids (e.g., T4 DNA ligase or T3 DNA ligase). In a particular example, the ligase is T4 DNA ligase.

[0053] Nuclease: an enzyme that cleaves phosphodiester bonds. Endonucleases are enzymes that cleave internal phosphodiester bonds within a nucleotide chain (as opposed to exonucleases, which cleave phosphodiester bonds at the ends of a nucleotide chain). Endonucleases include endoribonucleases (which cleave RNA at sequence-specific sites), restriction endonucleases such as RNase HII (e.g., to remove any ribonucleotides) or other site-specific endonucleases, or uracil-DNA glycosylase. Other examples of nucleases include DNase I, S1 nuclease, CEL I nuclease, mung bean nuclease, ribonuclease A (RNase A), ribonuclease T1 (RNase T1), ribonuclease H (RNase H), RNase I, RNase PhyM, RNase U2, RNase CLB, micrococcal nuclease, and apurinic / apyrimidinic endonucleases. Exonucleases include exonuclease I, exonuclease III, lambda exonuclease, exonuclease VII, and Bal31 nuclease. In particular examples herein, the nuclease is an RNA-specific nuclease such as RNase HII (e.g., to remove any ribonucleotides), or uracil-DNA glycosylase, or an exonuclease such as exonuclease I, exonuclease III, or lambda exonuclease.

[0054] Regulatory element: A segment of a nucleic acid molecule that can increase or decrease the expression of a particular gene. Exemplary regulatory elements include activators, such as promoters (e.g., a region of DNA that initiates transcription of a gene), and enhancers (e.g., a transcription factor or region of DNA that can interact with other molecules, such as proteins, to increase the likelihood of transcription of a particular gene), or repressors, such as silencers (e.g., a region of DNA that inhibits transcription of a DNA sequence into RNA when bound to a repressor protein or transcription factor).

[0055] Subject: Any multi-cellular vertebrate organism, such as human and non-human mammals (eg, veterinary subjects).

[0056] Vector: A nucleic acid (e.g., DNA or RNA) that is used as a vehicle to artificially transport exogenous genetic material into another cell. Exemplary types of vectors include plasmids, viral vectors, cosmids, and artificial chromosomes. Exemplary elements that are included in a vector are a replication origin, a regulatory element (e.g., a promoter or enhancer), a multicloning site, a marker, and / or a reporter. In certain examples, a vector may include at least a multicloning site; a regulatory element; for example, a promoter (e.g., a synthetic promoter such as a basal promoter and / or a supercore promoter), an enhancer, or a repressor; and a poly(A) tail.

[0057] Methods for constructing nucleic acid molecular reporter libraries The present disclosure describes a method for constructing a nucleic acid molecule reporter library. Thus, a method is provided that allows the determination of the presence or absence of a nucleic acid sequence of interest, such as a specific and / or functional sequence within a larger nucleic acid sequence, such as a genome (e.g., an animal genome or a human genome), and / or the expression of the nucleic acid sequence of interest. The present disclosure's method can be used with any nucleic acid sequence of interest, such as a functional nucleic acid sequence, for example, a nucleic acid sequence that regulates the expression of a gene (e.g., a regulatory element or module, such as a cis-regulatory element or module). In some examples, the disclosed method allows the identification or quantification of a nucleic acid sequence of interest. In some examples, the method includes isolating a plurality of nucleic acid sequences, such as a plurality of nucleic acid sequences that include a nucleic acid sequence of interest, and fusing the plurality of nucleic acid sequences with a reporter nucleic acid to produce a plurality of reporter constructs.

[0058] In some embodiments, the method includes isolating a plurality of nucleic acid molecules of a selected size range. Any nucleic acid molecule can be used, including genomic DNA (such as genomic DNA fragments) or synthetic DNA. In some examples, the nucleic acid is genomic DNA obtained from a cell of interest or a population of cells of interest. Any cell or population of cells can be used, such as an animal cell (e.g., a mammalian cell), a plant cell, a bacterial cell, a fungal cell, or an archaeal cell. In some examples, the mammalian cell includes at least one of a stem cell, a neuronal cell, a cardiovascular cell, a liver cell, an endothelial cell, an epithelial cell, an oral cell, a germ cell, an endocrine cell, a lens cell, an adipocyte, a secretory cell, a kidney cell, an extracellular matrix cell, a contractile cell, an immune cell, a blood cell, or an embryonic cell. In certain non-limiting examples, the mammalian cell is at least one of a cardiomyocyte, a neuron, a hepatocyte, an endothelial cell (e.g., a human umbilical vein endothelial cell, HUVEC, such as in an angiogenesis model), an embryonic stem cell, an induced pluripotent stem cell, a HepG2 cell, a LNCaP cell, a HeLa cell, a HCT116 cell, or a K562 cell. In some examples, the plant cell is at least one of a meristematic cell (including a meristematic derived cell), a parenchyma cell (such as a mesophyll cell, a transfer cell, or a chlorenchyma cell), a collenchyma cell, a scleren ... At least one of the following: a tissue cell (such as a sclerenchyma sclereid or sclerenchyma fiber), a tracheid, a vessel element, a phloem cell (such as a sieve tube, a companion cell, a phloem fiber, or a phloem sclereid), or an epidermal cell (such as a stomatal guard cell) In certain non-limiting examples, the plant cell is at least one of Arabidopsis, cannabis, corn, rice, barley, wheat, switchgrass, tomato, potato, Chlamydomonas, Hydrodictyon, Spirogyra, and Actebularia. In some examples, the bacterial cell comprises at least one of a gram-negative bacterial cell or a gram-positive bacterial cell, such as an Acidobacteria, Actinobacteria, Aquificae, Bacteroidetes, Caldiserica, Chlamydiae, Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus-Thermus, Dictyoglomi, Escherichia, Elusimicrobia, Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes, Lentisphaerae, Nitrospira, Planctomycetes, Proteobacteria, Spirochaetes, Synergistetes, Tenericutes, Thermodesulfobacteria, Thermotogae, or Verrucomicrobia cell. In some examples, the fungal cell comprises at least one of Trichoderma, Neurospora, Aspergillus, Monascus, Mucor, Saccharomyces, Pichia, or Rhizopus. In some examples, the archaeal cell comprises at least one of Cenarchaeum, Caldococcus, Ignisphaera, Acidilobus, Acidococcus, Aeropyrum, Desulfurococcus, Ignicoccus, Staphylothermus, Stetteria, Sulfophobococcus, Thermodiscus, Thermosphaera, Geogemma, Hyperthermus, Pyrodictium, Pyrolobus, Nitrosopumilus (candidatus), Acidianus, Metallosphaera, Stygiolobus, Sulfolobus,Sulfurisphaera, Thermofilum, Caldivirga, Pyrobaculum, Thermocladium, Thermoproteus, Vulcanisaeta, Aciduliprofundum, Archaeoglobus, Ferroglobus, Geoglobus, Haladaptatus, Halalkalicoccus, Haloalcalophilium, Haloarcula, Halobacterium, Halobaculum, Halobiforma, Halococcus, Haloferax, Halogeometricum, Halomicrobium, Halopiger, Haloplanus, Haloquadra, Halorhabdus, Halorubrum, Halosarcina, Halosimplex, Haloterrigena, Halovivax, Natrialba, Natrinema, Natronobacterium, Natronococcus, Natronolimnobius, Natronorubrum, Methanoregula (candidate M ethanomicrobium、Methanoplanus、Methanospirillaceae、Methanospirillum、Methanosaeta、Methanimicrococcus、Methanococcoides、Methanohalobium、Methanohalophilus、Methanolobus、Methanomethylovorans、Methanosalsum、Methanosarcina、Methanopyrus、Palaeococcus、Pyrococcus、At least one of Thermococcus, Ferroplasma, Picrophilus, Thermoplasma, Korarchaeota, Nanoarchaeota, or Nanoarchaeum cells.

[0059] The plurality of nucleic acid molecules of selected size range may be derived from any source, for example, genome or partial genome derived from a cell, including chromosomal DNA and mitochondrial DNA.Thus, in some examples, the isolated nucleic acid is isolated from a selected cell type or a population of cell types.DNA (e.g., genomic DNA) is fragmented, for example, by digestion, shearing, sonication, or combinations thereof.In some examples, the nucleic acid is synthetic DNA, such as random double-stranded DNA sequence of selected length or length range.Any DNA synthesis method can be used to produce synthetic DNA. In certain examples, synthetic DNA (e.g., DNA of a selected size range) may be generated by ligating two or more DNA molecules smaller than the DNA of the selected size range (e.g., for DNA of a selected size range of about 750-850 base pairs or about 800 base pairs, the smaller DNA may be at least about 25, 50, 100, 200, 300, or 400 base pairs, or about 25-50, 25-100, 25-200, 25-400, or 100-400 base pairs, or about 100 base pairs). An exemplary method for generating synthetic DNA nucleic acid molecules of a selected size range is shown in FIG. 13.

[0060] In some examples, the size range of the isolated nucleic acid is at least about 50, 100, 200, 300, 400, 500, 750, 800, 900, 1000, 1200, 1500, 2000, 2500, or 3000 base pairs in length, such as about 50-200, 100-200, 100-300, 300-500, 100-1500, 500-1200, 700-1000, 700-900, or 750-850 base pairs in length, or about 800 base pairs in length, such as about 50-3000 or 100-3000 base pairs in length. Any method can be used to select a plurality of nucleic acid molecules in the desired size range. In some examples, the plurality of nucleic acid molecules are selected using gel electrophoresis (e.g., using an agarose gel, such as a manually prepared agarose gel or an agarose gel cassette, such as a 1-5%, 1-2%, 2-3%, or 3-5% agarose gel, or a 1.2% agarose gel, such as at least a 1%, 1.2%, 1.5%, 2%, 3%, or 5% agarose gel, e.g., using a constant or variable voltage) or bead-based size selection (e.g., using solid phase reversible immobilization, SPRI, such as using paramagnetic beads, e.g., paramagnetic beads having a carboxyl coating).

[0061] In some examples, the method includes ligating a nucleic acid molecule (e.g., a plurality of isolated nucleic acid molecules of a selected size, also referred to herein as "insert") to an adapter sequence (e.g., at least one adapter sequence, such as at least one linear adapter sequence). Any adapter sequence can be used, such as a linear adapter sequence that can be ligated, such as with a plurality of isolated nucleic acid molecules, to form a circular nucleic acid molecule (e.g., a plurality of circular nucleic acid molecules). In some examples, the adapter sequence includes ribonucleotides and deoxyribonucleotides. In particular examples, the adapter sequence includes one ribonucleotide or at least two consecutive ribonucleotides (e.g., at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, or 100 nucleotides, such as about 2-5, 2-10, 2-25, 25-50, or 50-100 ribonucleotides, or about two ribonucleotides).In some examples, the adapter sequence comprises at least one deoxyribonucleotide at the 3' end (e.g., at least about 1, 2, 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 100, 250, 500, or 1000 deoxyribonucleotides, or about 5-45, 10-40, 15-35, 20-30, 1-50, 1-100, 1-250, 1-500, or 1-1000 deoxyribonucleotides, or about 21, 28, or 29, or about 15-35 or 20-30 deoxyribonucleotides at the 3' end). The ribonucleotides may comprise one ribonucleotide or at least two consecutive ribonucleotides flanked by two deoxyribonucleotides (e.g., at least about 1, 2, 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 100, 250, 500, or 1000 deoxyribonucleotides, or about 5-45, 10-40, 15-35, 20-30, 1-50, 1-100, 1-250, 1-500, or 1-1000 deoxyribonucleotides, or about 21, 28, or 29, or about 15-35 or 20-30 deoxyribonucleotides at the 5' end). In particular examples, the linear adapter sequence may comprise: CTGCTGAATCACTAGTGAATTATTACCCrUrUCAAGACACTACTCTCCAGCAGT (SEQ ID NO: 1) or CTGCTGGAGAGTAGTGTCTTGrArAGGGTAATAATTCACTAGTGATTCAGCAGT (SEQ ID NO: 2). In the sequence, "rU" and "rA" represent ribonucleotides. In a particular example, the adapter is a double-stranded linear adapter prepared by hybridization of the nucleic acids of SEQ ID NO: 1 and SEQ ID NO: 2.

[0062] A plurality of isolated nucleic acid molecules (such as a plurality of inserts) are ligated to an adapter sequence (e.g., at least one adapter sequence, such as at least one linear adapter sequence, e.g., SEQ ID NO:1 and / or SEQ ID NO:2) using any ligation method (e.g., ligase-mediated ligation or chemical ligation). In some examples, at least one ligase is used for ligation. Any nucleic acid or adapter sequence described herein can be used. In some examples, the ligation method is sufficient to form a circular nucleic acid molecule (e.g., a plurality of circular nucleic acid molecules) that includes an "insert" nucleic acid molecule and an adapter sequence (e.g., a double-stranded adapter that includes SEQ ID NO:1 and SEQ ID NO:2). Thus, in certain examples, such methods can be used to produce a plurality of circular nucleic acid molecules, each having an insert and an adapter sequence. In some examples, a DNA ligase is used. Any ligase (e.g., T4 DNA ligase) sufficient for ligating nucleic acids can be used. Examples of ligases that can be used include: DNA ligases (T4 DNA ligase, T3 DNA ligases include T7 DNA ligase, Taq DNA ligase (e.g., Taq DNA ligase, or high fidelity Taq DNA ligase such as HiFi Taq DNA ligase), thermostable DNA ligases (e.g., thermostable ligases that catalyze the formation of phosphodiester bonds between the 5' phosphate and 3' hydroxyl of two adjacent DNA strands that hybridize to complementary DNA strands without gaps and are precisely paired, such as 9°N® DNA ligase), and ligases that ligate adjacent single-stranded DNA splinted with complementary RNA strands (e.g., SPLINTR® ligase). In some examples, the ligase is sufficient for ligating the blunt ends of double-stranded nucleic acids (e.g., T4 DNA ligase or T3 DNA ligase). In a particular example, the ligase is T4 DNA ligase.

[0063] In some embodiments, the method further comprises contacting the plurality of circular nucleic acid molecules with at least one enzyme (e.g., at least about 1, 2, 5, or 10 enzymes, or about 1-2, 1-5, or 1-10 enzymes, or about 1 or 2 enzymes) specific for removing consecutive nucleotides from an end of a polynucleotide molecule (e.g., at least one exonuclease, such as at least about 1, 2, 5, or 10 exonucleases, or about 1-2, 1-5, or 1-10 exonucleases, or about 1 or 2 exonucleases) under conditions sufficient to remove linear nucleic acid from the circular nucleic acid molecule (e.g., any circular nucleic acid molecule described herein, such as a plurality of circular nucleic acid molecules). In some examples, the at least one exonuclease comprises exonuclease I, exonuclease III, and / or lambda exonuclease. In particular examples, the at least one exonuclease is exonuclease I and exonuclease III.

[0064] In some embodiments, the method includes contacting a plurality of circular nucleic acid molecules comprising an insert and an adaptor sequence with an enzyme specific for separating nucleotides within a polynucleotide chain (e.g., a nucleotide other than the 5'- or 3'-terminal nucleotide, an endonuclease, etc.) under conditions sufficient to produce linear nucleic acid molecules (e.g., a plurality of linear nucleic acid molecules) from the plurality of circular nucleic acid molecules comprising the insert and the adaptor. In some examples, the linear nucleic acid molecules produced each include, for example, at least one deoxyribonucleotide at the 5'-terminus and at least one deoxyribonucleotide at the 3'-terminus flanked by an insert (e.g., any insert described herein). In some examples, the linear nucleic acid molecules produced include an insert flanked by at least one deoxyribonucleotide at the 5'-terminus and at least one deoxyribonucleotide at the 3'-terminus. For example, the at least one deoxyribonucleotide at the 5' or 3' terminus may comprise at least one deoxyribonucleotide, such as at least about 1, 2, 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 100, 250, 500, or 1000 deoxyribonucleotides, or about 5-45, 10-40, 15-35, 20-30, 1-50, 1-100, 1-250, 1-500, or 1-1000 deoxyribonucleotides, or about 21, 28, or 29, or about 15-35 or 20-30 deoxyribonucleotides. In certain instances, the enzyme is specific for removing ribonucleotides within a double stranded nucleic acid (e.g., an endoribonuclease). For example, the enzyme can remove at least one ribonucleotide, such as at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, or 100 ribonucleotides, such as about 2-5, 2-10, 2-25, 25-50, or 50-100 ribonucleotides, or about 2 ribonucleotides, from a circular nucleic acid (e.g., any of the circular nucleic acid molecules described herein, such as a plurality of circular nucleic acid molecules).In particular examples, the enzyme (e.g., endoribonuclease) may include RNase HII (e.g., to remove any ribonucleotides) or uracil-DNA glycosylase (e.g., to remove uracil). Linearization of the circular nucleic acid produces a plurality of linear nucleic acid molecules that include the insert nucleic acid and at least one deoxyribonucleotide at the 3' end and at least one deoxyribonucleotide at the 5' end.

[0065] In some embodiments, the method includes fusing a plurality of linear nucleic acid molecules obtained by linearizing the circular nucleic acid comprising the insert and at least one deoxyribonucleotide at the 3' end and at least one deoxyribonucleotide at the 5' end with at least one reporter nucleic acid (e.g., to produce a plurality of reporter constructs, such as a nucleic acid molecule reporter library). Any reporter nucleic acid can be used, for example, a fluorescent reporter nucleic acid or a barcode reporter nucleic acid, such as a nucleic acid encoding a fluorescent protein and / or a nucleic acid comprising a barcode. In some examples, the at least one reporter is a nucleic acid encoding a fluorescent protein. Any fluorescent protein can be encoded, such as a blue, purple, green, yellow, orange, or red fluorescent protein, or a protein exhibiting any combination or variation of such fluorescence. In a particular example, the at least one reporter nucleic acid is a nucleic acid encoding a green fluorescent protein (GFP). In other examples, the at least one reporter nucleic acid is a nucleic acid comprising a barcode (e.g., a nucleic acid or a genetic marker). Any nucleic acid or genetic marker can be used as a barcode. In some examples, the barcode is a short nucleic acid or genetic marker, e.g., at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 250, 500, 1000, 2000, 3000, or 5000 nucleotides in length, or about 5-10, 10-20, 15-40, 20-30, 10-50, 10-75, 10-100, 100-250, 250-500, 500-1000, 1000-3000, or 1000-5000 nucleotides in length, or about 20, 25, 30, 15-40, or 20-30 nucleotides in length. In further examples, the reporter comprises at least one nucleic acid encoding a fluorescent protein and at least one barcode nucleic acid.

[0066] In certain examples, at least one reporter nucleic acid is a barcode nucleic acid. Any nucleic acid barcode can be used. For example, random, semi-random, or non-random barcodes can be used, such as those derived from a barcode library. In certain examples, the barcode is a random barcode. In some examples, the barcode is a random barcode. 3 , 10 3 ~10 4 , 10 4 ~10 6 , 10 6 ~10 7 , 10 7 ~10 8 , 10 8 ~10 9 , or 10 6 ~10 9 pcs barcodes, or approximately 10 7 ~2×10 7 pcs barcodes, or approximately 2 x 10 7 Barcodes, such as at least 10, 25, 50, 100, 250, 500, 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9 The barcodes are derived from a library of barcodes (e.g., a pre-existing or algorithmically generated barcode library), such as a library of barcodes. In a particular example, the barcodes are about 2×10 7 It is derived from a random library of barcodes.

[0067] In some embodiments, the method includes fusing a linear nucleic acid molecule including an insert nucleic acid having at least one deoxyribonucleotide at the 3' end and at least one deoxyribonucleotide at the 5' end and a reporter to a linear vector nucleic acid to produce a plurality of linear vectors. Any linear vector nucleic acid can be used. For example, the linear vector nucleic acid can include a nuclease cleavage site and a transcriptional or translational regulatory element (such as a promoter, enhancer, repressor, and / or poly(A) tail). In some examples, the linear vector nucleic acid can include at least one promoter, such as a basal promoter and / or a synthetic promoter. For example, the linear vector nucleic acid can include at least about 1, 2, 3, 4, 5, 6, 8, or 10 promoters, or about 1-4, 5-10, or 1-10 promoters. In some examples, at least one promoter, such as a basal and / or synthetic promoter, may include at least about 1, 2, 3, 4, 5, 6, 8, or 10 promoter motifs, or about 1-4, 5-10, or 1-10 promoter motifs, or about 4 promoter motifs, e.g., a synthetic promoter may include a TATA box, an initiator (Inr), a motif 10 element (MTE), a downstream promoter element (DPE), a B recognition element (BRE), an E-box, a CCAAT box, an NRF-1, a GABPA, a YY1, an ACTACAnnTCCC, and / or a decamer promoter motif. In certain examples, the at least one promoter is a synthetic promoter (e.g., a supercore promoter) that includes a TATA box, an Inr, a MTE, and a DPE motif. Additional exemplary promoters can be found in Morgan, addgene blog: “Plasmids 101: The Promoter Region - Let's Go!”, 2014, which is incorporated by reference in its entirety.

[0068] A linear nucleic acid molecule comprising an insert nucleic acid having at least one deoxyribonucleotide at the 3' end and at least one deoxyribonucleotide at the 5' end can be fused with a linear vector nucleic acid at any time, for example, during, before or after fusing the linear nucleic acid molecule with at least one reporter nucleic acid. In some examples, the linear vector nucleic acid comprises at least one reporter nucleic acid (e.g., at least one reporter nucleic acid encoding a fluorescent protein such as green fluorescent protein, or at least one reporter nucleic acid comprising at least one barcode), and thus, fusing the linear nucleic acid molecule with the linear vector nucleic acid comprises fusing with at least one reporter nucleic acid. In some examples, the method comprises fusing the linear nucleic acid molecule with a linear vector nucleic acid before fusing the linear nucleic acid molecule with at least one reporter nucleic acid (e.g., a nucleic acid encoding a fluorescent protein or a nucleic acid comprising a barcode). For example, fusing the plurality of linear nucleic acid molecules with at least one reporter nucleic acid may include fusing the plurality of linear vectors with a reporter nucleic acid (e.g., a fluorescent reporter nucleic acid) encoding a fluorescent protein to produce a plurality of fluorescent reporter constructs. In some examples, fusing the plurality of linear nucleic acid molecules with at least one reporter nucleic acid may include fusing the plurality of linear vectors with a reporter nucleic acid (e.g., a barcode reporter nucleic acid) comprising a barcode to produce a plurality of barcode reporter constructs. In other examples, the linear nucleic acid includes an insert nucleic acid having at least one deoxyribonucleotide at the 3' end and at least one deoxyribonucleotide at the 5' end, and a reporter nucleic acid, prior to fusion with the linear vector nucleic acid.

[0069] The method includes fusing any number of reporter nucleic acids to a nucleic acid molecule, e.g., a plurality of linear nucleic acid molecules or a plurality of linear vectors comprising at least about 1, 2, 3, 4, 5, 10, 15, 20, or 25, or about 1-2, 1-5, 1-10, 10-20, 15-25, or 1-25, or about 2 reporter nucleic acids. In some examples, the method includes fusing a plurality of linear nucleic acid molecules or a plurality of linear vectors comprising nucleic acid molecules to a fluorescent reporter nucleic acid (e.g., a reporter nucleic acid encoding GFP) to produce a plurality of fluorescent reporter constructs. In some examples, the method includes fusing a plurality of linear nucleic acid molecules or a plurality of linear vectors comprising nucleic acid molecules to a barcode reporter nucleic acid (e.g., a reporter nucleic acid comprising a short barcode, such as a barcode about 25 nucleotides in length) to produce a plurality of barcode reporter constructs. In some examples, the method includes fusing a plurality of linear nucleic acid molecules or a plurality of linear vectors comprising nucleic acid molecules with fluorescent reporter nucleic acids and barcode reporter nucleic acids (e.g., a reporter nucleic acid encoding GFP and a reporter nucleic acid comprising a short barcode, such as a barcode about 25 nucleotides long) to produce a plurality of fluorescent and barcode reporter constructs. In particular examples, the method includes fusing a plurality of linear vectors comprising nucleic acid molecules with fluorescent reporter nucleic acids and / or barcode reporter nucleic acids (e.g., a reporter nucleic acid encoding GFP and / or a reporter nucleic acid comprising a short barcode, such as a barcode about 25 nucleotides long) to produce a plurality of fluorescent and barcode reporter constructs.

[0070] In some embodiments, fusing the plurality of linear nucleic acid molecules or the plurality of linear vectors comprising the nucleic acid molecules with the barcode reporter nucleic acid comprises contacting the plurality of linear nucleic acid molecules comprising an insert nucleic acid having at least one deoxyribonucleotide at the 3' end and at least one deoxyribonucleotide at the 5' end, or the plurality of linear vectors comprising an insert nucleic acid having at least one deoxyribonucleotide at the 3' end and at least one deoxyribonucleotide at the 5' end, with a primer nucleic acid comprising a barcode reporter nucleic acid (e.g., a reporter nucleic acid comprising a short barcode, such as a barcode of about 25 nucleotides in length). In some examples, a polymerase chain reaction (PCR) is performed using the plurality of linear nucleic acid molecules or the plurality of linear vectors comprising the linear nucleic acid molecules and at least one primer nucleic acid comprising the barcode reporter nucleic acid, e.g., to extend the linear nucleic acid molecules or the plurality of linear vectors to produce a plurality of barcode reporter constructs or a plurality of linear vectors comprising the barcode reporter constructs. In a particular example, a polymerase chain reaction (PCR) is performed using a plurality of linear vectors comprising the nucleic acid molecules and a primer nucleic acid comprising a barcode reporter nucleic acid to produce a plurality of linear vectors comprising the barcode reporter constructs.

[0071] In some examples, the method includes using a ligase to ligate the ends of a plurality of linear vectors including reporter constructs (e.g., fluorescent reporter constructs and / or barcode reporter constructs) to produce a plurality of circular vectors including reporter constructs (e.g., fluorescent reporter constructs and / or barcode reporter constructs). In a particular example, the method includes using a ligase to ligate the ends of a plurality of linear vectors including barcode reporter constructs to produce a plurality of circular vectors including barcode reporter constructs. Any ligase described herein (e.g., a DNA ligase such as T4 DNA ligase) can be used. In some examples, the ligase is sufficient for ligating blunt ends of double-stranded nucleic acids (e.g., T4 DNA ligase or T3 DNA ligase). In a particular example, the ligase is T4 DNA ligase. In some examples, the method further comprises contacting the plurality of circular vectors comprising the barcode reporter construct with at least one exonuclease to remove linear nucleic acid molecules from the plurality of circular vectors. Any exonuclease described herein can be used (e.g., exonuclease I, exonuclease III, and / or lambda exonuclease). In a particular example, the at least one exonuclease is exonuclease I and exonuclease III.

[0072] Also, in some embodiments, the method includes determining genome coverage of the plurality of linear nucleic acid molecules, for example, the plurality of linear nucleic acid molecules includes genomic DNA. The genome coverage can be determined at any time. In some examples, the genome coverage is determined before fusing the insert nucleic acid and the plurality of linear nucleic acid molecules including at least one deoxyribonucleotide at the 3' end and at least one deoxyribonucleotide at the 5' end with the reporter nucleic acid. In certain examples, the coverage can be determined using a plurality of linear nucleic acid molecules (e.g., a linear nucleic acid molecule including a nucleic acid molecule and an adapter sequence). The genome coverage can be determined using any method. In certain examples, the genome coverage is determined by selecting at least one genomic region of interest (e.g., the entire genome or a partial genome), amplifying the plurality of linear nucleic acid molecules (e.g., using PCR such as quantitative PCR, QPCR), and determining whether the selected genomic region is present in the plurality of linear nucleic acid molecules. In some cases, such as when the linear nucleic acid molecule comprises a nucleic acid molecule and an adapter sequence, PCR is performed using a primer complementary to the adapter sequence (e.g., a primer that is complementary to all or a portion of the adapter sequence, such as all or a portion of the adapter sequence located 5' to the nucleic acid molecule).

[0073] In a particular example of a method for constructing a nucleic acid molecule reporter library, the method includes nucleotide sequences in a selected size range (e.g., at least about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4500, 5000, 5000, 6000, 7000, 7500, 850, 9000, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4000, 4000, 4000, 4000, ligating the plurality of nucleic acid molecules to at least one linear adaptor sequence using a ligase (e.g., T4 ligase), the linear adaptor sequence comprising at least one deoxyribonucleotide at the 3' end and at least one deoxyribonucleotide at the 5' end, such as SEQ ID NO:1 or SEQ ID NO:2; contacting the plurality of circular nucleic acid molecules with an exonuclease (e.g., exonuclease I and / or exonuclease III) under conditions sufficient to remove linear nucleic acid molecules from the plurality of circular nucleic acid molecules; contacting the plurality of circular nucleic acid molecules with an endoribonuclease (e.g., RNase I and / or exonuclease III) under conditions sufficient to produce a plurality of linear nucleic acid molecules each comprising at least one deoxyribonucleotide at the 3' end and at least one deoxyribonucleotide at the 5' end, flanked by an insert; (a) fusing the plurality of nucleic acid molecules with a linear vector nucleic acid, thereby producing a plurality of linear vectors comprising the nucleic acid molecules; (b) contacting each of the plurality of linear vectors comprising the nucleic acid molecule with a primer comprising a barcode nucleic acid;and (c) fusing the plurality of linear nucleic acid molecules with at least one reporter nucleic acid to produce a plurality of reporter constructs, such as by performing a polymerase chain reaction (PCR) to produce a plurality of circular vectors comprising the barcode reporter constructs; and contacting the plurality of circular vectors comprising the barcode reporter constructs with an exonuclease (e.g., exonuclease I and / or exonuclease III) under conditions sufficient to remove the linear nucleic acid molecules from the plurality of circular vectors comprising the barcode reporter constructs;

[0074] Compositions and kits for constructing nucleic acid molecule reporter libraries Contemplated herein is a nucleic acid molecule reporter library produced using any of the methods described herein. The reporter library may include any number of reporter constructs. In some examples, the number of reporter constructs may depend on the nucleic acid sequence or sequences of interest. For example, when the nucleic acid molecule reporter library includes nucleic acid molecules derived from a larger sequence, such as a genome (e.g., an animal or human genome, a plant genome, a bacterial genome, a fungal genome, or an archaeal genome), the number of reporter constructs may depend on the size of the larger sequence and / or the level of coverage by the library. In some examples, the number of reporter constructs may be about 10 to 100, 100 to 10 3 , 10 3 ~10 4 , 10 4 ~10 6 , 10 6 ~10 7 , 10 7 ~10 8 , 10 8 ~10 9 , or 10 6 ~10 9 , or about 10 7 ~2×10 7 Or about 2 x 10 7 (For example, 1.91×10 7 ) at least about 10, 25, 50, 100, 250, 500, 10 3, 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9 It is.

[0075] Contemplated herein is a library of reporter constructs comprising a reporter molecule and a nucleic acid molecule (e.g., insert). Also, the elements of the reporter constructs in the nucleic acid molecule reporter library produced using the methods herein may vary depending on the intended method of identification and / or quantification. For example, the libraries produced using the methods herein can be used in vivo or in vitro, and the identification and / or quantification may range from using visual-based reporters (e.g., fluorescent reporters, e.g., nucleic acids encoding blue, purple, green, yellow, orange, or red fluorescent proteins, for visual and / or spectroscopic based identification and / or quantification, etc.) to sequence-based reporters (e.g., barcode reporters, e.g., at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 210, 220, 230, 240, 250, 260, 270, 280, 290, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 610, 620, 630, 640, 650, 660, 670, Libraries may range from 0, 75, 100, 250, 500, 1000, 2000, 3000, or 5000 nucleotides in length, or from about 5-10, 10-20, 15-40, 20-30, 10-50, 10-75, 10-100, 100-250, 250-500, 500-1000, 1000-3000, or 1000-5000 nucleotides in length, or from about 20, 25, 30, 15-40, or 20-30 nucleotides in length (random, semi-random, or non-random barcodes). Libraries containing more than one reporter or type of reporter are contemplated herein. In some examples, libraries may include visual-based reporters and sequence-based reporters, such as libraries including fluorescent reporters and barcode reporters. In a particular example, the library includes a reporter construct having both a nucleic acid encoding GFP and a nucleic acid including a short barcode (e.g., a barcode of about 25 nucleotides in length). Also, the size of the intended insert of the reporter construct may vary depending on the intended method of identification and / or quantification.For example, the insert size range is at least about 50, 100, 200, 300, 400, 500, 750, 800, 900, 1000, 1200, 1500, 2000, 2500, or 3000 base pairs in length, such as about 50-200, 100-200, 100-300, 300-500, 100-1500, 500-1200, 700-1000, or 750-850 base pairs in length, or about 800 base pairs in length, such as about 50-3000 or 100-3000 base pairs in length.

[0076] Further contemplated herein are libraries of reporter constructs that include elements other than reporter molecules. For example, the reporter nucleic acid linear adapter sequence or a portion thereof (e.g., SEQ ID NO:1 and / or SEQ ID NO:2 or a portion thereof) may be included. Also, for example, the reporter construct may include any of the vectors and / or vector elements described herein, such as nuclease cleavage sites and transcriptional or translational regulatory elements, such as promoters (e.g., synthetic promoters such as basal promoters and / or supercore promoters), enhancers, repressors, and / or poly(A) tails.

[0077] Also contemplated herein is a kit for constructing a nucleic acid molecule reporter library. In some examples, the kit includes one or more linear adaptors, e.g., SEQ ID NO:1 and / or SEQ ID NO:2. In some examples, the kit includes any of the reporter nucleic acids described herein. For example, the kit may include a visual-based nucleic acid reporter (e.g., a fluorescent reporter, e.g., a nucleic acid encoding a blue, purple, green, yellow, orange, or red fluorescent protein, such as for visual-based and / or spectroscopic-based identification and / or quantification) and / or a sequence-based reporter (e.g., at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 250, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10 ... The kit may include a barcode reporter that includes a nucleic acid or genetic marker, such as a random, semi-random, or non-random barcode, that is about 5-10, 10-20, 15-40, 20-30, 10-50, 10-75, 10-100, 100-250, 250-500, 500-1000, 1000-3000, or 1000-5000 nucleotides in length, or about 20, 25, 30, 15-40, or 20-30 nucleotides in length. More than one reporter or type of reporter is contemplated. For example, the kit may include a visual-based reporter and a sequence-based reporter, such as a fluorescent reporter and a barcode reporter. In a particular example, the kit includes a nucleic acid reporter that encodes GFP and includes a short barcode (e.g., a barcode that is about 25 nucleotides in length).

[0078] Further contemplated herein are kits having reporter constructs that include other elements besides the reporter molecule. For example, a linear adapter sequence of the reporter nucleic acid may be included (e.g., SEQ ID NO:1 and / or SEQ ID NO:2). The kit may also include any of the vectors and / or vector elements described herein, such as nuclease cleavage sites and transcriptional or translational regulatory elements, for example, promoters (e.g., synthetic promoters such as basal and / or supercore promoters), enhancers, repressors, and / or poly(A) tails. Any enzyme for carrying out the methods described herein is contemplated. For example, the kit may include a DNA ligase (T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, Taq DNA ligase (e.g., Taq DNA ligase, or high fidelity Taq DNA ligase such as HiFi Taq DNA ligase), or a ligase that is capable of binding to a target gene (e.g., Taq DNA ligase, or high fidelity Taq DNA ligase such as HiFi Taq DNA ligase). at least one ligase, such as a thermostable DNA ligase (e.g., a thermostable ligase that catalyzes the formation of a phosphodiester bond between the 5' phosphate and the 3' hydroxyl of two adjacent DNA strands that hybridize to complementary DNA strands without gaps and are precisely paired, such as 9°N® DNA ligase), and a ligase that ligates adjacent single-stranded DNA splinted with a complementary RNA strand (e.g., SPLINTR® ligase); at least one exonuclease, such as at least about 1, 2, 5, or 10 exonucleases, or about 1-2, 1-5, or 1-10 exonucleases, or about 1 or 2 exonucleases (e.g., exonuclease I, exonuclease III, and / or lambda exonuclease); an endoribonuclease (e.g., RNase The nucleic acid may comprise a polymerase, including any polymerase suitable for PCR (e.g., a high fidelity polymerase), such as HII or uracil-DNA glycosylase, and / or any polymerase suitable for PCR (e.g., a high fidelity polymerase).

[0079] Methods for detecting functional nucleic acid regulatory elements and kits therefor The libraries of the present disclosure can be used for various purposes, including identifying cis-regulatory elements in a genome of interest. In some examples, the libraries of the present disclosure can be used to directly measure the functional differences of CRMs from different individuals of the same species. The libraries and methods of the present disclosure can directly measure the functional consequences of sequence variations in cell-based procedures (e.g., cardiomyocytes, neurons, hepatocytes). In other examples, the libraries and methods of the present disclosure can be used to identify biomarker CRMs, such as CRMs that mediate drug cytotoxicity, CRMs that maintain pathological conditions of cells, and / or CRMs that maintain healthy cell conditions.

[0080] For example, the disclosed libraries and methods can identify CRMs that respond to the cytotoxicity of a drug. A collection of biomarker CRMs that detect multiple different cytotoxic effects can be generated. This collection of biomarkers can be used to test the toxicity of a drug in a single screen. The disclosed libraries and methods can also identify CRMs that are specific to a pathological cell state of a patient-derived cell (e.g., iPSC-derived cardiomyopathy cells). Furthermore, the disclosed libraries and methods can be used to identify CRMs that are specific to a healthy cell state of a control cell (e.g., iPSC-derived control cardiomyocytes). Furthermore, by pooling all three types of biomarker CRMs, drugs that can turn a pathological cell state to a normal state can be screened in a single screen without causing a cytotoxic effect.

[0081] In another embodiment, the libraries and methods of the present disclosure can screen for artificial CRMs possessing any desired activity, including potent drivers of selectable markers in any cell type, such as drivers for precise control of gene expression (e.g., enzymes) in engineered cells (bacterial, fungal, plant, archaeal, mammalian cells).

[0082] In other embodiments, the libraries and methods of the present disclosure can screen for enrichment motifs of non-expressed transcription factors in host cell types, such as to detect gene regulatory interactions in various cell types (e.g., mutually exclusive cell types formed from stem cells, such as embryonic stem cells or induced stem cells). Exemplary applications include, for example, tissue engineering to generate specific cell types. For example, one cell type can be suppressed and another cell type can be promoted (e.g., in applications where one cell type can be transformed into another cell type, such as an application where a desired or desired cell type can be transformed into an undesired or undesired cell type).

[0083] Disclosed herein are methods for detecting functional nucleic acid regulatory elements (e.g., CRMs such as promoters, enhancers, and / or repressors). In some examples, the methods may include transfecting at least one cell of interest with a nucleic acid molecule reporter library disclosed herein. In some examples, the methods include selecting a cell of interest. Any cell of interest can be used and / or selected, such as an animal cell (e.g., a mammalian cell), a plant cell, a fungal cell, a bacterial cell, or an archaeal cell. In some examples, the mammalian cell includes at least one of a stem cell, a neuronal cell, a cardiovascular cell, a liver cell, an endothelial cell, an epithelial cell, an oral cell, a germ cell, an endocrine cell, a lens cell, an adipocyte, a secretory cell, a kidney cell, an extracellular matrix cell, a contractile cell, an immune cell, a blood cell, or an embryonic cell. In certain non-limiting examples, the mammalian cell is at least one of a cardiomyocyte, a neuron, a hepatocyte, an endothelial cell (e.g., human umbilical vein endothelial cell, HUVEC, such as in an angiogenesis model), an embryonic stem cell, an induced pluripotent stem cell, a HepG2 cell, a LNCaP cell, a HeLa cell, a HCT116 cell, or a K562 cell. In some examples, the plant cell comprises at least one of a meristematic cell (including a meristematic derived cell), a parenchyma cell (such as a mesophyll cell, a transport cell, or a chlorophyll cell), a sclerenchyma cell, a sclerenchyma cell (such as a sclerenchyma cell or a sclerenchyma fiber), a tracheid, a vessel element, a phloem cell (such as a sieve tube, a companion cell, a phloem fiber, or a phloem sclerenchyma cell), or an epidermal cell (such as a stomatal guard cell). In certain non-limiting examples, the plant cell is at least one of Arabidopsis, cannabis, corn, rice, barley, wheat, switchgrass, tomato, potato, Chlamydomonas, Hydrodictyon, Spirogyra, and Actebularia. In some examples, the bacterial cell is a gram-negative bacterial cell or a gram-positive bacterial cell, such as Acidobacteria, Actinobacteria, Aquificae, Bacteroidetes, Caldiserica, Chlamydiae, Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres,The fungal cells include at least one of Deinococcus-Thermus, Dictyoglomi, Elusimicrobia, Escherichia, Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes, Lentisphaerae, Nitrospira, Planctomycetes, Proteobacteria, Spirochaetes, Synergistetes, Tenericutes, Thermodesulfobacteria, Thermotogae, or Verrucomicrobia cells. In some examples, the fungal cells include at least one of Trichoderma, Neurospora, Aspergillus, Monascus, Mucor, Saccharomyces, Pichia, or Rhizopus. In some cases, archaeal cells include Cenarchaeum, Caldococcus, Ignisphaera, Acidilobus, Acidococcus, Aeropyrum, Desulfurococcus, Ignicoccus, Staphylothermus, Stetteria, Sulfophobococcus , Thermodiscus, Thermosphaera, Geogemma, Hyperthermus, Pyrodictium, Pyrolobus, Nitrosopumilus(candidatus), Acidianus, Metallosphaera, Stygiolobus, Sulfolobus, Sulfur isphaera, Thermofilum, Caldivirga, Pyrobaculum, Thermocladium, Thermoproteus, Vulcanisaeta, Aciduliprofundum, Archaeoglobus, Ferroglobus, Geoglobus, Haladaptatus, Ha lalkalicoccus, Haloalcalophilium, Haloarcula, Halobacterium, Halobaculum, Halobiforma, Halococcus, Haloferax, Halogeometricum, Halomicrobium, Halopiger, Haloplanus,Haloquadra, Halorhabdus, Halorubrum, Halosarcina, Halosimplex, Haloterrigena, Halovivax, Natrialba, Natrinema, Natronobacterium, Natronococcus, Natronolimnobius, Natronorubrum, Methanoregula(candidatus), Methanocalculus, Methanobacterium, Methanobrevibacter, Methanosphaera, Methanothermobacter, Methanothermus, Methanocaldococcus, Methanotorris, Methanococcus, Methanothermococcus, Methanocorpusculum, Methanoculleus, Meth anofollis、Methanogenium、Methanolacinia、Methanomicrobium、Methanoplanus、Methanospirillaceae、Methanospirillum、Methanosaeta、Methanimicrococcus、Methanococcoides、Methanohalobium、Methanohalophilus、Methanol obus、Methanomethylovorans、Methanosalsum、Methanosarcina、Methanopyrus、Palaeococcus、Pyrococcus、Thermococcus、Ferroplasma、Picrophilus、Thermoplasma、Korarchaeota、Nanoarchaeota、or Nanoarchaeum containing at least 1 cell.、

[0084] In some examples, the method includes collecting at least one cell of interest (e.g., from at least one subject). In some examples, cells are collected from at least two subjects, such as at least one subject with a disease or condition and at least one subject without a disease or condition. In other examples, cells are collected from cells or subjects under different conditions (e.g., before or after administration of a reagent or protocol, such as a drug or treatment protocol). Any of the libraries described herein may be used. The method also includes measuring at least one reporter. In some embodiments, the method also includes identifying and / or quantifying at least one reporter. In certain embodiments, identifying and / or quantifying at least one reporter indicates the presence of one or more CRMs linked to the reporter. The CRM can be further characterized, for example, by isolating the nucleic acid linked to the reporter and sequencing the nucleic acid. The isolated nucleic acid can be further tested to identify the CRM contained in the nucleic acid.

[0085] In some embodiments, the method includes isolating RNA from a cell of interest transfected with a nucleic acid reporter library, thereby producing an isolated RNA. Any method can be used to isolate RNA, including extraction and precipitation methods (e.g., Tan et al. Journal of biomedicine & biotechnology (2009): 574398-574398, which is incorporated herein by reference in its entirety). In some cases, additional steps can be included, such as to enhance the purity of the isolated RNA. Any additional RNA isolation step can be included, such as contacting the RNA with a DNA-specific enzyme, such as DNase (e.g., DNase I) and / or exonuclease (e.g., exonuclease I and / or exonuclease III).

[0086] In some embodiments, identifying the reporter comprises synthesizing cDNA. In some examples, synthesizing cDNA comprises reverse transcribing isolated RNA (e.g., RNA isolated using any of the methods described herein), thereby producing cDNA. Any reverse transcription method can be used. In some examples, the method comprises contacting isolated RNA with at least one reverse transcriptase. Any reverse transcriptase can be used. In some examples, recombinant Moloney murine leukemia virus (rMoMuLV) reverse transcriptase and / or avian myeloblastosis virus (AMV) reverse transcriptase can be used. An optional additional cDNA synthesis step may be included. In certain examples, the additional cDNA synthesis step comprises further contacting the RNA and at least one reverse transcriptase with an RNA-dependent and DNA-dependent DNA polymerase. In some examples, the additional cDNA synthesis step comprises contacting an RNase (e.g., RNase I f The method includes adding an RNase specific for single-stranded RNA, such as RNase I,

[0087] In some embodiments, the method includes detecting and / or identifying cDNA (e.g., cDNA synthesized using any of the methods described herein). Any method for detecting and / or identifying cDNA can be used (e.g., sequencing-based, microarray-based, and / or PCR-based methods, such as next-generation sequencing, microarray and hybridization, and / or quantitative PCR). In some examples, the cDNA includes at least one unique barcode reporter. In some examples, detecting the cDNA includes amplifying the cDNA, such as a barcode reporter cDNA (e.g., a barcode reporter cDNA) (e.g., using PCR, such as high-fidelity PCR, by contacting the cDNA with a high-fidelity polymerase and / or at least one primer, such as a pair of universal primers). In certain examples, amplifying the cDNA includes selecting primers specific to nucleotides that include at least one unique nucleic acid barcode (e.g., a pair of primers, e.g., at least one primer, such as a pair of universal primers). In some examples, the primers include a pair of universal primers that amplify a pool of barcodes in the cDNA. In some examples, amplifying the cDNA further comprises contacting a primer with the cDNA and performing PCR (e.g., using the primer and the cDNA). Thus, in some examples, the method can be used to produce an amplified DNA (e.g., cDNA), such as an amplified barcode DNA. In some examples, the method includes identifying the cDNA, such as by identifying a reporter (e.g., a nucleic acid barcode). In some examples, the method includes identifying the nucleic acid barcode using a sequencing-based method, a microarray-based method, and / or a PCR-based method, such as next-generation sequencing, microarray and hybridization, and / or quantitative PCR. In certain examples, the cDNA is identified by sequencing the nucleic acid barcode (e.g., using next-generation sequencing).Exemplary methods may further include a quantification step (e.g., quantifying at least one unique nucleic acid barcode).

[0088] In some examples, the method described herein is a high-throughput method. In some examples, the plurality of nucleic acid molecules in the library described herein covers at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 98%, or 100%, or about 10-20%, 20-40%, 25-50%, 50-75%, 75-85%, 80-90%, 85-90%, 85-100%, or 90-100%, or about 93%, 93.4%, or 94% of the selected genome of interest (e.g., animal genome or human genome). In other examples, the plurality of nucleic acids in the library provides greater than 1X coverage of the genome (e.g., 1X, 1.5X, 2X, 2.5X, 3X, 3.5X, 4X, 4.5X, 5X, 8X, 10X, or greater coverage). In some examples, the plurality of nucleic acid molecules comprises at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 98%, or 100%, or about 10-20%, 20-40%, 25-50%, 50-75%, 75-85%, 80-90%, 85-90%, 85-100%, or 90-100%, or about 85%, 90%, or 95% of the cis-regulatory elements in a selected genome of interest.

[0089] Further contemplated herein is a kit for detecting functional nucleic acid regulatory elements.In some examples, the kit can be used for identifying and / or quantifying functional nucleic acid regulatory elements.In some examples, the kit can be used for high-throughput detection, identifying and / or quantifying functional nucleic acid regulatory elements.In some examples, the kit can include any nucleic acid reporter library described herein. In some examples, the library covers at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 98%, or 100%, or about 10-20%, 20-40%, 25-50%, 50-75%, 75-85%, 80-90%, 85-90%, 85-100%, or 90-100%, or about 93%, 93.4%, or 94% of a selected genome of interest (e.g., an animal genome or a human genome). In some examples, the library comprises at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 98%, or 100%, or about 10-20%, 20-40%, 25-50%, 50-75%, 75-85%, 80-90%, 85-90%, 85-100%, or 90-100%, or about 85%, 90%, or 95% of the cis-regulatory elements in a selected genome of interest (e.g., an animal genome or a human genome).

[0090] In some examples, the kit further comprises at least one reverse transcriptase (e.g., recombinant Moloney murine leukemia virus (rMoMuLV) reverse transcriptase, avian myeloblastosis virus (AMV) reverse transcriptase). fAdditional cDNA synthesis elements may be included, such as an RNase specific for single-stranded RNA, such as a ribonuclease (RIA) or a ribonuclease (RIA) specific for single-stranded RNA. In some examples, the kit includes elements for amplification, such as by PCR, of cDNA, such as a cDNA that includes at least one unique barcode. In particular examples, the kit includes PCR primers and a DNA polymerase, such as a high-fidelity DNA polymerase. EXAMPLES

[0091] The following examples are provided to illustrate certain specific features and / or embodiments. These examples should not be interpreted as limiting the present disclosure to the specific features or embodiments described. In these examples, a genome-scale reporter assay method for cis-regulatory modules (CRMs) is described. GRAMc can reliably measure the cis-regulatory activity of nearly 90% of the human genome in 200 million HepG2 cells with randomly fragmented inserts of about 800 bp. A library of reporter constructs was generated that covers the human genome about four times (4x coverage) using ≧15M randomly fragmented inserts of about 800 bp.

[0092] Example 1 This example describes the methods and materials used in Examples 1-7.

[0093] GRAMc library construction Fusion adapter preparation: The GRAMc preparation contains a custom designed fusion adapter to minimize the formation of unwanted ligations (Figure 6). Two complementary hybrid oligomers were synthesized by Integrated DNA Technologies (IDT): p-AD4_F (5'- / p / CTGCTGAATCACTAGTGAATTATTACCCrUrUCAAGACACTACTCTCCAGCAGT-3'; SEQ ID NO: 1) and p-AD4_R (5'- / p / CTGCTGGAGAGTAGTGTCTTGrArAGGGTAATAATTCACTAGTGATTCAGCAGT-3'; SEQ ID NO: 2). The ribonucleotide sites are labeled "rU" and "rA." Fusion adapters were prepared by diluting p-AD4_F and p-AD4_R to 4 pmol / μL in 1× T4 DNA ligase buffer (NEB® B0202S) followed by annealing at 95° C. for 2 min, followed by temperature ramping at a rate of −0.5° C. / 20 sec cycles for 160 cycles. The annealed adapters were aliquoted in 3 μl volumes and kept at −80° C. until use.

[0094] GRAMc vector preparation: The GRAMc vector was constructed by replacing the sea urchin nodule basal promoter upstream of the GFP ORF of an existing vector (Nam, et al. PLoS One 7.4 (2012): e35934) based on pGEM-T Easy vector (PROMEGA®) with the super core promoter 1 (SCP) (Juven-Gershon, et al. Developmental biology 339.2 (2010): 225-229). The GFP ORF was inserted into pGREEN LANTERN® (GIBCO BRL®) (Arnone, et al. Development 124.22 (1997): 4649-4659). The vector was cloned with AflII / The fragments were linearized by overnight digestion with HindIII and amplified as two separate cassettes from 20 ng of linearized template in 10 cycles of PCR (Figure 7). The SCP-GFP cassette was amplified using primers NJ-95 and NJ-145 for the SCP-GFP cassette and NJ-146 and NJ-96 for the vector backbone in a 50 μL Q5® High Fidelity DNA Polymerase reaction (NEB® M0491) with an annealing temperature of 62°C and an extension of 2 minutes. A sequence of six phosphorothioated bases at the 5' end of NJ145 and NJ146 prevents primer site loss during subsequent GIBSON ASSEMBLY®.

[0095] Genomic insert preparation: 20 micrograms of NG16408 genomic DNA (Coriell Institute) was randomly fragmented in 200 μL of water using a QSONICA® Q125 at 20% amperage for three cycles of 15 sec pulse / 10 sec rest. The DNA was column cleaned using Zymo-25 columns (Zymo Research) and approximately 800 bp fragments were size selected on a 1.2% agarose gel. A portion of the gel purified gDNA was size checked on a 2% agarose E-gel (THERMOFISHER® G501802). The remaining purified fragments were repaired in 25 μL of PreCR reaction (NEB® M0309) containing 1× THERMOPOL® buffer, 100 μM dNTPs, 1× NAD+, and 0.5 μL of PreCR enzyme for 30 minutes at 37°C. The PreCR-treated fragments were purified using a Zymo-6 column and End Repair / dA Tailing was performed in a 32.5 μL reaction mixture. The ligated DNA was treated with the TA Ligation Module (NEB® E7370) followed by treatment with annealed AD4 fusion adapters at a 10:1 adapter to insert molar ratio in a 41 μL reaction in a TA Ligation Module (NEB E7370). Unligated adapters and genomic inserts were removed with 20 U each of Exonuclease I (NEB M0293) and Exonuclease III (NEB® M0206) in a 50 μL reaction supplemented to 1× with CutSmart buffer. The ligated DNA was column cleaned up (Zymo-6) and then linearized with 15 U of RNase HII (NEB® M0288) in a 30 μL reaction of 1× THERMOPOL® buffer at 37° C. for 90 min. RNase HII also cleaves AD4 adapter concatemers to approximately 60 bp units, which can be removed by subsequent magnetic bead purification. The linearized insert was purified using 20 μL of AXYGEN® magnetic beads (AXYGEN®) supplemented to a final concentration of 17% PEG8000 and 10 mM MgCl2, followed by washing three times with 70% ethanol and elution with 30 μL of water.

[0096] Stepwise synthesis of long random DNA sequences from short random oligomers: Since de novo synthesis of a large number of long random DNA sequences remains challenging, in some cases, pools of long random DNA sequences were generated from commercially available short random single-stranded DNA (ssDNA; FIG. 13). First, 2 μg of ssDNA was phosphorylated using polynucleotide kinase and subsequently converted to double-stranded DNA (dsDNA) with random hexamers, dNTPs, and Klenow enzyme. In parallel, 1 μg of unphosphorylated ssDNA was converted to dsDNA using random hexamers, dNTPs, and Klenow enzyme. Second, a reaction tube was prepared with 200 ng of unphosphorylated dsDNA and T4 DNA ligase in 1× T4 DNA ligase buffer. The unphosphorylated dsDNA was ligated to the phosphorylated dsDNA. Third, 50 ng of phosphorylated dsDNA (or a portion of the unphosphorylated DNA, such as about 1 / 4) was added to the ligation reaction tube to initiate ligation. Because there was an excess of unphosphorylated DNA in the reaction, most of the phosphorylated DNA was ligated to the unphosphorylated DNA. Each molecule of unphosphorylated DNA can accept up to two molecules of phosphorylated DNA (one molecule at each end). The ligation product contains an unphosphorylated 5' end. The ligation process was repeated for at least one cycle (e.g., at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 25, 30, 45, 50, 60, 75, 90, or 100 cycles, or about 1-5, 1-10, 1-15, 1-20, 5-20, 10-25, 25-50, or 50-100 cycles, or about 16 cycles). The number of cycles (X) is expected to be ≧2×L / I, where L and I are the desired length of the generated random DNA and the length of the starting nucleic acid, respectively. For example, to synthesize a pool of DNA molecules with a length of about 800 bp with a nucleic acid with a length of 100 bp, X should be about ≧16. Fourth, the nicks of the ligation product were repaired with a DNA repair enzyme (NEB® PreCR Repair Mix, catalog number M0309S).Fifth, DNA molecules of desired length were enriched by gel-based or bead-based size selection. The eluted DNA was now ready to be used for GRAMc library construction or other applications. Using this method, we generated a GRAMc library containing approximately 1M random DNA sequences of about 800bp in length.

[0097] Genome coverage estimate: To determine the amount of adaptor-ligated insert that represents 1X genome coverage, dilutions of insert were prepared at 0.5ng / μl, 0.25ng / μl, 0.1ng / μl, 0.05ng / μl, and 0.025ng / μL. Each dilution was amplified with two adaptor-specific primers, NJ-213 and NJ-214, with annealing at 61°C and extension for 1 minute as determined in cycle testing. Q5® High Fidelity DNA Polymerase Kit (NEB® M0491) was used. Amplicons were cleaned up with AXYGEN®. Eight nanograms per well of each amplification dilution and NG16408 stock DNA were used for QPCR against the following single copy targets: ACTA1, ADM, ADAM12, AXL, CFB, DLX5, Kiss1, NCOA6, Notch2, RPP30, and TOP1. For each dilution sample, targets with dCT>5 were counted as absent compared to stock genomic DNA.

[0098] The Poisson probability (P) of a genomic region being present in a library is given as P = 1-(1-p)XN, where p = (insert size) / (genome size), N = number of partitions of the genome for a given insert size, and X = intended genome coverage. The proportion of targets identified as present by QPCR was compared to the value of P. Based on this model, a sample with genome coverage of approximately 1X had a P of approximately 0.6. When a dilution of 0.1 ng / µL was tested, 6 of 11 targets were positive, i.e., a proportion of positives of 0.545, representing a coverage of 0.5X to 1X. Therefore, it was determined that 0.2 ng of insert represents approximately 1X genome coverage. Equimolar amounts of independently amplified replicates were mixed to obtain a pool of inserts with 5X genome coverage.

[0099] Insert cloning and N25 barcoding of GRAMc libraries: Thirty nanograms of 5X genomic insert was cloned into a 1:1:1 molar ratio of two pieces of linearized GRAMc vector, SCP-GFP, and backbone cassette in 16 μL of NEBUILDER® HiFi assembly reaction (NEB® E2621) at 50° C. for 20 min. The assembled linear DNA was column purified and eluted in 20 μL of water. To prepare assembled libraries for barcoding, four replicates of 8 ng of purified assembly were amplified in a PCR with primers NJ-101 and NJ-126 for 9 cycles as determined in the cycle test, using an annealing temperature of 62° C. and an extension time of 5 min. The replicates were combined and column cleaned up.

[0100] To add the N25 barcode downstream of the GFP ORF, 150 ng of the library was used for single cycle PCR with NJ-127, which contains a random 25 bp barcode sequence, a core poly(A) signal (Nag, et al. RNA 12.8 (2006): 1534-1544), and 5' biotinylation, in a 50 μL Q5 high fidelity DNA polymerase reaction with an annealing temperature of 60° C. for 40 seconds and an extension time of 15 minutes. NJ-126 was used as a competitor in the PCR to reduce the possibility of template crossover due to opposite strand occupancy and extension. Primers were removed by AXYGEN® bead purification using 50 μL beads and 20 μL water elution as described. The barcoded library was isolated using 20 μL of DYNABEADS® MyOne C1 beads (INVITROGEN® 65001) by preparing, binding, and washing the beads according to the manufacturer's protocol.

[0101] After isolation, the C1 beads were washed with 20 μL water and then resuspended in 50 μL water. Half of the barcoded library was amplified in 24×20 μL Duplicate Q5® High-Fidelity DNA Polymerase reactions using NJ-128 and NJ-129, annealing at 61° C., and extension for 5 minutes for 9 cycles as determined in the cycle test. The replicates were combined and cleaned up with AXYGEN® beads, then gel purified (Zymo Research) and subjected to an additional AXYGEN® bead cleanup.

[0102] The barcoded GRAMc library was then self-ligated. To reduce intermolecular ligation, 125 ng of the barcoded library was ligated with 14,000 U of high-concentration T4 DNA ligase (NEB®M0202T) in 600 μL of 1×T4 ligase buffer (NEB®B0202) for 4 hours at 20° C. The ligation product was supplemented with 67 μL of lambda exonuclease buffer and 30 U each of exonuclease I (NEB®M0293) and lambda exonuclease (NEB®M0262S) for 1 hour at 37° C., and then spiked with 1 μL of proteinase K (THERMOFISHER®) for 15 minutes at 37° C. Proteinase K treatment reduces the viscosity of the ligation mix and increases DNA yield by almost 2-fold. The library was purified using 25 μL of magnetic beads (AXYGEN®) supplemented with a final concentration of 15% PEG8000 and 10 mM MgCl2, followed by washing four times with 70% ethanol and elution in 6.5 μL of water. The output of this process is a pure population of circularized GRAMc libraries.

[0103] Transformation and size estimation of the GRAMc library: To determine the scale of electroporation, 1 μl of ligation product was electroporated into 25 μL of ELECTROMAX® DH10B® competent cells (THERMOFISHER® 18290015). Transformants were immediately resuspended in 1 ml pre-warmed SOC medium and 1 / 500 of the transformants were used for 10-fold serial dilutions and plated without recovery to estimate the number of colonies in the whole pool. Based on this test, the scale of transformation to reach the target colony number is determined. Electroporation of 4-10 ng of ligation product produces approximately 40M colonies.

[0104] To generate a complete GRAMc library with 200M colony targets, duplicate electroporation steps were performed using 30ng of library ligate (12ng / μL) per each of 2×25 μL ELECTROMAX® DH10B® competent cells. Immediately after electroporation, each replicate was resuspended in 1 ml SOC medium, and then the replicates were combined. To estimate the size of the GRAMc library, 1 / 2000 of the transformants were used for 10-fold serial dilutions and plated without recovery. The remaining transformants were immediately used to inoculate 180 ml LB, to which 100 μg / ml ampicillin was added after 20 min recovery, followed by overnight growth. The plasmid library was prepared using the ZYMOPURE® II Plasmid Maxiprep Kit (Zymo Research). Hereafter, this library is referred to as the Hs800_GRAMc library.

[0105] As a quality control step, 12 colonies were selected from the plate and plasmids were extracted to check insert size and barcodes using Sanger sequencing. The plasmid from each colony should contain an insert (~800 bp) and a barcode. If the ligation products contain a high degree of barcode diversity, the barcode sequences identified from the colonies should not be present in the final library. Example sequences of the GRAMc vectors and oligomers used are shown in Table 3. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6]

[0106] GRAMc library characterization by ILLUMINA® paired-end sequencing

[0107] Sequencing libraries: Paired-end sequencing on the NextSeq500 platform was used to identify the inserts and associated barcodes of the individual reporter constructs. Sequencing the Hs800_GRAMc library on the ILLUMINA® platform was problematic for two reasons: i) the length of the reporter constructs was too long for paired-end sequencing, and ii) the adapter sequences lacked diversity and were therefore incompatible with the ILLUMINA® platform. To address the length issue, the construct length was reduced by deleting either the SCP-GFP region or the vector backbone by inverse PCR and self-ligation to bring the inserts closer to the N25 barcodes. To address the issue of low sequence diversity, a set of phasing primers (Wu, et al. BMC microbiology 15.1 (2015): 125) was used to artificially increase sequence diversity. Generation of two distinct populations of sequencing libraries lacking any of the target backbones also increases sequence diversity in the adapter region (Figure 8).

[0108] In this example, construction of the sequencing library begins with cleavage of 500 ng of maxi-prepared plasmid with Cas9 (NEB® M0386) using sgRNAs against either the vector backbone or the GFP ORF. Both sgRNAs were predicted to have seven off-target sites in the human genome (crispr.mit.edu). Primer pairs, NJ-179 / NJ-183 and NJ-180 / NJ-183, were used to generate templates for in vitro transcription of sgRNAs targeting the backbone and GFP, respectively. Primer sequences are shown in Table 3. The CRISPR cleaved plasmid library was mixed with an equimolar amount of the uncut plasmid library. Inverse PCR of 5 ng of the GFP cleaved linear library mixture was performed using NJ-209 and NJ-141 (denoted as "Hs800_23") to remove the SCP-GFP region. Inverse PCR of 5ng of the backbone-truncated linear library mixture was performed using NJ-208 and NJ-142 (designated "Hs800_14") to remove the vector backbone. Q5® High-Fidelity DNA Polymerase (NEB®) was used for PCR. A total of 20 replicates were prepared for each template / primer pair. Each replicate was combined, column concentrated, gel isolated, and cleaned up with AXYGEN® beads. Each amplification was self-ligated overnight at 20°C with 3μL of concentrated T4 ligase at a concentration of 75ng in 350μL of 1×T4 DNA ligase buffer, supplemented with 20U each of exonuclease I and exonuclease III for 1 hour at 37°C, followed by incubation with proteinase K for 10 minutes at 37°C. The ligates were cleaned up with AXYGEN® beads and eluted in 30μL of water.

[0109] To amplify the insert::N25 cassette, four replicates containing 2ng of Hs800_14 ligate were amplified from the circularized first round PCR products using NJ-209 and NJ141 (hereafter referred to as Hs800_1423), and four replicates containing 2ng of Hs800_23 ligate were amplified using NJ-208 and NJ142 with a total of 8 cycles of annealing temperature of 60° C. and extension time of 90 seconds (hereafter referred to as Hs800_2314). Products were column cleaned, gel isolated, and bead cleaned for subsequent PCR amplification to add PE adapter sequences for ILLUMINA® sequencing.

[0110] To increase the diversity of the Hs800_1423 and Hs800_2314 sequencing libraries for sequencing on the ILLUMINA® platform, each library (Hs800_1423 and Hs800_2314) was amplified using seven different phased PE1-containing primers. For the Hs800_1423 library, 2 ng of template was used in each separate reaction with the PE2-containing primer NJ-401, and each of the following partial PE1-containing primers: NJ-400, NJ-504, NJ-505, NJ-506, NJ-507, NJ-508, and NJ-509, with an annealing temperature of 60° C. and an extension time of 90 seconds for a total of 7 cycles. For the Hs800_2314 library, 2 ng of template was used in each separate reaction with the PE2-containing primer NJ-403 and each of the following partial PE1-containing primers: NJ-402, NJ-498, NJ-499, NJ-500, NJ-501, NJ-502, and NJ-503, with an annealing temperature of 60° C. and an extension time of 90 seconds for a total of 7 cycles. The phased PE1 primers can be pooled before PCR amplification to simplify the procedure. Individual amplifications were cleaned up on columns, gel isolated, and cleaned up with AXYGEN® beads. Each of the seven phased Hs800_1423 libraries was amplified using NJ-497 and NJ-401 to complete the PE1 adapter sequence. Each of the seven phased Hs800_2314 libraries was amplified using NJ-497 and NJ-403 to complete the PE1 adapter sequence. For each amplicon, 2 ng of the corresponding library template was amplified with 6 cycles of PCR with an annealing temperature of 60° C. and an extension time of 90 seconds. The libraries were purified again, gel isolated, and cleaned up with AXYGEN® beads. Equimolar amounts of the 14 phased libraries (seven from each direction) were used for paired-end sequencing in combination with 90% sequencing pool + 10% PhiX control. The sequences of the primers are shown in Table 3.

[0111] Trimming adapter sequences from inserts and barcodes: The 5' and 3' ends of the insert and its associated N25 barcode were extracted from each pair of sequence reads. Adapter sequences were removed using Trimmomatic (Bolger, et al. Bioinformatics 30.15 (2014): 2114-2120) and the barcodes were extracted using seqtk (github.com). The complementary sequences were reversed using the 5′ and 3′ ends of the inserts. To extract the 5′ and 3′ ends of the inserts, the P1 and P2 adapters were trimmed, respectively. To extract the N25 barcodes, depending on the orientation of the sequence reads, the P3 or P4 adapters were first trimmed, the trimmed sequences were reverse complemented, and the P4 or P3 adapters were trimmed. Paired-end reads that failed to trim any adapter sequence were discarded. Note that for N25 barcode sequences, 1 bp from each adapter was retained, resulting in 27 bp reads. The adapter sequences used for trimming are shown in Table 3.

[0112] Mapping sequence reads in the human genome and identifying inserts: To identify inserts, the extracted 5' and 3' ends of the inserts were mapped against the GRCh38 / hg38 assembly (downloaded from genome.ucsc.edu). The Burrows-Wheeler alignment tool (BWA) (Li, et al. Bioinformatics 25.14 (2009): 1754-1760) was used with the following command: "bwa me Sequences were mapped with the 'm -W1500'. Mapped read pairs spanning >1,500 bp or <300 bp were discarded. If two mapped inserts overlapped, with their midpoints within 20 bp and both ends within 50 bp, they were combined into a single insert, taking the coordinates that maximized its length.

[0113] Clustering of N25 barcodes: To identify reads from the same barcode, the extracted barcode reads were clustered based on the following steps: i) representative reads were generated by filtering redundant reads using the Khmer software package (Crusoe, et al. F1000 Research 4 (2015)) with the command "normalize-by-median.py -C1 -k 25 -N 5-x 2.5e9"; ii) the entire set of barcode reads was clustered using the BWA software (Li, et al. Bioinformatics 25.14 (2009): 1754-1760) with the command "bwa aln -n 2 -o 2 -e -1 -M 3 -O 11 -E 8 -k 1 -l 6" were used to compare against the representative reads. Barcode reads that did not match any of the representative reads were added to the representative reads file and the BWA search was repeated. Reads with the same barcode were clustered using single-linkage clustering. We identified and assigned a unique barcode cluster (bcl) number to each cluster. A new file of representative reads with bcl numbers was generated for future use (see below, GRAMc assay in HepG2: comparison of barcoded reads to barcoded clusters).

[0114] Association of genomic inserts with barcode clusters (bcls): Although each barcode read was inherently connected to a read derived from an insert in the paired-end reads, a minority of bcls were associated with more than one of the identified genomic inserts. This ambiguity arises mainly because there are highly similar overlapping regions in the genome. The assignment of a bcl was constrained to the insert that had the most reads for that bcl. If ≥ 2 inserts had the same number of reads for a bcl, the bcl was not assigned to any insert.

[0115] GRAMc assay in HepG2 Cell culture: HepG2 cells (ATCC HB-8065) ​​were grown without antibiotics under the supplier's recommended conditions in EMEM supplemented with 10% fetal bovine serum. For all experiments, HepG2 cells were used within 16 passages of receipt. All experiments were performed with cells that had undergone a minimum of 5 passages since thawing, as reporter expression in cells with <5 passages was different compared to cells with ≥5 passages.

[0116] Genome-scale transfection and lysate collection: For each genome-scale transfection batch, 10 7 Cells were seeded (100M cells) in 30 ml medium in each of 10×150 mm culture dishes and allowed to attach for 30 hours. 100 μg of Hs800_GRAMc library was transfected into cells in 4 ml of OPTI-MEM® (THERMOFISHER®) prepared in 2×2 mL siliconized tubes using 100 μL of DNA-IN® Reagent for HepG2 (MTI-Globalstem) according to the manufacturer's protocol. A total of 10 sets of 10×150 mm dishes were used to harvest approximately 200M cells per batch.

[0117] For harvesting, cells were washed with 1x PBS 26 hours after transfection and harvested by scraping in 2.4 mL of RNA-STAT-60 (AMSBIO®) per plate. Lysates were combined and prepared according to the manufacturer's protocol with the addition of a second 70% ethanol wash.

[0118] RNA preparation and cDNA synthesis: In this protocol, we focused on two parameters: i) thoroughly removing contaminating DNA in the RNA sample, and ii) maximizing the efficiency of reverse transcription (RT) using a large amount (~4 mg) of total RNA. Because DNase I is less efficient on single-stranded DNA, DNase I was supplemented with a cocktail of exonucleases I and III to thoroughly remove both double-stranded and single-stranded contaminating DNA. To cost-effectively maximize RT without compromising the cDNA yield of the RT reaction, 15-fold more RNA was used than the maximum input RNA recommended by the manufacturer. A schematic of this procedure is shown in Figure 9.

[0119] To remove contaminating DNA, isolated total RNA (approximately 4 mg) was resuspended in 1.7 mL nuclease-free water and digested in a 2 mL reaction containing 1×DNase I buffer, 100 U DNase I (NEB® M0303), and 900 U each of exonuclease I (ExoI) and exonuclease III (ExoIII) for a minimum of 4 hours at 37° C. Progress of DNA removal was monitored by QPCR for GFP ORFs (NJ-443 and NJ-444). For this quality control step, diluted samples of RNA were heat inactivated at 80° C. for 20 minutes and loaded at an equal volume of approximately 1000 cells / well. If necessary, DNase digestion was allowed to proceed overnight until QPCR Ct values ​​were greater than 30. After digestion, nucleases were removed by extraction with phenol:chloroform:isoamyl alcohol (25:24:1) and ethanol precipitation overnight at −20° C., followed by two washes with 75% ethanol. RNA was resuspended in 1 mL of RNase-free water.

[0120] As a quality control for reverse transcription (RT), an equal volume of total RNA containing approximately 4000 cells (approximately 1 μg) was used for cDNA synthesis using the High Capacity cDNA Reverse Transcription Kit (APPLIED BIOSYSTEMS® 4368813) according to the manufacturer's protocol, with the addition of 5 pmol of GRAMc library-specific RT oligo (NJ-489) to serve as a standard for maximum cDNA synthesis from the transcripts.

[0121] The remaining total RNA (approximately 4 mg) was diluted to 1.420 mL and 2000 pmol of GRAMc_RT_oligo (NJ-489) was added. The RNA / primer mixture was incubated at 65°C for 1 min and cooled on ice before adding 200 μL of 10x High Capacity buffer, 80 μL of 10 mM dNTPs, and 100 μL of Multiscribe without random oligomers. The reaction was incubated at room temperature for 10 min and then at 37°C for 4 h. The progress of genome-scale cDNA synthesis was monitored by QPCR for GFP compared to a reference RT control using an equal volume of 100 cells / well. The reaction was allowed to proceed until the Ct values ​​were similar to the reference RT reaction. If necessary, the reaction was spiked with M-MuLV reverse transcriptase (NEB® M0253) and additional dNTPs and allowed to proceed overnight.

[0122] Upon completion of the RT reaction, the samples were ethanol precipitated to reduce the volume. The RNA / cDNA was resuspended and digested with 1000U of RNase If (NEB® M0243) in a 500 μL reaction with 1× NEBUFFER® 3 overnight at 37° C. To remove excess protein, 1 μL of Proteinase K solution was added to the reaction and incubated at 37° C. for 15 min. The cDNA was ethanol precipitated overnight at −20° C. with glycogen as a carrier and washed three times with 80% ethanol. The cDNA pellet was resuspended in 200 μL of water and heated to 95° C. for 10 min to destroy residual Proteinase K. A sample of the cDNA library was subjected to quality control by QPCR.

[0123] Preparation of expressed N25 barcodes for NGS: The entire pool of expressed N25 was amplified using primers NJ-141 and NJ-142 in eight replicates of 50 μl Q5® PCR reactions with an annealing temperature of 62° C. and an extension time of 1 min for a total of eight cycles. The replicates were combined for each batch. A 50 μL aliquot of each batch was treated as follows: unwanted long DNA was bound using 0.5× volume of AXYGEN® beads for 20 min at room temperature. The desired short amplicons (65 bp) from the supernatant were further purified for each batch using duplicate Zymo columns, each eluted in 20 μL of water. To prepare amplicons for sequencing the expressed barcodes, 2 ng of the first round amplified and cleaned up N25 barcodes were subjected to another nine cycles of amplification with NJ-141 and NJ-142. To prepare amplicons for sequencing the input library, 2 ng of the input library was amplified from a mixture of uncut / CRISPR backbone-cut / CRISPR GFP-cut plasmid library templates with NJ-141 and NJ-142 primers for 9 cycles of PCR.

[0124] Sequencing libraries were prepared for both IONTORRENT® Proton sequencing (Batch 1: NJ197 and NJ-523; Batch 2: NJ-198 and NJ-523) and ILLUMINA® NextSeq500 sequencing (14 phased libraries using NJ-400 / NJ-504 / NJ-505 / NJ-506 / NJ-507 / NJ-508 / NJ-509 with NJ364 or NJ-402 / NJ-498 / NJ-499 / NJ-500 / NJ-501 / NJ-502 / NJ-503 with NJ-399). All of these amplifications used an annealing temperature of 65° C. and an extension time of 20 seconds for a total of 6 cycles. Primer sequences are shown in Table 3.

[0125] Comparison of barcode reads to barcode clusters (bcl): The goal of this step is to count the number of barcode reads originating from either the expression barcode or the input library for each barcode cluster (bcl). The adapter trimmed barcode reads were compared to the representative barcode reads established above using a BWA search with the same command as above. If a barcode read matched more than one bcl, each match was counted to the corresponding bcl. The same procedure was applied for both the expression barcode and the input library, so the effect of multiple counting of barcode reads is neutralized.

[0126] Calculation of CRM activity: In this step, the cis-regulatory activity of each insert is calculated based on the expression barcode and the number of reads of each bcl counted from the input library. If an insert is associated with ≥2 bcls (99% of the inserts), the read counts of all bcls of this insert were combined. First, to avoid false positive CRMs due to too few input counts, inserts with ≥10 counts from the input library or expression barcodes with ≥50 counts for both batches of the experiment were retained. This filtering resulted in 9,339,996 inserts that met the retention criteria. Then, the read counts of the expression barcodes were divided by the read counts of the input library, and the resulting numbers were ranked. The middle 30% of the data was used to calculate background activity (bg) (e.g., 26). Furthermore, CRM activity was normalized to background activity. An insert was considered as a CRM if at least one batch showed ≥5×bg and another batch showed ≥4.5×bg (90% of 5×bg). A total of 54,115 inserts were identified that passed the criteria. After removing inserts with ≥95% sequence identity to other parts of the genome and merging overlapping CRMs, the final set contained 41,216 unique non-overlapping CRMs. A scatter plot is shown in Figure 2A. This scatter plot was generated using 500,000 randomly selected inserts and ggplot2 (Wickham. ggplot2: Elegant Graphics for Data Analysis, Springer-Verlag New York, 2009) from the R package (cran.r-project.org).

[0127] Genomic distribution of CRMs To compare the genomic locations of CRMs and genes, we used the publicly available gene annotation file "GRCh38.89.gff3" from ftp.ensembl.org, and the RNA-seq data "ENCFF861GCR and ENCFF640ZBJ" from encodeproject.org for HepG2 cells. Genes with FPKM ≥ 1 in both RNA-seq data were considered "expressed". To generate the maps shown in Figure 2C and Figures 10A-10F, the Grid Graphics package in R (Murrell. R graphics. CRC Press, 2016) was used to plot the 1 Mb bins. It was used in

[0128] To calculate the enrichment of CRMs in genomic regions relative to genes (Figure 2D), inserts / CRMs spanning windows larger than 2 kb were assigned to the window that most overlapped with the insert. Genomic coordinates of the 5' and 3' ends of genes were extracted from the GRCh38.89.gff3 file. Inserts / CRMs were counted only once per gene, but multiple counts were allowed for different genes.

[0129] One-by-one reporter assays for validation Creation of individual reporter constructs: 20 genomic regions (11 CRMs, 5 weakly active regions, and 4 inactive regions) were individually PCR amplified and integrated into pre-barcoded SCP-GRAMc vectors (Guay, et al. Developmental biology 422.2 (2017): 92-104). Primers were used to amplify inserts containing flanking sequences that overlap with adapter sequences present in the vector. Each assembly was performed using 2 μL of NEBUILDER® HiFi assembly reaction. The assembly reaction was used to mix and go DH10B competent cells (Zymo Research T3019) were transformed and positive clones were identified by colony PCR. Endotoxin-free plasmid was prepared (Zymo Research D4208T).

[0130] The pre-barcoded SCP-GRAMc vector was further used to generate an EGFP internal control vector for use in QPCR of GFP reporter expression of individual clones. In this step, the vector was amplified by inverse PCR with NJ731 and NJ732. The EGFP ORF of pEGFP-C1 was amplified using NJ729 and NJ730 and assembled into the SCP-GRAMc vector using GIBSON ASSEMBLY® at a 2:1 ratio using NEBUILDER® HiFi Assembly Master Mix. The GFP ORF used in the GRAMc vector is different from the commonly used EGFP ORF, allowing for differential detection of the two GFPs by QPCR. The sequences of the primers are shown in Table 3.

[0131] Individual reporter assays to validate GRAMc results: HepG2 cells were seeded at approximately 60K cells per well in 500 μL of EMEM supplemented with 10% FBS in 24-well plates. For consistency with the genome-scale assay, cells that were 12-15 passages after receipt from ATCC and at least 7 passages after recovery were used. Cells were allowed to attach for 24 h and transfected with a mixture of 50 μL of OPTI-MEM®, 200 ng of individual test plasmid containing GFP, 200 ng of SCP-EGFP control vector, and 1.2 μL of DNA-IN® reagent. After 26 h (approximately 80-85% confluency consistent with the genome-scale assay), cells were washed twice in DPBS and collected in 300 μL of DNA / RNA lysis buffer (ZymoResearch), and gDNA and total RNA from each sample were combined and washed according to the manufacturer's protocol and purified using ZymoII columns. RNA was eluted in 34 μL water. Half of the total RNA from each sample was treated in 20 μL Turbo DNase reaction (THERMOFISHER®) for 1 hour at 37° C. The reaction was stopped with 2 μL DNase inactivation reagent (THERMOFISHER®). Half of the DNase-treated RNA was used in 20 μL 1× High-Capacity cDNA synthesis reaction with an additional 10 pmoles of GRAMc_RT_oligo (NJ-489) and RNase inhibitor. QPCR was performed on GFP and EGFP on total gDNA representing 1 / 40,000 of the original sample, non-RT control representing 1 / 40 of the total RNA sample, and cDNA representing 1 / 160 of the original sample. GFP expression driven by individual test fragments was normalized to an internal control (EGFP expression, NJ404 / NJ405). The sequences of the QPCR primers are shown in Table 3.

[0132] Relative enrichment of ENCODE annotations in CRMs compared to inactive inserts ENCODE ChIP-seq files were obtained from encodeproject.org. Overlap between CRMs and individual ENCODE data was analyzed using bedtools (Quinlan, et al. Bioinformatics 26.6 (2010): 841-842) with the command "be dtools jaccard -f 1E-09 -F 1E-09". Relative enrichment of ENCODE annotations in CRMs was calculated as follows: i) First, the genomic fraction of base pairs overlapping between CRMs and ENCODE annotations was calculated. ii) Random expected overlap was calculated by multiplying the genomic fractions of the two datasets. iii) Enrichment was calculated by dividing the result of i) by the result of ii). iv) Following the same procedure, enrichment of the same ENCODE annotations in inactive regions (L1 group) was calculated. v) Relative enrichment was calculated by taking the ratio of iii and iv.

[0133] Motif enrichment and predicted strong enhancers in CRMs Selection of GRAMc inserts: ChromHMM (Ernst, et al. Nature 473.7345 (2011): 43; Ernst, et al. Nature biotechnology 28.8 (2010): 817) The predicted potent enhancers of HepG2 were compared with the GRAMc data for CRM activity and motif enrichment. Genomic coordinates of the chromatin state were converted to hg38 with liftOver (Hinrichs, et al. Nucleic acids research 34. suppl_1 (2006): D590-D598). First, non-overlapping GRAMc inserts that overlapped in length with predicted potent enhancers by ≥90% were randomly selected. This selection process yielded 18,898 GRAMc inserts that corresponded to predicted potent enhancers. This data was used to generate Figure 3A.

[0134] To compare motif enrichment, we randomly sampled another 18,898 non-overlapping GRAMc CRMs (≥5xbg or G5) without considering predicted enhancers. As negative controls, we also sampled 37,796 non-overlapping inactive (≤1xbg or L1) inserts.

[0135] Motif enrichment survey: 75,592 sampled inserts were simultaneously analyzed to search for putative transcription factor binding site (TFBS) motifs. Using the HOCOMOCOv10 database (Kulakovskiy, et al. Nucleic acids research 44. D1 (2015): D116-D125) and FIMO software (Cuellar-Partida, et al. Bioinformatics 28.1 (2011): 56-62;Bailey, et al. Nucleic acids research 37 (2009): W202-W208) were used with an E-value cutoff of 1E-5. The abundance of each motif was determined by the given Figure 1 shows the proportion of motif-containing inserts per set. Relative motif enrichment was calculated by dividing the abundance of a motif or predicted enhancer in the CRM by the abundance of the same motif in the negative control set.

[0136] Comparison of motif enrichment in CRMs with ChIP-seq peaks: 58 transcription factors shared by name between HOCOMOCOv10 and ENCODE ChIP-seq data were identified. The calculated relative enrichment scores were used to generate Figure 4B.

[0137] Measuring the effect of gene ectopic expression on CRM Preparation of random subsets of the GRAMc library: To obtain small subsets of the GRAMc library for perturbation experiments with ectopic expression of pitx2 or ikzf1, approximately 50 μL of frozen glycerol stock was diluted into 2 ml of LB medium and allowed to recover for 20 minutes at 37° C. with orbital shaking at 250 RPM. A series of two-fold dilutions was prepared, 1 / 100 of which was used for two ten-fold dilutions for plating and colony counting, and the remainder of each two-fold diluted culture was used to inoculate 150 ml LB-Amp cultures and grown overnight. Cultures estimated to contain approximately 80,000 colonies (80K library) were processed using the ZYMOPURE® Plasmid Maxiprep Kit.

[0138] Perturbation assay of 80K construct library: Three co-transfections were performed: 80K library + CMV::pitx2 (Genscript OHu17480D), 80K library + CMV::IKZF1 (Genscript OHu28016D), and 80K library + CMV::EGFP (Clontech pEGFP-C1) with each of the following: 10 cm cells. 2 Approximately 2M cells were seeded in duplicate per plate for transfection. Cells were cultured for approximately 24 hours prior to transfection. Cells were co-transfected with 9 μg of the 80K library and 3 μg of each expression vector using 36 μL of DNA-IN® Reagent for HepG2 (MTI-Globalstem) and 1.2 ml of OPTI-MEM® (THERMOFISHER®) prepared according to the manufacturer's protocol.

[0139] 24 hours after transfection, cells were harvested by trypsinization and 1xDPBS washing. 1 / 10 portion of cells was reserved for Western blot analysis to confirm Pitx2 and IKZF1 expression. The remaining cells were lysed and both DNA and RNA were processed through IIICG columns using Zymo-Duet kit without on-column DNase I treatment. DNA was eluted in 100 μL and RNA was eluted in 80 μL and treated with DNase I (8U) / ExoI (100U) / ExoIII (100U) in a total reaction volume of 100 μL 1xDNase I buffer for a minimum of 4 hours at 37°C. Assuming approximately 10M cells per sample, the equivalent of gDNA from approximately 10,000 cells and nuclease-treated RNA from approximately 5000 cells were tested using QPCR with GFP as a target to confirm the quality of transfection and completeness of DNA removal in RNA, respectively. If necessary, another 2U of DNase I was spiked into the reaction. RNA was column cleaned using a Zymo-IIIC column and eluted in 50 μL of water. The equivalent of approximately 4000 cells was used as a quality control measure of the standard RT reaction as described in the genome-scale protocol. The remaining RNA was incubated for quality control QPCR 2 hours after RT in 80 μL 1× High-Capacity cDNA synthesis reaction with 8 μL Multiscribe and 3.2 μL dNTPs but no random primers at 37°C for 4 hours to overnight with 80 pmoles of GRAMc_RT_oligo (NJ-489) used for cDNA synthesis. Once DNA digestion was complete, 4 μL of NEBUFFER® 3 and 2 μL of RNase If were added to the reaction and incubated at 37° C. for 2 hours, then spiked with Proteinase K and incubated at 37° C. for 15 minutes, heat inactivated at 95° C. for 10 minutes, then ethanol precipitated overnight and resuspended in 30 μL of water.

[0140] N25 barcodes were pre-amplified as described above, but using a single 50 μL Q5® high fidelity DNA polymerase reaction with 6 cycles, and IX barcoding for IONTORRENT® Proton sequencing was used with the following primer pairs: NJ-197 / NJ523 for control-1; NJ-198 / NJ523 for control2; NJ-200 / NJ523 for Pitx2-1; NJ-132 / NJ523 for Pitx2-2; NJ-133 / NJ523 for IKZF1-1; and NJ-134 / NJ523 for IKZF1-2. Data analysis was performed as described above. Primer sequences are shown in Table 3.

[0141] Confirmation of ectopic transcription factor expression by Western blot: Aliquots of each transfection condition (80K library + CMV::pitx2, 80K library + CMV::IKZF1, and 80K library + CMV::EGFP) were lysed in 80 μL of RIPA buffer (150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl pH 8.0, 5 mM EDTA) spiked with a 1:100 dilution of Halt protease inhibitor cocktail (THERMOFISHER®) for 30 minutes on ice with intermittent flicking. Lysates were centrifuged at 12,000 RPM for 10 minutes at 4°C and quantified using BCA reagent.

[0142] Approximately 25 ng of each sample was loaded in duplicate sets (expression and control), separated on a 12% polyacrylamide gel, transferred to a PVDF membrane, and blotted with antibodies against FLAG (1:500, Santa Cruz sc-166355) or GAPDH (1:1000, Santa Cruz sc-25778). Bands were detected on a Bio-Rad ChemiDoc MP system using horseradish peroxidase-conjugated secondary antibodies (1:5000) and enhanced chemiluminescence reagents (GE Healthcare).

[0143] Example 2 This example describes the construction of a GRAMc library. In this example, the GRAMc library was generated by the following steps (Figure 1A-1D). First, random genomic DNA fragments were size-selected, adapter-ligated, and serially diluted to reach the intended genome coverage (Figure 1A). To improve the accuracy of adapter ligation, adapters (Figure 6) were fused to form circular ligation products that could be resistant to exonuclease I / III treatment on linear DNA, including non-ligated DNA and linear ligates. After treatment with exonuclease, the circular ligation products were linearized with RNase HII, which cleaves ribonucleotide sites (UU / AA) within the fused adapters. The linearized ligates were then serially diluted and PCR amplified using adapter-specific primers. Dilutions of the intended genome coverage were identified by QPCR by counting the presence or absence of 11 randomly selected genomic regions. For a dilution containing approximately 4M randomly sampled genomic DNA fragments of approximately 800 bp in length (average 1× genome coverage), the expected abundance of the target region is 0.6. A 5× dilution (or any desired genome coverage) was assembled with two common DNA pieces to generate a linear DNA product containing the genomic test fragment, the basal promoter, the GFP ORF (Arnone, et al. Development 124.22 (1997): 4649-4659), and the vector backbone. A library was constructed (Figure 7). The vector system used was the pan-bilaterian Super Core Promoter 1 (SCP) (Juven-Gershon, et al. Developmental biology 339.2 (2010): 225-229).

[0144] Second, the resulting genomic DNA library was barcoded with an excess number of random 25-mers (N25s) by PCR using a pair of common primers that can amplify the entire library, including the vector backbone (Figure 1B). One of the common primers, primer_R, contains a random N25 in the middle and a core-polyadenylation signal (polyA) (Nag, et al. RNA 12.8 (2006): 1534-1544). The barcoded library was self-ligated, treated with exonuclease I / III, and electroporated into E. coli for library amplification and plasmid extraction. A small percentage of unrecovered transformants (e.g., 1 / 1,000) were used to measure colony forming units (cfu), and the remainder were used for library amplification in liquid culture and subsequent plasmid extraction. Because PCR-mediated barcoding introduces excess barcodes, virtually every individual transformant contains a unique barcode. For example, barcodes present in transformants used for colony counting were not identified in the final library. The number of unique barcode reporters in the GRAMc library can be controlled by the scale of electroporation. In the protocol used herein, 4-10 ng of circular ligation product with an insert of approx. 800 bp consistently generated approx. 40M cfu. This is comparable to the advertised efficiency of commercially available competent cells. As long as the number of unique barcodes harvested is significantly greater than the number of unique inserts, the genome coverage of the library determined in the first step is maintained. Purified plasmids were used for library characterization. Library characterization included identifying the genomic inserts and the pairs of inserts and barcode reporters by ILLUMINA® paired-end sequencing (see Example 1 and Figure 8).

[0145] Using this method, we generated a human GRAMc library of approximately 800 bp long inserts. The intended number of unique genomic DNA inserts and unique barcodes in this library were 20M (5x genome coverage) and 200M (10 barcodes / insert), respectively. Analysis of 479.1M pairs of sequences mapped to the hg38 assembly (out of 519M paired-end reads) identified 15.6M genomic regions. The total number of unique barcodes associated with these genomic regions was 191M. This library covered 93.4% of the human genome at least once (Table 1). [Table 1-1] [Table 1-2]

[0146] Although obtaining more sequencing reads would improve these numbers, these numbers are already close to the intended number of inserts and barcodes in the library. Of the 15.6M genomic regions detected, 13.8M inserts were sequence unique (<95% sequence identity with other genomic regions). In addition, the genomic distribution of unique inserts was somewhat uniform (Figure 2C). In the case of unique inserts (Figure 1C), 71% of the inserts were in the range of 750-850 bp, indicating that size selection was effective. Furthermore, when considering the number of barcodes per insert (Figure 1D), the barcode number of the majority of inserts significantly deviated from the expected number of 10, while 99% and 55% of unique inserts were connected to ≥2 and ≥10 barcodes, respectively. Thus, barcode-specific effects on reporter expression were insignificant in the GRAMc library. A list of the genomic coordinates of the inserts and their associated barcodes is shown in Figure 6. Example 3

[0147] In this example, the application of GRAMc in HepG2 cells is described. The GRAMc library was tested in two batches of 100M HepG2 cells at the time of seeding, or 200M cells at the time of transfection. In comparison, a previous genome-scale enhancer screen was performed using 300M LNCaP cells (Liu, et al. Genome biology 18.1 (2017): 219) and 800M HeLa cells (Muerdter, et al. Nature Genome-scale promoter screening was performed using the Sigma-Aldrich methods 15.2 (2018): 141. For the sequencing, 100M K562 cells were used (van Arensbergen, et al. Nature biotechnology 35.2 (2017): 145). The GRAMc library was transfected into the cells. After injection, total RNA was extracted, reverse transcribed, and expression barcodes were PCR amplified. To avoid loss of reporter transcripts (Muerdter, et al. Nature methods 15.2 (2018): 141) or reporter transcripts (Tewhey, et al. Cell 165.6 (2016): 1519-1529) during secondary enrichment of mRNA, total RNA and GRAMc-specific oligomers were used for reverse transcription. Expression barcodes were amplified by PCR, and reporter expression levels were measured by ILLUMINA® sequencing. A schematic for processing RNA into sequencing libraries, together with associated quality control steps, is shown in Figure 9. Reporter expression was double-normalized to the relative copy number of inserts in the input GRAMc library and to background activity, which is the average activity of the middle 30% of ranked reporter expression (Nam, et al. PNAS USA 107.8 (2010): 3930-3935). The background activity determined is very similar to the leakage activity of known inactive fragments in sea urchin embryos (Nam, et al. PNAS USA 107.8 (2010): 3930-3935, Guay, et al. Developmental biology 422.2 (2017): 92-104).

[0148] Approximately 200M reads were obtained from each batch of expression barcodes. 78-79% of the barcodes matched barcodes with associated genomic regions. Approximately 450M barcode reads were obtained from the input plasmid to account for copy number variation. Read counts for multiple barcodes on the same insert were combined because 99% of inserts drove ≥2 barcodes. Approximately 7.5M inserts with ≥10 reads derived from the input plasmid were used for data analysis. A total of 50,993 inserts derived from 41,216 non-overlapping genomic regions displayed activity ≥5-fold greater than background (bg) activity (red dots, ≥5×bg) in two independent experiments (Figure 2A). Replicate GRAMc data showed a Pearson correlation coefficient (r) of 0.95, with a probability of 0.80 for a CRM from one batch to be considered a CRM from another batch (80% CRM reproducibility). When the cutoff was lowered to 3-fold above background (orange and red dots, ≧3×bg), the number of active regions increased to 150,011 (CRM reproducibility of 62%).

[0149] To verify the accuracy of GRAMc, 11 CRMs (≥5 × bg, red dots), 5 weakly active fragments (3–5 × bg, orange dots), and 4 inactive fragments (≤1 × bg, black dots) were randomly selected and their regulatory activity was tested individually in a single reporter assay (Figure 2B). The level of GFP transcript relative to transfected DNA copies was measured by QPCR. Reporter expression was further normalized to background activity (bg), the average level of the four inactive reporter constructs. The average level of the four independent assays is shown by the black bar for each individual insert. Of the 11 CRMs tested, 8 inserts were ≥5 × bg, whereas 2 and 1 inserts were 2.8 × bg and 1.9 × bg, respectively. This result is comparable to the 80% CRM reproducibility in GRAMc (Figure 2A). For the five slightly active inserts, one insert was 10 × bg, three inserts were within the expected range of 3–5 × bg, and one insert was 1.4 × bg. Overall, cis-regulatory activity measured with GRAMc was reproducible in independent assays (R2 = 0.83). These results indicate that GRAMc is a reliable and efficient tool for genome-scale discovery of CRMs.

[0150] Example 4 In this example, GRAMc-identified CRMs that possess expected CRM features are described. Because GRAMc is based on a standard configuration of reporter constructs, GRAMc-identified CRMs should possess known CRM features that have been identified in conventional reporter assays. First, CRMs should be located primarily near genes expressed in HepG2. When comparing the genomic locations of genes expressed in HepG2, CRMs, and the input library, the expressed genes and CRMs had similar patterns, whereas the input library was roughly evenly distributed (Figure 2C and Figures 10A-10F).

[0151] Second, CRMs are known to be enriched in the 5'-proximal region of genes (promoters); however, the majority are located outside the proximal region (distal enhancers) (26). When the percentage of CRMs was calculated for the number of inserts examined that were within a moving 2-kb window upstream or downstream of expressed genes, the 5'-proximal 2-kb region showed the highest enrichment (0.03) (Figure 2D). The 3'-proximal 2-kb region showed the second highest peak, although CRMs were slightly depleted in gene regions. Despite this regional variation, CRMs were consistently enriched around expressed genes within at least a 100-kb region in each direction, compared to the genome average of 0.0067. A similar pattern was observed near non-expressed genes, although the degree of enrichment was lower than near expressed genes. These results indicate that GRAMc can efficiently identify both proximal promoters and distal enhancers.

[0152] Third, CRMs are predicted to be associated with the binding of transcription factors and other proteins that positively affect CRM function. The relative enrichment (total shared base pairs relative to random expectation) of narrow peaks in CRMs compared to inactive fragments was calculated from 167 ENCODE ChIP-seq or DNase-seq data from HepG2 (Figure 2E). 153 data showed ≥2-fold enrichment in CRMs compared to inactive regions. These include general transcription factors (e.g., GTF2F1, TAF1, and TBP), transcriptional coactivators (P300), and histone modifying enzymes (e.g., H3K4me3 and H3K9ac). ChIP-seq peaks that were not enriched or even depleted in CRMs include transcription factors (TCF12 and BCLAF1), spliceosomal components (PLRG1 and SNRNP70), and histone methylases (H3K27me3, H3K36me3, and H3K9me3). Interestingly, despite the overall enrichment, only 32% of GRAMc-identified CRMs overlapped with 153 ENCODE data showing ≥2-fold enrichment of CRMs, and 58% of CRMs did not overlap with any of the ENCODE data used in this analysis. Although overlap can be increased by obtaining ChIP-seq data for more transcription factors, reporter assays may detect CRMs that are not active in the genome due to chromatin silencing or that may evade detection by ChIP-seq.

[0153] Example 5 In this example, motif enrichment is shown to explain the differential activity of ChromHMM-predicted enhancers. Previous studies have shown that chromatin mark-based CRM predictions are enriched for functionally validated CRMs, but the vast majority of predicted CRMs do not drive significant expression in reporter assays (Liu, et al. Genome biology 18.1 (2017): 219;Muerdter, et al. Nature methods 15.2 (2018): 141;van Arensbergen, et al. Nature biotechnology 35.2 (2017): 145). Consistent with these observations, motif enrichment is shown to explain the differential activity of ChromHMM-predicted enhancers in HepG2 (Ernst, et al. Nature methods 9.3 (2012): 215) that show ≥90% overlap with ChromHMM-predicted strong enhancers in HepG2 (Ernst, et al. Nature methods 9.3 (2012): 215). Assays of cis-regulatory activity of GRAMc test fragments using GRAMc showed that approximately 80% of predicted enhancers showed ≤2-fold background GRAMc activity (Figure 3A). If the predicted enhancers were true enhancers, enrichment for transcription factor binding site (TFBS) motifs would be expected. Here we focused on predicted strong enhancers because promoters are naturally enriched in motifs and predicted weak enhancers may increase ambiguity.

[0154] The enrichment of 601 HOCOMOCO_v10 HUMAN motifs within predicted enhancers (Kulakovskiy, et al. Nucleic Acids Research 44.D1 (2015): D116-D125), GRAMc-identified CRMs, and inactive fragments were compared using FIMO software (Cuellar-Partida, et al. Bioinformatics 28.1 (2011): 56-62; Bailey, et al. Nucleic Acids Research 37 (2009): W202-W208). Overall, GRAMc-identified CRMs showed stronger motif enrichment than predicted enhancers (Figure 3B). Predicted enhancers that were active or slightly active in GRAMc (Figure 3C-3D) displayed motif enrichment or depletion comparable to that of GRAMc-identified CRMs. In contrast, motif enrichment was gradually diluted in predicted enhancers that showed weaker reporter expression (Figures 3E-3G). Given the inability to drive significant reporter expression and the weak motif enrichment, it is likely that the majority of predicted enhancers are not true enhancers. However, this does not exclude the possibility that chromatin marks may indicate the vicinity of enhancers rather than their exact location, and that predicted enhancers may harbor other types of cis-regulatory activities that cannot be measured by reporter assays.

[0155] Activation of the interferon pathway leads to the erroneous identification of interferon-responsive enhancers upon DNA transfection (Muerdter, et al. Nature methods 15.2 (2018): 141), and such artifacts are not identified by GRAMc-identified CRMs and Chro However, consistent with our original finding that HepG2 cells do not activate this pathway, motifs for interferon-stimulated transcription factors, including IRF1-9 and hMX1, were not enriched in GRAMc-identified CRMs.

[0156] Example 6 In this example, enriched motifs in CRMs are shown to predict potentially new types of gene regulatory interactions. The pattern of reporter expression measured by mini-reporter constructs is a direct readout of the trans-regulatory environment of the host cell. Because CRM DNA sequences contain binding sites for transcription factors, computational motif analysis has been widely used to infer gene regulatory programs (e.g., Xie, et al. Nature 434.7031 (2005): 338;Mariani, et al. Cell systems 5.3 (2017): 187-201;Enuameh, et al. Genome research (2013): gr-151472;Markstein, et al. Development 131.10 (2004): 2387-2394;Halfon, et al. BMC genomics 12.1 (2011): 578). Based on 601 HOCOMOCO_v10 HUMAN motifs computationally predicted by FIMO in CRMs and inactive fragments (negative controls) (Kulakovskiy, et al. Nucleic acids research 44.D1 (2015): D116-D125), abundance (proportion of motif-positive CRMs or inactive fragments) and relative enrichment of motifs (relative abundance of motifs in CRMs compared to inactive fragments) were calculated (Figure 4A). The results show that 176 of the 601 motifs were enriched ≥2-fold in the CRM compared to the inactive fragment. The majority of enriched motifs (65%) were for expressed (FPKM ≥1) transcription factors, but interestingly the remainder were for transcription factors that were not expressed or very poorly expressed (FPKM <1) (3).

[0157] Enriched motifs of expressed transcription factors should predict positive regulators of CRMs identified in HepG2. To assay for regulators, we compared the results of motif analysis with ENCODE ChIP-seq data from HepG2 cells (3). If the predicted transcription factors based on motif enrichment were correct, the ChIP-seq peaks of the same transcription factors should also be enriched. A total of 58 transcription factors were common between the two datasets. Of the 58 factors, 31 motifs and 56 ChIP-seq peaks were enriched ≥2-fold in CRMs compared to inactive fragments (Figure 4B). Considering that all but one of the enriched motifs was also enriched in the ChIP-seq data, the prediction of positive regulators based on motif enrichment shows a very low false positive rate (<<0.1). The other approximately 50% of transcription factors showed motif enrichment <2-fold, but the ChIP-seq peaks were still highly enriched. Although more detailed analysis is required, in a conservative scenario, our motif-based predictions exhibit a false negative rate of approximately 0.5.

[0158] Motif enrichment of non-expressed transcription factors indicates that they regulate HepG2-CRM as either activators or repressors in other cell types or conditions (Figure 4C). Ectopic expression of transcription factor candidates in HepG2 was used to assay such regulators. Two transcription factor genes, pitx2 (homeobox gene) and ikzf1 (Ikaros homolog), were investigated. In mice, pitx2 is expressed in fetal liver and is required for fetal liver hematopoietic function, and blocking both pitx2 and hematopoietic function in fetal liver is essential for fetal liver to adult liver differentiation (Kieusseian, et al. Blood 107.2 (2006): 492-500). Similarly, ikzf1 is a key regulator of hematopoietic development (Davis. Therapeutic advances in hematology 2.6 (2011): 359-368) and is expressed in fetal liver (Roy, et al. PNAS USA (2012): 201211405), but its function in liver development is unknown. Plasmids capable of constitutively expressing pitx2 (CMV::pitx2) or ikzf1 (CMV::ikzf1) mRNA were co-transfected with a set of approximately 80,000 GRAMc reporter constructs randomly selected from the complete GRAMc library. As a control experiment, a plasmid capable of constitutively expressing GFP mRNA (CMV::gfp) was co-transfected with the same set of reporter constructs. All three experimental replicates were highly reproducible (Pearson's r ≥ 0.99) (Figure 14). Ectopic expression of pitx2 in HepG2 downregulated the majority of CRMs by ≥2-fold, and this downregulation was more pronounced for pitx2 motif-positive CRMs (two-sample t-test, P=4.4E-16) (Figure 4D). For ikzf1, only nine CRMs were downregulated by ≥2-fold, and six of the nine downregulated CRMs were positive for the IKZF1 motif (two-sample t-test, P=2.5E-4) (Figure 4E). Protein expression of both recombinant genes was confirmed by Western blot (Figure S11).These results indicate that pitx2 (and to a lesser extent ikzf1) maintains HepG2-CRM repression in fetal liver and that clearance of pitx2 is important for activation of HepG2-CRM and gene expression in adult liver. These results indicate that CRMs are useful not only for predicting host cell regulatory programs but also for predicting regulatory interactions between temporally and spatially separated cells.

[0159] Example 7 In this example, SINE / Alu elements are shown to be enriched in CRMs. Early models of eukaryotic gene regulation proposed that repetitive elements play an important role in controlling gene expression (McClintock. PNAS USA 36.6 (1950): 344-355; Britten, et al. Science 165.3891 (1969): 349-357). These predictions were subsequently supported by multiple examples of Alu and ERV elements contributing to gene regulation and development (Britten. PNAS USA 93.18 (1996): 9374-9377). Furthermore, genomic surveys of chromatin signatures show that SINE / Alu elements are enriched in putative CRMs (Su, et al. Cell reports 7.2 (2014): 376-385;Trizzino, et al. BMC genomics 19.1 (2018): 468). Hanser (Muerdter, et al. Nature methods 15.2 (2018): 141) or Promo (van Arensbergen, et al. Nature biotechnology 35.2 (2017): 145) Our genome-scale reporter assay detected enrichment of LTR / ERV1 and LTR / ERVL-MaLR, but not SINE / Alu, in CRMs. To assay such enrichment of GRAMc-identified CRMs, we compared our data with annotated repetitive elements in the human genome (Smit, et al. "RepeatMasker Open-4.0" (2015)). Three families of repetitive elements, satellite / telomere, SINE / Alu, and LTR / ERV1, were detected to be enriched ≥2-fold in CRMs (G5 set in Figure 5A), whereas LTR / ERVL-MaLR was not enriched in CRMs. The three elements were also enriched, to a lesser extent, in the slightly active G3L4 and G4L5 sets. Interestingly, alpha satellites were depleted in CRMs by approximately 8-fold, indicating a repressive function or incompatibility with other CRMs in HepG2. However, no depletion of retroposon / SVA elements predicted to be transcriptional repressors in the liver was detected (Trizzino. Genome research 27.10 (2017): 1623-1633).

[0160] Using GRAMc-identified CRMs, we assayed the evolution of Alu elements toward enhancers as a function of time (Su, et al. Cell reports 7.2 (2014): 376-385). The enrichment of Alu elements in CRMs should positively correlate with age. However, when we investigated the three major subfamilies of Alu (Figure 5B), the youngest subfamily (AluY) and intermediate subfamily (AluS) showed ≥3-fold enrichment in CRMs, whereas the oldest subfamily (AluJ) showed only moderate enrichment (1.3-fold). This discrepancy can be explained by the different cell types, since the original study was based on chromatin annotation in HeLa cells. Thus, 19 subfamilies of Alu elements tested using luciferase assays in HeLa cells were summarized (Su, et al. Cell reports 7.2 (2014): 376-385). Consistent with these results, 8 / 10 AluY or AluS elements were active, but only 4 / 9 AluJ elements were active, and our results are therefore consistent with an alternative model in which Alu elements lose regulatory activity with age.

[0161] These results demonstrate that GRAMc data can be useful for testing multiple evolutionary genomic hypotheses and that GRAMc data can lead to different conclusions compared to data generated by earlier genome-scale reporter assays or chromatin annotation. Furthermore, the discrepancies observed between GRAMc and earlier reporter assays can be attributed in large part to the different cell types used. The enrichment of the entire list of repetitive elements is shown in Table 2.

[0162] [Table 2-1] [Table 2-2] [Table 2-3] NOTE: Enrichment scores are on a log2 scale.

[0163] It should be recognized that there are numerous possible embodiments to which the principles of the present disclosure may be applied, and that the described embodiments are merely examples and should not be construed as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. Accordingly, the inventors claim as their invention all that comes within the scope and spirit of such claims. The present invention further provides the following aspects. (Item 1) 1. A method for constructing a nucleic acid molecule reporter library, comprising: isolating a plurality of nucleic acid molecules of a selected size range; ligating the plurality of isolated nucleic acid molecules of the selected size range to at least one linear adaptor sequence using a ligase, wherein the linear adaptor sequence comprises at least two consecutive ribonucleotides flanked by at least one deoxyribonucleotide at a 3' end and at least one deoxyribonucleotide at a 5' end, thereby producing a plurality of circular nucleic acid molecules comprising inserts and adaptors; contacting the plurality of circular nucleic acid molecules, including inserts and adaptors, with an exonuclease under conditions sufficient to remove linear nucleic acid molecules from the plurality of circular nucleic acid molecules; contacting the plurality of circular nucleic acid molecules comprising an insert and an adaptor with an endoribonuclease under conditions sufficient to produce a plurality of linear nucleic acid molecules flanked by the insert, each of the linear nucleic acid molecules comprising at least one deoxyribonucleotide at the 3' end and at least one deoxyribonucleotide at the 5' end; and fusing each of said plurality of linear nucleic acid molecules with at least one reporter nucleic acid to generate a plurality of reporter constructs, thereby generating said nucleic acid molecule reporter library. A method comprising: (Item 2) 2. The method of claim 1, wherein the ligase comprises a DNA ligase. (Item 3) 3. The method of claim 1 or 2, wherein the ligase comprises T4 DNA ligase. (Item 4) 4. The method of any one of items 1 to 3, wherein the plurality of nucleic acid molecules of a selected size range are about 100 to 3000 base pairs in length. (Item 5) 5. The method of claim 4, wherein the plurality of nucleic acid molecules of a selected size range are about 750 to 850 base pairs in length. (Item 6) 7. The method of any one of claims 1 to 5, wherein the plurality of isolated nucleic acid molecules of a selected size range are selected using gel electrophoresis or bead-based size selection. 7. The method of any one of items 1 to 6, wherein the plurality of nucleic acid molecules of a selected size range comprises genomic DNA or synthetic DNA. (Item 8) 8. The method of claim 7, wherein the genomic DNA is derived from a mammalian cell, a plant cell, a bacterial cell, a fungal cell, or an archaeal cell. (Item 9) 9. The method of claim 8, wherein the genomic DNA is derived from a mammalian cell. (Item 10) 9. The method of claim 8, wherein the genomic DNA derived from a mammalian cell is derived from at least one of a cardiomyocyte, a neuron, a hepatocyte, an endothelial cell, an embryonic stem cell, a skin cell, a cancer cell, a kidney cell, an immune cell, a bone cell, an organoid-derived cell, or an induced stem cell. (Item 11) 9. The method according to item 8, wherein the genomic DNA is derived from a plant cell. (Item 12) 9. The method of claim 8, wherein the genomic DNA is derived from a bacterial cell. (Item 13) 9. The method of claim 8, wherein the genomic DNA is derived from a fungal cell. (Item 14) 9. The method of claim 8, wherein the genomic DNA is derived from an archaeal cell. (Item 15) 15. The method of any one of items 1 to 14, wherein contacting the plurality of circular nucleic acid molecules comprising inserts and adapters with the endoribonuclease comprises contacting the plurality of circular nucleic acid molecules comprising inserts and adapters with an endoribonuclease specific for a ribonucleotide in a DNA duplex. (Item 16) 16. The method of claim 15, wherein the endoribonuclease is RNase HII or uracil-DNA glycosylase. (Item 17) 17. The method of any one of items 1 to 16, further comprising determining a genome coverage of the plurality of linear nucleic acid molecules comprising at least one deoxyribonucleotide at the 3' end and at least one deoxyribonucleotide at the 5' end flanked by the insert. (Item 18) The step of determining genome coverage comprises: selecting at least one genomic region of interest; amplifying said plurality of linear nucleic acid molecules comprising at least one deoxyribonucleotide at said 3' end and at least one deoxyribonucleotide at said 5' end flanked by said inserts; determining whether the selected genomic region is present in the plurality of linear nucleic acid molecules. Item 18. The method according to item 17, comprising: (Item 19) 19. The method of any one of items 1 to 18, wherein the at least one reporter nucleic acid comprises a nucleic acid encoding a fluorescent protein and / or comprising a barcode nucleic acid. (Item 20) 20. The method of any one of items 1 to 19, further comprising fusing the plurality of linear nucleic acid molecules comprising at least one deoxyribonucleotide at the 3' end and at least one deoxyribonucleotide at the 5' end flanked by the inserts with a linear vector nucleic acid, thereby producing a plurality of linear vectors. (Item 21) 21. The method of claim 20, wherein the linear vector nucleic acid comprises a basal promoter. (Item 22) the at least one reporter nucleic acid comprises a nucleic acid encoding a fluorescent protein, and fusing the plurality of linear nucleic acid molecules comprising at least one deoxyribonucleotide at the 3' end and at least one deoxyribonucleotide at the 5' end flanked by the inserts with at least one reporter nucleic acid comprises fusing the plurality of linear vectors with a fluorescent reporter nucleic acid, thereby producing a plurality of fluorescent reporter constructs; or the at least one reporter nucleic acid comprises a barcode nucleic acid, and fusing the plurality of linear nucleic acid molecules comprising at least one deoxyribonucleotide at the 3' end and at least one deoxyribonucleotide at the 5' end flanked by the insert with at least one reporter nucleic acid comprises fusing a plurality of reporter linear vectors with the barcode nucleic acid, thereby producing a plurality of barcode reporter constructs; or The at least one reporter nucleic acid comprises a barcode nucleic acid and a nucleic acid encoding a fluorescent protein, and fusing the plurality of linear vectors with the at least one reporter nucleic acid fuses the plurality of reporter constructs with a barcode nucleic acid and a nucleic acid encoding a fluorescent protein, thereby forming a plurality of fluorescent and barcode reporter constructs. 22. The method of claim 20 or 21, comprising producing (Item 23) contacting each of the plurality of linear vectors with a primer nucleic acid comprising a barcode reporter construct; performing a polymerase chain reaction (PCR) thereby producing a plurality of amplified vectors comprising said barcode reporter constructs; ligating the amplified vector containing the barcode reporter construct, thereby producing a plurality of circular vectors containing the barcode reporter construct; and contacting the plurality of circular vectors comprising the barcode reporter constructs with an exonuclease under conditions sufficient to remove linear nucleic acid molecules from the plurality of circular vectors comprising the barcode reporter constructs. 23. The method according to any one of items 20 to 22, further comprising: (Item 24) 1. A method for constructing a nucleic acid molecule reporter library, comprising: (i) isolating a plurality of nucleic acid molecules of a selected size range; ligating the plurality of isolated nucleic acid molecules of the selected size range to at least one linear adaptor sequence using a ligase, wherein the linear adaptor sequence comprises at least two consecutive ribonucleotides flanked by at least one deoxyribonucleotide at a 3' end and at least one deoxyribonucleotide at a 5' end, thereby producing a plurality of circular nucleic acid molecules comprising inserts and adaptors; (ii) contacting the plurality of circular nucleic acid molecules, including inserts and adaptors, with an exonuclease under conditions sufficient to remove linear nucleic acid molecules from the plurality of circular nucleic acid molecules; (iii) contacting the plurality of circular nucleic acid molecules comprising inserts and adaptors with an endoribonuclease under conditions sufficient to produce a plurality of linear nucleic acid molecules flanked by the inserts, each of the linear nucleic acid molecules comprising at least one deoxyribonucleotide at the 3' end and at least one deoxyribonucleotide at the 5' end; (iv) determining genome coverage of the plurality of linear nucleic acid molecules comprising at least one deoxyribonucleotide at the 3' end and at least one deoxyribonucleotide at the 5' end flanked by the insert, (a) selecting at least one genomic region of interest; (b) amplifying the plurality of linear nucleic acid molecules comprising at least one deoxyribonucleotide at the 3' end and at least one deoxyribonucleotide at the 5' end flanked by the insert; and (c) determining whether the selected genomic region is present in the plurality of linear nucleic acid molecules. and (v) fusing the plurality of linear nucleic acid molecules comprising at least one deoxyribonucleotide at the 3' end and at least one deoxyribonucleotide at the 5' end flanked by the inserts with at least one reporter nucleic acid to produce a plurality of reporter constructs, (a) fusing the plurality of linear nucleic acid molecules, comprising at least one deoxyribonucleotide at the 3' end and at least one deoxyribonucleotide at the 5' end flanked by the insert, with a linear vector nucleic acid, thereby producing a plurality of linear vectors; (b) contacting each of the plurality of linear vectors with a primer that comprises a barcode nucleic acid; and (c) performing a polymerase chain reaction (PCR) to isolate the insert and barcode; generating a plurality of circular vectors comprising a barcode reporter construct comprising at least one deoxyribonucleotide at the 3' end and at least one deoxyribonucleotide at the 5' end flanked by a deoxyribonucleotide; and (d) contacting the plurality of circular vectors comprising the barcode reporter constructs with an exonuclease under conditions sufficient to remove linear nucleic acid molecules from the plurality of circular vectors comprising the barcode reporter constructs. Including steps A method comprising: (Item 25) 25. The method of any one of items 1 to 24, wherein the exonuclease is exonuclease I, exonuclease III, and / or lambda exonuclease. (Item 26) 26. The method of any one of items 1 to 25, wherein the at least one linear adapter sequence comprises SEQ ID NO:1 and / or SEQ ID NO:2. (Item 27) 27. The method according to any one of items 1 to 26, wherein the linear adapter sequence comprises a double-stranded duplex of SEQ ID NO: 1 and / or SEQ ID NO: 2. (Item 28) 28. A nucleic acid molecule reporter library produced using the method of any of items 1 to 27. (Item 29) 1. A method for detecting a functional nucleic acid regulatory element, comprising: Transfecting at least one cell of interest with the library of item 28; and Measuring at least one reporter A method comprising: (Item 30) 30. The method of claim 29, further comprising identifying and / or quantifying the at least one reporter. (Item 31) 31. The method of any one of items 29 or 30, further comprising isolating RNA from the cells of interest to produce isolated RNA. (Item 32) The step of measuring the reporter comprises: reverse transcribing the isolated RNA to produce cDNA; and Detecting the cDNA 32. The method according to any one of items 29 to 31, comprising: (Item 33) 33. The method of claim 32, wherein the step of reverse transcribing the isolated RNA comprises using recombinant Moloney murine leukemia virus (rMoMuLV) reverse transcriptase or avian myeloblastosis virus (AMV) reverse transcriptase. (Item 34) The method of claim 32 or 33, further comprising using an RNA-dependent and a DNA-dependent DNA polymerase. (Item 35) 35. The method of any one of items 29 to 34, wherein the at least one reporter is at least one unique barcode nucleic acid. (Item 36) The step of detecting the cDNA comprises: amplifying the cDNA; and Identifying the at least one unique nucleic acid barcode. 36. The method according to claim 35, comprising: (Item 37) The step of amplifying the cDNA comprises: selecting a primer specific to a nucleotide comprising at least one unique nucleic acid barcode; contacting the primer with the cDNA; and performing PCR using said primers and said cDNA to produce amplified DNA. 37. The method according to claim 36, comprising: (Item 38) 38. The method of claim 37, wherein identifying the at least one unique nucleic acid barcode comprises sequencing the amplified DNA. (Item 39) 39. The method of any one of items 35 to 38, further comprising a step of quantifying the at least one unique nucleic acid barcode. (Item 40) 40. The method of any one of items 29 to 39, wherein the at least one cell is a mammalian cell, a plant cell, a fungal cell, a bacterial cell, or an archaeal cell. (Item 41) 41. The method of claim 40, wherein the cell is a mammalian cell. (Item 42) 42. The method of claim 41, wherein the mammalian cell is at least one of a cardiomyocyte, a neuron, a hepatocyte, an endothelial cell, an embryonic stem cell, a skin cell, a cancer cell, a kidney cell, an immune cell, a bone cell, an organoid-derived cell, or an induced stem cell. (Item 43) 41. The method of claim 40, wherein the cell is a plant cell. (Item 44) 41. The method of claim 40, wherein the cell is a bacterial cell. (Item 45) 41. The method of claim 40, wherein the cell is a fungal cell. (Item 46) 41. The method of claim 40, wherein the cell is an archaeal cell. (Item 47) at least two subjects, including at least one subject having a disease or condition and at least one subject not having a disease or condition; or At least one subject from which a plurality of cells are collected under different conditions 47. The method of any one of items 29 to 46, further comprising the step of collecting the at least one cell of interest collected from. (Item 48) 48. The method of any one of items 29 to 47, which is high throughput. (Item 49) 49. The method of any one of items 1 to 48, wherein said plurality of nucleic acid molecules comprises at least 80% of a selected genome of interest. (Item 50) 50. The method of any one of items 1 to 49, wherein the plurality of nucleic acid molecules comprises at least 80% of the cis-regulatory elements in a selected genome of interest. (Item 51) 29. A nucleic acid comprising at least one reporter nucleic acid according to any one of items 1 to 28. A kit for constructing molecular reporter libraries. (Item 52) 52. The kit of item 51, wherein the linear adapter sequence of the reporter nucleic acid comprises SEQ ID NO:1 and / or SEQ ID NO:2. (Item 53) 53. The kit of item 51 or item 52, further comprising at least one ligase, exonuclease, endoribonuclease, and / or polymerase. (Item 54) 29. A kit for high-throughput identification and / or quantification of functional nucleic acid regulatory elements, comprising the library of item 28, said library covering at least 80% of the genome of interest. (Item 55) 55. The kit of item 54, further comprising at least one reverse transcriptase. (Item 56) 56. The kit of item 54 or item 55, further comprising PCR primers and a high-fidelity DNA polymerase.

Claims

[Claim 1] The invention described in the specification.

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