Systems, devices and methods for randomly fragmenting high-molecular weight DNA

Abstract

Embodiments of the present disclosure present systems, devices and methods for fragmenting high-molecular weight (HMW) genomic DNA (HMW DNA). In some embodiments, a HMW DNA sample is provided, then, one or more reagents are added to the HMW DNA sample to produce a HMW DNA sample mixture. Thereafter, a Cas9 preparation is added to the HMW DNA sample mixture establishing a HMW DNA – Cas9 mixture where it is then incubated, the Cas9 enzyme is inactivated, and the fragmented DNA purified.

Description

Via Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025 IN THE UNITED STATES PATENT AND TRADEMARK OFFICE SYSTEMS, DEVICES AND METHODS FOR RANDOMLY FRAGMENTING HIGH-MOLECULAR WEIGHT DNA RELATED APPLICATIONS

[0001] This disclosure claims benefit of and priority to U.S. provisional patent application no. 63 / 703,865, filed October 4, 2024, and entitled “SYSTEMS AND METHODS FOR RANDOMLY FRAGMENTING HIGH-MOLECULAR WEIGHT DNA,” the entire disclosure of which is incorporated herein by reference. BACKGROUND

[0002] Many workflows for genomic DNA sequencing and genomic mapping involve preparation of high-molecular-weight (HMW) genomic DNA followed by fragmentation to generate samples with a size distribution that matches the capabilities of the analysis method (i.e., short-read sequencing, long-read sequencing, optical or electronic genome mapping). Popular fragmentation methods include shearing by passage of the sample through a narrow orifice, sonication, and enzymatic treatment with nucleases. Physical fragmentation methods typically require specialized equipment such as the Diagenode Megaruptor, Covaris Sonicators, or automated pipetting devices (for pipette tip shearing).

[0003] Enzymatic methods are attractive because they do not require specialized equipment. However, they can be problematic if the fragmentation enzymes have high sequence specificity, or if they produce samples with single-strand nicks. However, fragmentation enzymes with high sequence specificity can produce DNA products with poor sequence representation in genomic regions resulting in too many or too few enzymatic cleavage sites. To avoid sequence specificity, some commercial “random” fragmentation products use a cocktail of enzymes which include at least one relatively non-specific nickase, and at least one “resolvase,” definedVia Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025 here as an enzyme that cleaves the intact DNA strand opposite from the position of a single- strand nick. Since the cleavage of each strand is not performed in a concerted fashion, such cocktails can produce DNA products with substantial single-stranded nicks, especially if the cocktail is diluted in an effort to produce large products. Nicked DNA inputs will compromise the read length and yield in long-read sequencing experiments. Moreover, randomly-nicked DNA will contain many noisy labeling sites in a mapping workflow that depends on labeling of sequence-specific nicks, such as that currently used by the Nabsys OhmX electronic mapping system (Oliver et al., 2017; Nabsys website accessed July2024). SUMMARY

[0004] Inventions and embodiments thereof according to the present disclosure address issues of the state of the art (including, in some embodiments, those set out above).

[0005] In some embodiments, Cas9 complexed with guide RNAs are used to target common dinucleotide repeats that are abundant and evenly dispersed throughout mammalian genomes, such as, for example, 5’-CA(10)NGG-3’ and 5’-AT(10)NGG-3’. uHMW (i.e.., ultra-high- molecular-weight, generally considered greater than 100kb) human DNA samples fragmented with such Cas9 formulations produce long (i.e., generally greater than 10kb) Oxford Nanopore (“Nanopore”) reads with even coverage over the non-centromeric regions of the genome.

[0006] In some embodiments, a method for fragmenting high-molecular weight (HMW) genomic DNA (HMW DNA) is provided and includes providing a HMW DNA sample (optional), adding one or more reagents to a / the HMW DNA sample to produce a HMW DNA sample mixture, adding an RNA- guided DNA endonuclease to the HMW DNA sample mixture establishing a first mixture, incubating the first mixture to establish a fragmented DNA sample, inactivating the RNA-guided DNA endonuclease, and purifying the fragmented DNA sample.

[0007] Such embodiments (as well as other disclosed embodiments) may include at least one of the following additional features, functionality, structure, steps and clarifications (and in some embodiments, a plurality of, and in some embodiments, a majority of, and in some embodiments, substantially all of, and in some embodiments all of): - the one or more reagents are configured to provide a buffer condition suitable for digestion of the HMW DNA sample by the RNA-guided DNA nuclease; - the RNA-guided endonuclease comprises a Cas9 enzyme assembled with one or more guideVia Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025 RNAs molecules (gRNAs); - a / the Cas9 enzyme is configured to target Cas9 cleavage in the HMW DNA sample; - a / the Cas9 cleavage is configured to cleave DNA at a plurality of repeated genomic sequences within the DNA; - incubating the first mixture is performed under conditions and time for producing a desired extent cleavage of the HMW DNA; - a / the conditions (e.g., for incubation) comprise a temperature; o the temperature is selected from the group consisting of: between 20 and 40 deg. C, between 20 and 35 deg. C, between 20 and 30 deg. C, between 20 and 25 deg. C, between 25 and 40 deg. C, between 30 and 40 deg. C, between 35 and 40 deg. C, and ranges therebetween; - a / the time is selected from the group consisting of: between 15 minutes and 2 hours, between 20 minutes and 2 hours, between 30 minutes and 2 hours, between 45 minutes and 2 hours, between 1 and 2 hours, between 1.25 hours and 2 hours, between 1.5 hours and 2 hours, between 1.75 hours and 2 hours, between 15 minutes and 1.75 hours, between 15 minutes and 1.5 hours, between 15 minutes and 1.25 hours, between 15 minutes and 1 hour, between 15 minutes and 45 minutes, between 15 minutes and 30 minutes, between 15 minutes and 20 minutes, between 20 minutes and 1.75 hours, between 20 minutes and 1.5 hours, between 20 minutes and 1.25 hours, between 20 minutes and 1 hour, between 20 minutes and 45 minutes, between 20 minutes and 30 minutes, between 25 minutes and 2 hours, between 25 minutes and 1.75 hours, between 25 minutes and 1.5 hours, between 25 minutes and 1.25 hours, between 25 minutes and 1 hour, between 25 minutes and 45 minutes, between 25 minutes and 30 minutes, between 30 minutes and 1.75 hours, between 30 minutes and 1.5 hours, between 30 minutes and 1.25 hours, between 30 minutes and 1 hour, between 30 minutes and 45 minutes, between 45 minutes and 2 hours, between 45 minutes and 1.75 hours, between 45 minutes and 1.5 hours, between 45 minutes and 1.25 hours, between 45 minutes and 1 hour, between 1 hour and 2 hours, between 1 hour and 1.75 hours, between 1 hour and 1.5 hours, between 1 hour and 1.25 hours, between 1.25 hours and 2 hours, between 1.25 hours and 1.75 hours, between 1.25 hours and 1.5 hours, between 1.5 hours and 2 hours, between 1.5 hours and 1.75 hours, between 1.75 hours and 2 hours, and ranges therebetween; - a / the RNA-guided DNA endonuclease is configured to recognize one or more classes of repeated genomic sequences; andVia Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025 - a / the RNA-guided endonucleases are selected from the group consisting of: Streptococcus pyogenes Cas9 (SpyCas9), Acidaminococcus sp. Casl2a (AsCas 12a), or derivatives thereof.

[0008] In some embodiments, a method for fragmenting high-molecular weight (HMW) genomic DNA (HMW DNA) is provided and includes providing a HMW DNA sample (optional), adding one or more reagents to a / the HMW DNA sample, the one or more reagents configured to provide a buffer condition suitable for digestion of the HMW DNA sample by a Cas9 nuclease, adding a Cas9 preparation to the HMW DNA sample establishing a HMW DNA – Cas9 mixture, where the Cas9 preparation comprises a Cas9 enzyme assembled with one or more guide RNA molecules, the Cas9 preparation configured to cleave at a plurality of repeated genomic sequences that are dispersed throughout the genome to produce a fragmented genomic DNA sample, incubating the HMW DNA - Cas9 mixture under conditions and time for producing a desired extent of Cas9 cleavage, inactivating the Cas9 enzyme, and purifying the fragmented DNA.

[0009] In some embodiments, a kit for enzymatic fragmentation of high molecular weight (HMW) genomic DNA is provided which includes one or more RNA-guided DNA endonucleases, and one or more gRNA molecules for recognizing one or more classes of repeated genomic sequences.

[0010] In such embodiments, the kit of claim 10, further comprising at least one of one or more reagents, an apparatus, and a device for extraction of HMW DNA, and / or at least one of the RNA- guided endonucleases comprises Streptococcus pyogenes Cas9 (SpyCas9) or Acidaminococcus sp. Casl2a (AsCasl2a), or derivatives thereof.

[0011] In some embodiments, a system for performing the any of the methods embodiments according to the present disclosure.

[0012] These and other embodiments, elements, examples, objects and advantages thereof are further described with reference to the following additional details and figures associated with the subject disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 is a histogram graph of fragment sizes predicted to be produced by SpyCas9 cleavage with dinucleotide repeat gRNAs, T2T-CHM13 genome, according to some embodiments of the present disclosure.

[0014] FIGs. 2A-B show photographs of a cassette (FIG. 2A) and instrument (FIG. 2B) for performing methods according to some embodiments of the present disclosure.

[0015] FIGs. 3A1-A6, and 3B1-B8 are schematic diagrams of a DNA extraction andVia Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025 enzymatic fragmentation workflow (using, e.g., cassette / instrument of FIGs.2A-B), according to some embodiments of the present disclosure.

[0016] FIG.4 shows a graph of DNA output from an extraction device / system (see FIGs.2A- B) for extraction using fragmentation with SpyCas9 complexes according to some embodiments of the present disclosure, which targe certain repeat sequences.

[0017] FIG. 5 illustrates an image of a pulsed field gel analysis of the DNA products of resulting from methods according to some embodiments of the disclosure. DETAILED DESCRIPTION

[0018] FIG. 1 is a histogram graph of fragment sizes predicted to be produced by SpyCas9 cleavage with dinucleotide repeat gRNAs, T2T-CHM13 genome. Specifically, CHM13 genome reference sequence produced by the T2T consortium (CHM13v2.0 / hs1 – Assembly accession ID GCA009914755.4) was queried for gRNA target recognition sequences (CA)10NGG, (AC)10NGG, (AT)10NGG, and (TA)10NGG, using the “findMotif” program (available from the Utilities section of the UC Santa Cruz Genome Browser; (see https: / / hgdownload.soe.ucsc.edu / admin / exe / linux.x86_64; accessed July 2024). The histogram shows the distribution of fragment lengths between gRNA recognition sites over the entire CHM13 genome sequence.

[0019] FIGs. 2A-B show photographs of a cassette 200 (FIG. 2A) for use in a system 250 (FIG.2B) (e.g., Sage Science SageHLS cassette and system / instrument. One of skill in the art will appreciate that any quantity of structure listed is merely exemplary, i.e., one or more. The cassette(s) 200 can include two electrophoresis compartments, each containing (1) an agarose gel column (channel 202), (2) a set of six membrane-delimited elution modules 204 in contact with the right side of the gel column, (3) an electrophoresis buffer compartment around the perimeter of the gel columns and the elution modules, (4) a pair electrode ports at the ends of the gel column, and (5) a pair of long electrode ports at the left and right boundaries of the electrophoresis compartments. The gel column has two input ports at the upper end. The smaller input well 206 (“Sample well”) is used to load a suspension of nuclei for electrophoretic extraction, and the larger input well 208 (“Reagent well”) is used to load an SDS-based lysis reagent. Extraction is a carried out by electrophoresing the SDS lysis reagent down through the sample well, where the nuclei are lysed and the HMW DNA is driven into the agarose wall ofVia Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025 the sample well, where it becomes immobilized due to its large size (typically >>5Mb). After electrophoretic extraction and enzymatic digestion (as described in Example 2), size selection electrophoresis of the digested DNA is carried out, thereby moving the products down the agarose column. After size selection electrophoresis, the products are electroeluted into the elution modules at the right side of the gel column, using long wire electrodes that are inserted into the ports on the left and right boundaries of the electrophoresis compartment. After electroelution, the size-selected DNA products (in electrophoresis buffer) are collected from the elution modules using pipetting devices. See also, e.g., U.S. patent nos. 11,867,661, 11,542,495 and 10,738,298, together “the Patents”, the entire disclosures of which is incorporated herein by reference.

[0020] The instrument 250 includes compartments split between the lid and the base (ref.252 and 254 being arranged in the lid and including electrodes integrated therein (not shown) for applying electrical currents to the gel / wells of the cassettes 200.

[0021] FIGs. 3A-B are schematic diagrams of exemplary DNA extraction and enzymatic fragmentation workflows (e.g., SageHLS DNA extraction), in which more details are provided in Example 2 below. In FIGs.3A1-A6 are high level flow / structure diagrams where each image represents a single lane of an HLS cassette at various stages of the workflow, for obtaining fragmented HMW DNA and cleaving it using SpyCas9 cleavage at genomic repeats (see also, Id., the above-identified incorporated by reference disclosures, including U.S. patent no. 11,867,662, FIGS. 7A-10B, and written description, including col. 5, lines 23-38 for other general characteristics). As shown in FIG. 3A1, a sample can be loaded into the sample well 302a, and a regent(s) added to reagent well 304a. The reagent is driven by electrophoresis into / through the gel 306a and into / through the sample well (see FIG. 3A2). In FIG. 3A3, SpyCas9 cleavage is added to the sample well 302a, and at FIG. 3A4, SDS purification as added to the reagent well 304a. In FIG. 3A5, final purification and size selection via electrophoresis is performed, and the cleaved fragments are then driven laterally via electrophoresis into elution modules 308a.

[0022] FIGs. 3B1-B8 illustrate a cross-sectional / side views of agarose column within a gel cassette (see FIG. 2A). In FIG. 3B1, a regent well 304b (SDS) having at least one reagent is arranged adjacent a cell sample well 302b containing a cell sample. Through electrophoresis, the reagent(s) in 304b are driven into and through well 302b, as shown in FIG.3B2 and 3B3, resulting in lysis without shear force. As shown in FIG.3B4, 3B5, since DNA 306b (i.e., HMWVia Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025 DNA) too large to undergo electrophoresis becomes immobilized in the wall of the sample well, and SDS-coated contaminates 308b are removed.

[0023] Accordingly, as shown in FIG. 3B5-3B6, in the empty sample well (where the HMW DNA is immobilized in the wall thereof), a Cas9 preparation is added to the sample well 302b (which in some embodiments includes a Cas9 enzyme assembled with one or more guide RNA molecules). As shown in FIG.3B7, limited DNA digestion is allowed to occur. Accordingly, thereafter, in FIG. 3B8, electrophoretic purification, size selection and elution are performed. As shown, the fragmented DNA 312b is within the gel, and the cleavase / reagents 310b in the gel as well.

[0024] Figure 4 shows a graph of DNA output from an extraction device / system (e.g., SageHLS) for extraction using fragmentation with SpyCas9 complexes targeting dinucleotide repeat sequences: (CA)10NGG, (AC)10NGG, (AT)10NGG, and (TA)10NGG. Details of the experiment are outlined in Example 2.

[0025] Figure 5 illustrates an image of a pulsed field gel (CHEFMapper, Bio-Rad, Richmond, CA, USA) analysis of the DNA products of the experiment described in Example 2 and Figure 4. “w” =Size markers used were H. wingei chromosome ladder (Bio-Rad). “L” = Phage lambda concatemer ladder (Bio-Rad), “1k” = 1kb Extend ladder (New England Biolabs). Fragmentation via RNA-guided endonuclease cleavage at genomic repeat sequences

[0026] The major classes of human repeat sequences were catalogued in the original draft of the human genome (Lander et al., 2001). A large portion of the human genome is composed of transposon-derived sequences, including SINES, LINES, LTR retrotransposons, and DNA transoposons. Other types of highly repeated sequences in the human genome are microsatellite sequences, also known as simple-sequence repeats, such as mono-, di-, and tri-nucleotide repeating sequences. These repeat types are also observed in virtually all other organisms, procaryotes and eucaryotes, but the abundance of specific repeat types in any given species can vary widely. All of these types of repeats can be used in the disclosed methods (according to some embodiments), the subject disclosure focuses on use of dinucleotide repeats of the human genome as non-limiting examples to describe and exemplify such embodiments.

[0027] Accordingly, in some embodiments, high specificity of RNA-guided endonucleases, such as SpyCas9, are used to cleave at highly repeated sequences in genomic DNA under study. Thus, such embodiments can be adapted to generate genomic DNA product with different size distributions in several ways.Via Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025

[0028] In some embodiments, by targeting repeat elements with a high copy number in the genome, fragmented DNA products with a relatively small size can be produced. Examples of highly repeated sequence elements in the human genome include SINE and LINE elements which are present at several hundred copies per megabase (Lander et al., 2001). Similarly, targeting repeat elements with a lower copy number in the genome can generate a population of larger DNA fragments. Examples of repeated sequence elements in the human genome with a lower copy number include the simple sequence dinucleotide repeats, which are present at about 20 copies per megabase (Lander et al., 2001), as discussed in the next section. In either case, the size of the digestion product can be fine-tuned by adjusting the endonuclease to DNA ratio.

[0029] In general, in some embodiments, complete digestion of specific SpyCas9 target sites can be achieved using a slight molar excess of SpyCas9 to the molar concentration of specific target sites in the input DNA (in some embodiments, approximately 5-10x excess in SpyCas9 concentration over the molar concentration of input DNA target sites, and ranges therebetween). In situations where partial cleavage is desired, an optimum SpyCas9 / input DNA ratio can be determined in test digestions using decreasing enzyme / DNA ratios, since the efficiency of cleavage varies for different gRNA recognition sequences. Fine-tuning product size by limiting amounts of endonuclease is a convenient method when using SpyCas9, since that enzyme does not turn over after cleavage, as it remains tightly bound to the cleaved DNA substrate (Yourik et al., 2019; Jinek et al., 2012).

[0030] In general, in some embodiments, the use of RNA-guided endonucleases that recognize genomic repeat elements to fragment HMW DNA into a size distribution suitable for a particular type of genomic analysis is provided. The desired product size can vary according to such an analysis method. For example, for genomic DNA sequencing using the PacBio Revio platform, a desired size range for genomic sequencing libraries is from approximately 10kb to 25kb, with the peak of DNA mass at approximately 20kb. In contrast, some sequencing applications using the Oxford Nanopore sequencing platforms benefit from using DNA greater than 10kb in size, while other Nanopore methods benefit from using DNA samples greater than 100kb in size. Long range optical or electronic mapping methods such as those marketed by Bionano Genomics and Nabsys benefit from DNA samples with a minimum size of 50kb with the bulk of the fragments greater than 100kb.

[0031] To generate DNA products with the above-noted size ranges, the input HMW for theVia Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025 fragmentation process should be several-fold larger than the desired product size. For example, to generate PacBio libraries with a median 20kb size, the input HMW DNA for fragmentation using the inventive method should ideally be greater than 60kb in size. To generate DNA product for the long-range mapping methods of Bionano Genomics and Nabsys, the input HMW DNA should ideally be greater than 200-300kb in size.

[0032] The catalog of human repeats in the original human genome draft show that the most abundant dinucleotide repeats (with lengths >50bp) were (CA)n and (AT)n, at an abundance of 27.7 and 19.4 copies per 1Mb of genomic sequence, respectively. A fraction of these sites will be bound by a short sequence, the PAM sequence, which can be recognized by an RNA-guided nuclease. For the SpyCas9 enzyme, with a PAM sequence of 5’NGG3’, based on random sequence distribution, it is expected that 6.25% of these repeats would be bound by an NGG PAM (P = 1 x 0.25 x0.25 = 0.0625). On this basis, it can be predicted that gRNAs targeting (CA)n or (AT)n repeats should be present at a frequency of approximately 2.94 copies per 1Mb (see FIG. 1). The predicted average spacing between SpyCas9 cleavage sites using such gRNAs, in some embodiments, is approximately 340,000bp. Genomic DNA samples with this size are recommended for long-read nanopore sequencing (Oxford Nanopore platform) and genomic mapping experiments using optical or electronic sensing (Bionano Genomics and Nabsys platforms).

[0033] To assess this prediction, the CHM13 sequence produced by the T2T consortium (CHM13v2.0 / hs1 – Assembly accession ID GCA009914755.4) was queried for gRNA target recognition sequences (CA)10NGG, (AC)10NGG, (AT)10NGG, and (TA)10NGG, using the “findMotif” program (available from the Utilities section of the UC Santa Cruz Genome Browser; (see, e.g., https: / / hgdownload.soe.ucsc.edu / admin / exe / linux.x86_64; accessed July 2024). The four (4) gRNA target sites divide the CHM13 genome into 2927 fragments. 2847 of the fragments have a size <4Mb and contain approximately 83% of the CHM13 genome sequence. 80 fragments are >4Mb in size and contain about 17% of the genome. The average spacing between all of the dinucleotide target sites was 1Mb, in rough agreement with the statistics of the previous paragraph. However, previous studies on use of SpyCas9 in vitro for targeted isolation of large genomic fragments have demonstrated that off-target cutting typically exceeds on-target cutting by at least a factor of 10 (Shin et al., 2019). Off-target cutting can produce a larger fraction of the genome in fragments of practical size (1Mb or less for long range mapping or Nanopore sequencing) (Zhou et al., 2024).Via Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025

[0034] To achieve smaller average fragment sizes for long-read libraries used by the PacBio platform, i.e., approximately ~20,000 (for example), the gRNA pool can be supplemented with gRNAs that target more abundant repeat families such as the human DNA MER1-Charlie transposons which are present at approximately 182,000 copies per genome (Lander et al., 2001).

[0035] Since repeat sequences are not evenly spaced throughout the genome, it is expected that there will be some genomic regions with closely spaced repeat sequence targets and some regions that lack repeat sequence targets. Accordingly, in some embodiments, two strategies can be blended to address such challenges. In particular, a partial digest strategy can be used to address the problem of sequence loss due to close spacing of repeat targets. Digestion using less specific RNA-guided endonucleases or using reaction conditions or gRNA designs that increase the off-target cutting by the RNA-guided endonuclease, can improve recovery of sequence from genomic regions that lack exact copies of the repeat sequence gRNA targets.

[0036] In some embodiments, an advantage of using RNA-guided endonucleases such SpyCas9 and AsCas12a for genomic fragmentation is that the enzymes make concerted double- stranded breaks with very little production of single-stranded cleavages (Yourik et al. 2019; Jinek et al, 2012, Swartjes et al., 2019). The lack of single-stranded cleavages increases read length and quality in long-read sequencing, and it also decreases background labeling in workflows that introduce labels with site-specific nickases, such as the labeling process used by the Nabsys OhmX electronic mapping workflow (Oliver et al.2017).

[0037] The following examples help detail processes according to some embodiments of the disclosure. Example 1: Preparation of Nuclei from Coriell cell line GM24385 (HG002)

[0038] Cells were decanted from culture flask into 50mL conical centrifuge tubes and washed 2-3 times by centrifugation and resuspension in 25-40mL of Ca-Mg-free phosphate-buffered saline (PBS) to remove media. Centrifugation was conducted at 200xg for 5 minutes at 4C. After washing, cells were resuspended in cold PBS at approximately 1 mL per 1e09 cells. Nine (9) mL of cold nuclear prep buffer (NPB) was added rapidly to the concentrated cells in PBS, and the cells were then vortexed at medium speed (70% of maximum rpm, Fisherbrand benchtop vortex mixer) to rapidly disperse and lyse the cells and liberate the nuclei therefromVia Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025 (NPB = 25 mM Tris, pH 8.0, 5mM EDTA, 0.5% Triton X100, 500 mM sucrose, 4mM spermidine-3 HCl).

[0039] A second 10mL volume aliquot of NPB was added to the tube and it was mixed again thoroughly by medium speed vortexing. After this mixing step, the mixture was centrifuged for 2 minutes at 5,000 x g to pellet the nuclei. The supernatant was decanted, and the bottom of the tube was flicked to loosen the nuclear pellet. The loosened pellet was resuspended in approximately1-2ml NPB. The nuclear suspension was pipetted up and down to using a regular orifice 1000ml pipette tip to ensure complete resuspension. Nine (9) mL of cold NPB was added to the suspension, and it was mixed again by medium speed vortexing. The nuclei were pelleted again by centrifugation (2 minutes at 5,000 x g), the supernatant was decanted, and the final nuclear pellet was resuspended by flicking and pipetting in 1 mL of cold NPB. Nuclei were held at 4C until DNA extraction.

[0040] Measurement of the genomic DNA concentration of the nuclear suspension

[0041] 10uL aliquots or the nuclear suspension were then transferred in duplicate to 1.5 ml microcentrifuge tubes. 190uL of Qubit Lysis Buffer (Sage Science, 51mM Tris base, 29mM TAPS acid, 5mM EDTA, 1% SDS, 50mM NaCl, pH 8.7) was added rapidly and mixed by vigorously pipetting up and down several times. The tubes were vortexed at top speed for 30 seconds.600uL of TE (10 mM TrisHCl, 1mM EDTA, pH 7.5 to 8.0) was added to each tube, and the tubes were vortexed again at maximum speed for 30 seconds. Five (5) μl of the diluted lysed samples were assayed for DNA using the Qubit HS reagent kit (Invitrogen). The “tube concentration” reading obtained from the Qubit fluorimeter was used to calculate the DNA concentration of the original nuclear suspension, using the following dilution factors: - [Qubit tube conc, ng / ml] X (800 / 10) X (200 / 5) = [DNA conc original cell suspension, ng / ml]

[0042] For HMW DNA extraction (see below), the nuclear suspension was diluted to a DNA concentration of 143 ng / μL in NPB. Example 2: Semi-automated HMW DNA extraction using SageHLS instrument with dinucleotide repeat gRNAs and SpyCas9.

[0043] Preparation of the dinucleotide gRNA-SpyCas9 complexes:

[0044] The procedure below provides enough gRNA-SpyCas9 complex for 1 HLS sampleVia Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025 lane. Two-part gRNAs were purchased from IDT (Coralville, IA). The four crRNAs were designed to recognize alternating CA and AT dinucleotide repeats that are common in the human genome: (CA)10NGG, (AC)10NGG, (AT)10NGG, and (TA)10NGG. All gRNA and tracrRNA were dissolved at 100uM concentration in IDT Duplex buffer (IDT, Coralville, IA).

[0045] Two-part gRNA annealing:

[0046] The following components were mixed in a 200ul PCR tube: - 7.7 μl IDT Duplex buffer - 2 μl of a pooled equimolar mixture of all 4 dinucleotide crRNAs. - 1.3 μl of tracrRNA (100 μM).

[0047] The 11uL mixture was vortexed briefly and spun down in a microfuge. Concentrations after this step = 11.8 μM tracrRNA, 18 μM total pooled crRNA. The crRNA-tracrRNA mixture was heated for 5 minutes at 95C in a thermal cycler with a heated lid and allowed to cool on the lab bench at room temperature for 5 minutes. After cooling the tube was centrifuged for 30 seconds to collect condensation.

[0048] Assembly of annealed gRNA with SpyCas9:

[0049] Wild-type Cas9 was obtained from New England Biolabs (Ipswich, MA. Sage 1X Enzyme Buffer is 51mM Tris base, 29mM TAPS acid, 0.1mM EDTA acid, 10mM MgCl2, 32mg / ml Hydroxypropyl-beta-cyclodextrin, 50 ug / ml BSA.). The following components were mixed thoroughly by gentle pipetting (the components are viscous). - 5 μl Sage HLS 4X Enzyme Buffer - 11 μl annealed IDT guide RNAs - 4 μl wild-type S. pyogenes Cas9 enzyme (20uM, New England Biolabs #M0386M).

[0050] The mixture was incubated at 37C for 5 minutes. Concentrations at this stage were 4uM SpyCas9, 6.5uM annealed gRNA. The assembled enzyme mixture (20uL) was diluted with 60uL 1X Sage HLS Enzyme Buffer and mixed well by gentle pipetting. At this point, the Cas9 diluted mixture was stored at 4C until the time to initiate Cas9 digestion. 80ul of the diluted enzyme mixture was used in each HLS lane.

[0051] HMW extraction in the HLS system using SpyCas9 with dinucleotide repeat gRNAs

[0052] HMW DNA was extracted from aliquots of GM24385 nuclei containing 10ug ofVia Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025 genomic DNA (70uL of nuclear suspension in NPB at 143 ng / μl) using the electrophoretic SageHLS system (Sage Science, Inc., Beverly, MA, USA; see also the Patents, herein incorporated by reference) using the manufacturer’s instructions for the HLS-CATCH procedure (see “HLS-CATCH Workflow Guide”, available at https: / / sagescience.com / product- support / sagehls-support / ; accessed July 2024). Pictures of the HLS system which can be used are shown in Figures 2A-B. An HLS-CATCH workflow is also depicted schematically in FIGs.3A-3B.

[0053] Briefly, the procedure was as follows: - An aliquot of the nuclei suspension (70uL, 10ug total DNA) was loaded into the sample well; - 200uL of 1% SDS lysis buffer was loaded into the reagent well, upstream from sample well; and - DNA extraction was performed by electrophoresing the SDS lysis buffer downward through the sample well and gel column for three (3) hours using a constant field of 55V; - The SDS lyses the nuclei as it passes through the sample well, encapsulates non-DNA cellular components into negatively charged particles, and causes them to move down the gel column into lower electrode chamber.

[0054] During the three (3) hour extraction electrophoresis, the HMW genomic DNA was trapped and immobilized just inside the downstream wall of the sample well, due to its large size and the absence of any viscous shear during the extraction process.

[0055] At the end of the 3hr extraction period, the sample well was emptied and refilled with the SpyCas9-gRNA complexes (at a total concentration of 1uM). The complexes were electrophoresed into the sample well wall for 2 minutes at 80V constant field to ensure good mixing of the SpyCas9 complexes with the immobilized genomic DNA. The sample well was emptied and refilled with 1X HLS Enzyme buffer, and the genomic DNA in the sample well wall was digested for 30 minutes (without electrophoresis) at room temperature.

[0056] After digestion, the sample was subjected to size-selection electrophoresis down the agarose column using constant field 55V for 1.5 hours, followed by electroelution for 1.5 hours (at 50V constant field) into the six buffer-filled elution modules to the right side of the gel column.Via Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025

[0057] The eluted samples were evaluated by Qubit DNA assays and pulsed-field agarose gel analysis (Bio-Rad CHEFMapper).

[0058] For the data shown in FIG. 4, HMW extractions on GM24385 nuclei (10ug genomic DNA per lane) in quadruplicate (2 HLS cassettes were used, each cassette has two extraction lanes). SpyCas9-gRNA complexes using all four of the crRNAs recognizing (CA)10NGG, (AC)10NGG, (AT)10NGG, and (TA)10NGG, were used in each lane. The DNA products in the elution modules of lanes 2 and 4 were removed and sheared by 10 cycles of rapid up and down pipetting with a narrow bore 200uL pipette tip, in order to thoroughly homogenize any HMW DNA present. DNA concentration measurements were performed using duplicate 10uL aliquots in the Qubit HS fluorometer system (Invitrogen). The data is shown in FIG. 3. The yield of DNA was greater than 0.5ug per elution module (EM) in EMs 2-4 of lane 2 (total yield EMs 2- 4 = 4.9 ug) and EMs 3-5 of lane 4 (total yield EMs 3-5 = 4.2ug).

[0059] To examine the length of the DNA in the peak fractions, we analyzed the unsheared EM contents from the same cassette (HLS lanes 1 and 2 are located in the same cassette; lanes 3 and 4 are located in the same cassette by pulsed field electrophoresis. As seen in FIG.5, peak fractions of lanes 1 and 3 showed HMW DNA ranging in size from 30kb to just under 1Mb, with maximum fluorescence coming from fractions between 100 and 400kb in size. Such preparations are well suited for long-read Oxford Nanopore sequencing and genomic mapping using optical (Bionano Genomics) or electronic readouts (Nabsys). Example 3: Fragmentation of HMW DNA prepared using the gel-plug method

[0060] The CATCH protocol of Gabrieli et al., 2018 was used to (1) purify human peripheral blood mononuclear cells (PBMCs) from human whole blood, (2) embed the isolated PBMCs in low melting temperature agarose gel plug, and (3) extract and purify HMW DNA in the gel plugs by repetitive washing and incubation with detergent solutions and digestion with Proteinase K. After purification and washing of the gel plugs, they were incubated with SpyCas9 complexed with gRNAs containing crRNAs recognizing (CA)10NGG, (AC)10NGG, (AT)10NGG, and (TA)10NGG. After SpyCas9 digestion, the gel plugs were redigested with Proteinase K to remove the SpyCas9, and then washed in TE buffer to remove the Proteinase K. Finally, the gel plugs were melted by brief incubation at 70C, followed by digestion of the agarose at 43C using agarase (ThermoFisher Scientific). After agarose digestion, the fragmented HMW DNA was precipitated with isopropanol, and the HMW DNA pellet wasVia Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025 washed with 80% ethanol, dried, and resuspended in 10mM Tris-HCl, 1mM EDTA, pH 8. Example 4. Fragmentation of HMW DNA prepared by an optimized glass bead method

[0061] Ultra-HMW DNA was prepared from human whole blood using the Monarch HMW DNA Extraction Kit for Cells & Blood using the manufacturer’s protocol (New England Biolabs, Ipswich, MA, USA). Aliquots containing 10ug of extracted HMW DNA were digested with SpyCas9 complexed with gRNAs targeting (CA)10NGG, (AC)10NGG, (AT)10NGG, and (TA)10NGG recognition sites (see, e.g., Figure 1). Reactions containing 1 to 10 ug of HMW DNA were carried out in 100ul of 1X NEBuffer r3.1 at 37C for 1 hour, using SpyCas9 concentrations ranging between 30nM to 1uM, depending on whether partial or extensive digestion was desired. After digestion, 1ul of Proteinase K (20mg / ml, NEB) is added to the reaction and incubation at 37C is continued another hour to remove the SpyCas9. After Proteinase K digestion, the fragmented HMW DNA was precipitated with isopropanol, and the HMW DNA pellet was washed with 80% ethanol, dried, and resuspended in 10mM Tris-HCl, 1mM EDTA, pH 8. General Considerations

[0062] While various inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means, steps, components, and / or structures for performing the function of the embodiments (and elements thereof) disclosed herein, and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, amounts, dimensions, materials, steps, and configurations described herein are meant to be merely an example and that the actual parameters, amounts, dimensions, materials, steps, and configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is therefore to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of claims supported by the subject disclosure and equivalents thereto, and inventive embodimentsVia Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025 may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, device, system, article, material, kit, step, function / functionality, and method described herein. In addition, any combination of two or more such features, devices, systems, articles, materials, kits, steps, functions / functionality, and methods, if such features, systems, articles, materials, kits, steps, functions / functionality, and methods are not mutually inconsistent, is included within the inventive scope of the present disclosure and considered embodiments.

[0063] Embodiments disclosed herein may also be combined with one or more features, components, materials, parameters, as well as complete systems, devices, and / or methods, to yield yet other embodiments and inventions. Moreover, some embodiments, may be distinguishable from the prior art by specifically lacking one and / or another feature disclosed in the particular prior art reference(s); i.e., claims to some embodiments may be distinguishable from the prior art by including one or more negative limitations.

[0064] Also, as noted, various inventive concepts may be embodied as one or more methods, of which one or more examples have been provided. The acts performed as part of the method(s) may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include performing some acts simultaneously, even though shown as sequential acts in illustrative embodiments.

[0065] Any and all references to publications or other documents, including but not limited to, patents, patent applications, articles, webpages, books, etc., presented anywhere in the present application, are herein incorporated by reference in their entirety. Moreover, all definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0066] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” The terms “can” and “may” are used interchangeably in the present disclosure, and indicate that the referred to element, component, structure, function, functionality, objective, advantage, operation, step, process, apparatus, system, device, result, or clarification, has the ability to be used, included, or produced, or otherwise stand for the proposition indicated in the statement for which the term is used (or referred to) for a particular embodiment(s).

[0067] The phrase "and / or" as used herein in the specification and in the claims, should beVia Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025 understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and / or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and / or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0068] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of' or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e. "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of" "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0069] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and / or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at leastVia Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025 one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0070] In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of' and "consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. References:

[0071] Lander, et al.2001. Nature 409: 860-921.

[0072] Yourik, et al.2019. RNA 25:35-44.

[0073] Jinek, et al.2012. Science 337:816-821.

[0074] Swartjes, et al.2019. Biochemical Society Transactions 48 :207-219.

[0075] Oliver et al.2017. bioRxiv. https: / / doi.org / 10.1101 / 139840.

[0076] Zhou et al.2024. Proc Natl Acad Sci USA. https: / / doi.org / 10.1073 / pnas.2322834121.

[0077] Shin et al.2019. Nucleic Acids Res. https: / / doi:10.1093 / nar / gkz661.

[0078] Gabrieli et al.2018. Nucleic Acids Res. https / / doi:10.1093 / nar / gky411.

Claims

Via Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025 What is currently claimed:

1. A method for fragmenting high-molecular weight (HMW) genomic DNA (HMW DNA) comprising the steps: providing a HMW DNA sample; adding one or more reagents to the HMW DNA sample to produce a HMW DNA sample mixture; adding an RNA-guided DNA endonuclease to the HMW DNA sample mixture establishing a first mixture; incubating the first mixture to establish a fragmented DNA sample; inactivating the RNA-guided DNA endonuclease; and purifying the fragmented DNA sample.

2. The method of claim 1, wherein the one or more reagents are configured to provide a buffer condition suitable for digestion of the HMW DNA sample by the RNA-guided DNA nuclease.

3. The method of claims 1 or 2, wherein the RNA-guided endonuclease comprises a Cas9 enzyme assembled with one or more guide RNAs molecules (gRNAs).

4. The method of claim 3, wherein the Cas9 enzyme is configured to target Cas9 cleavage in the HMW DNA sample.

5. The method of claim 4, wherein the Cas9 cleavage is configured to cleave DNA at a plurality of repeated genomic sequences within the DNA.

6. The method of any of claims 1-5, wherein incubating the first mixture is performed under conditions and time for producing a desired extent cleavage of the HMW DNA.

7. The method of claim 6, wherein the conditions comprise a temperature, and wherein the temperature is selected from the group consisting of: between 20 and 40 deg. C, between 20 and 35 deg. C, between 20 and 30 deg. C, between 20 and 25 deg. C, between 25 and 40 deg. C, between 30 and 40 deg. C, between 35 and 40 deg. C, and ranges therebetween.Via Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025 8. The method of claims 6 or 7, wherein the time is selected from the group consisting of: between 15 minutes and 2 hours, between 20 minutes and 2 hours, between 30 minutes and 2 hours, between 45 minutes and 2 hours, between 1 and 2 hours, between 1.25 hours and 2 hours, between 1.5 hours and 2 hours, between 1.75 hours and 2 hours, between 15 minutes and 1.75 hours, between 15 minutes and 1.5 hours, between 15 minutes and 1.25 hours, between 15 minutes and 1 hour, between 15 minutes and 45 minutes, between 15 minutes and 30 minutes, between 15 minutes and 20 minutes, between 20 minutes and 1.75 hours, between 20 minutes and 1.5 hours, between 20 minutes and 1.25 hours, between 20 minutes and 1 hour, between 20 minutes and 45 minutes, between 20 minutes and 30 minutes, between 25 minutes and 2 hours, between 25 minutes and 1.75 hours, between 25 minutes and 1.5 hours, between 25 minutes and 1.25 hours, between 25 minutes and 1 hour, between 25 minutes and 45 minutes, between 25 minutes and 30 minutes, between 30 minutes and 1.75 hours, between 30 minutes and 1.5 hours, between 30 minutes and 1.25 hours, between 30 minutes and 1 hour, between 30 minutes and 45 minutes, between 45 minutes and 2 hours, between 45 minutes and 1.75 hours, between 45 minutes and 1.5 hours, between 45 minutes and 1.25 hours, between 45 minutes and 1 hour, between 1 hour and 2 hours, between 1 hour and 1.75 hours, between 1 hour and 1.5 hours, between 1 hour and 1.25 hours, between 1.25 hours and 2 hours, between 1.25 hours and 1.75 hours, between 1.25 hours and 1.5 hours, between 1.5 hours and 2 hours, between 1.5 hours and 1.75 hours, between 1.75 hours and 2 hours, and ranges therebetween.

9. The method of any of claims 1-2, wherein the RNA-guided DNA endonuclease is configured to recognize one or more classes of repeated genomic sequences.

10. The method of any of claims 1-9, wherein RNA-guided endonucleases are selected from the group consisting of: Streptococcus pyogenes Cas9 (SpyCas9), Acidaminococcus sp. Casl2a (AsCas 12a), or derivatives thereof.

11. A method for fragmenting high-molecular weight (HMW) genomic DNA (HMW DNA) comprising the steps: providing a HMW DNA sample; adding one or more reagents to the HMW DNA sample, the one or more reagents configured to provide a buffer condition suitable for digestion of the HMW DNA sample by a Cas9 nuclease;Via Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025 adding a Cas9 preparation to the HMW DNA sample establishing a HMW DNA – Cas9 mixture, wherein the Cas9 preparation comprises a Cas9 enzyme assembled with one or more guide RNA molecules, the Cas9 preparation configured to cleave at a plurality of repeated genomic sequences that are dispersed throughout the genome to produce a fragmented genomic DNA sample; incubating the HMW DNA - Cas9 mixture under conditions and time for producing a desired extent of Cas9 cleavage; inactivating the Cas9 enzyme; and purifying the fragmented DNA.

12. A kit for enzymatic fragmentation of high molecular weight (HMW) genomic DNA comprising: one or more RNA-guided DNA endonucleases; and one or more gRNA molecules for recognizing one or more classes of repeated genomic sequences.

13. The kit of claim 10, further comprising at least one of one or more reagents, an apparatus, and a device for extraction of HMW DNA.

14. The kit of claims 10 or 11, wherein at least one of the RNA-guided endonucleases comprises Streptococcus pyogenes Cas9 (SpyCas9) or Acidaminococcus sp. Casl2a (AsCasl2a), or derivatives thereof.

15. A system for performing the method according to any of claims 1-11.

16. A system, device, or method according to any of the disclosed embodiments.

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