Genomic library preparation and targeted epigenetic assays using CAS-gRNA ribonucleoproteins

Abstract

Provided herein are genomic library preparations and targeted epigenetic assays using Cas-gRNA RNPs. Some compositions include substantially only single-stranded polynucleotides from a first species and substantially only double-stranded polynucleotides from a second species, an amplification primer ligated to an end of the second double-stranded polynucleotide and substantially not ligated to either end of the first double-stranded polynucleotide. Some compositions include first and second molecules of a target polynucleotide having a sequence, the first molecule having a first end at a first subsequence, the second molecule having a first end at a second subsequence, and the first subsequence only partially overlapping with the second subsequence. Some examples provide compositions including a target polynucleotide and a first fusion protein including a Cas-gRNA RNP coupled to a transposase to which an amplification adaptor is coupled. The Cas-gRNA RNP can be hybridized to a subsequence in the target polynucleotide.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the following applications, the entire contents of each of which are incorporated herein by reference: U.S. Provisional Patent Application No. 63 / 158,492, filed March 9, 2021, entitled “Genomic library preparation and targeted epigenetic assays using Cas-gRNA ribonucleoproteins”; U.S. Provisional Patent Application No. 63 / 162,775, filed on March 18, 2021, entitled “Genomic library preparation and targeted epigenetic assays using Cas-gRNA ribonucleoproteins”; U.S. Provisional Patent Application No. 63 / 163,381, filed on March 19, 2021, entitled “Genomic library preparation and targeted epigenetic assays using Cas-gRNA ribonucleoproteins”; U.S. Provisional Patent Application No. 63 / 228,344, filed on August 2, 2021, entitled “Genomic library preparation and targeted epigenetic assays using Cas-gRNA ribonucleoproteins”; U.S. Provisional Patent Application No. 63 / 246,879, filed on September 22, 2021, entitled “Genomic library preparation and targeted epigenetic assays using Cas-gRNA ribonucleoproteins”; and U.S. Provisional Patent Application No. 63 / 295,432, filed on December 30, 2021, entitled “Genomic library preparation and targeting epigenetic assays using Cas-gRNA ribonucleoproteins.”

[0002] The present application relates to compositions and methods using Cas-gRNA RNPs for genomic library preparation and targeted epigenetic assays.

[0003] Description of sequence listing The sequence listing associated with this application is provided in text format in lieu of a paper copy and is incorporated herein by reference. The name of the text file containing the sequence listing is 8549102416_SL.txt. The text file is approximately 1.29KB, was created on Mar. 3, 2022, and has been submitted electronically via EFS-Web. [Background technology]

[0004] Clustered regularly interspaced short palindromic repeats (CRISPR) are involved in interference pathways that protect cells from bacteriophages and conjugative plasmids in many bacteria and archaea; see, e.g., Marraffini et al., "CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea," Nat Rev Genet. 11(3):181-190 (2010). CRISPR sequences contain an array of short repeat sequences spaced by similarly sized unique variable DNA sequences called spacers, which are often derived from phage or plasmid DNA. See, for example, the following references, the entire contents of which are incorporated herein by reference: Barrangou et al., "CRISPR provides acquired resistance against viruses in prokaryotes", Science 315:1709-1712 (2007); Bolotin et al., "Clustered regularly interspersed short palindrome repeats (CRISPR) have spacers of extrachromosomal origin", Microbiology 151:2551-1561 (2005); and Mojica et al., "Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements", J Mol Evol. 60:174-82 (2005). Thus, CRISPR sequences provide an adaptive heritable record of past infections and can be transcribed into CRISPR RNA (crRNA) - a small RNA that targets invasive polynucleotides (see, for example, Marraffini et al., cited above). CRISPRs are often associated with CRISPR-associated (Cas) genes, which code for proteins associated with CRISPR.Cas proteins can provide a mechanism for destroying invading foreign polynucleotides targeted by the crRNA. CRISPR, together with Cas genes, provide an adaptive immune system that provides acquired resistance to invading foreign polynucleotides in bacteria and archaea (see, e.g., Barrangou et al., cited above).

[0005] Single molecule sequencing studies have suggested CRISPR targeted methods for direct methylation sequencing using Cas9; see, for example, Gilpatrick et al., "Targeted nanopore sequencing with Cas9 for studies of methylation, structural variants and mutations," https: / / doi.org / 10.1101 / 604173, 1-14 (2019), the entire contents of which are incorporated herein by reference. However, beyond DNA methylation, there is an unmet need for methods that allow for sensitive characterization of epigenetic changes at targeted DNA loci. Chromatin accessibility (by ATAC-seq) and proteins associated with DNA loci (by ChIP-seq) are examples of epigenetic elements that are difficult to target with existing hybrid capture techniques. In general, assays that enrich for DNA sequences are associated with epigenetic features. However, because these sequences are not known a priori, it is difficult to design appropriate hybrid capture oligonucleotides to efficiently enrich the output of epigenetic assays for specific genomic regions (e.g., genomic loci) of interest.

[0006] Previous methods using inactivated Cas (dCas9) for targeted locus-specific protein isolation to identify histone gene regulators have been presented: see, e.g., Tsui et al., "dCas9-targeted locus-specific protein isolation method identifies histone gene regulators," PNAS 115(2):E2734-E2741 (2018), the entire contents of which are incorporated herein by reference. Such methods demonstrated that dCas9-based locus enrichment can isolate chromatin that can be subsequently assayed by mass spectrometry. However, this method can only assay a single chromatin locus in each experiment. Furthermore, this prior work provides two separate results (i.e., the sequence of the DNA locus, and mass spectrometry to identify DNA-associated proteins). Improved methods for gene coordinate epigenetic analysis are needed. Summary of the Invention

[0007] Provided herein are genomic library preparation and targeted epigenetic assays using Cas-gRNA ribonucleoproteins (RNPs).

[0008] Some examples herein provide a method for treating a mixture of a first double-stranded polynucleotide derived from a first species and a second double-stranded polynucleotide derived from a second species. The method may include protecting the ends of the first double-stranded polynucleotide and any ends of the second double-stranded polynucleotide. The method may include selectively generating free ends in the first double-stranded polynucleotide after protecting the ends of the first and second double-stranded polynucleotides. The method may include degrading the first double-stranded polynucleotide from the free end toward the protected end.

[0009] In some examples, selectively generating free ends in the first double-stranded polynucleotide comprises hybridizing a CRISPR-associated protein guide RNA ribonucleoprotein (Cas-gRNA RNP) to a sequence present in the first double-stranded polynucleotide and absent in the second double-stranded polynucleotide, and cleaving the sequence with the Cas-gRNA RNP. In some examples, the sequence comprises a mammalian-specific repeat element. In some examples, the mammalian-specific repeat element comprises a human-specific repeat element. In some examples, the second species is a bacterium, a fungus, or a virus. In some examples, the first double-stranded polynucleotide comprises a plurality of chromosomes from the first species.

[0010] In some examples, protecting the ends of the first and second double-stranded polynucleotides comprises ligating hairpin adaptors to the ends. In some examples, protecting the ends of the first and second double-stranded polynucleotides comprises 5' dephosphorylating the ends. In some examples, protecting the ends of the first and second double-stranded polynucleotides comprises adding modified bases to the ends. In some examples, the modified bases comprise phosphorothioate linkages. In some examples, the modified bases are added using a terminal transferase.

[0011] In some examples, degrading the first double-stranded polynucleotide is performed using an exonuclease.

[0012] In some examples, the free end comprises a 3' end. In some examples, degrading the first double-stranded polynucleotide is performed using exonuclease III. In some examples, the free end comprises a 5' end. In some examples, degrading the first double-stranded polynucleotide is performed using lambda exonuclease.

[0013] In some examples, the method further comprises subsequently ligating an amplification adaptor to the end of any remaining double-stranded polynucleotides in the mixture. In some examples, the amplification adaptor comprises a unique molecular identifier (UMI). In some examples, the method further comprises subsequently amplifying and sequencing the double-stranded polynucleotides.

[0014] In some examples, the first double-stranded polynucleotide comprises double-stranded DNA. In some examples, the second double-stranded polynucleotide comprises double-stranded DNA. In some examples, the second double-stranded polynucleotide comprises circular DNA.

[0015] In some examples, the Cas comprises Cas9.

[0016] Some examples herein provide a composition. The composition may comprise a first double-stranded polynucleotide derived from a first species. The end of the first double-stranded polynucleotide may be protected. The composition may comprise a second double-stranded polynucleotide derived from a second species. Any end of the second double-stranded polynucleotide may be protected. The composition may also comprise a CRISPR-associated protein guide RNA ribonucleoprotein (Cas-gRNA RNP) hybridized to a sequence present in the first double-stranded polynucleotide and not present in the second double-stranded polynucleotide. The Cas-gRNA RNP may be for cleaving a sequence to selectively generate a free end in the first double-stranded polynucleotide.

[0017] In some examples, the sequence comprises mammalian-specific repetitive elements. In some examples, the mammalian-specific repetitive elements comprise human repetitive elements. In some examples, the second species is a bacterium, a fungus, or a virus.

[0018] In some examples, the ends of the first and second double-stranded polynucleotides are protected using hairpin adaptors. In some examples, the ends of the first and second double-stranded polynucleotides are protected using 5' dephosphorylation. In some examples, the ends of the first and second double-stranded polynucleotides are protected using modified bases. In some examples, the modified bases include phosphorothioate bonds.

[0019] In some instances, the free end comprises a 3' end. In some instances, the free end comprises a 5' end.

[0020] In some examples, the first double-stranded polynucleotide comprises double-stranded DNA. In some examples, the second double-stranded polynucleotide comprises double-stranded DNA. In some examples, the second double-stranded polynucleotide comprises circular DNA.

[0021] In some examples, the Cas comprises Cas9.

[0022] Some examples herein provide a method for processing a mixture of a first double-stranded polynucleotide derived from a first species and a second double-stranded polynucleotide derived from a second species. The method may include selectively making the first double-stranded polynucleotide in the mixture single-stranded. The method may then include selectively ligating an amplification primer to any remaining double-stranded polynucleotide in the mixture. The method may then include amplifying any double-stranded polynucleotide in the mixture to which the amplification primer is ligated.

[0023] Some examples herein provide a composition.The composition may comprise substantially only single-stranded polynucleotides from a first species.The composition may comprise substantially only double-stranded polynucleotides from a second species.The composition may comprise an amplification primer that is ligated to the end of the second double-stranded polynucleotide and is not substantially ligated to either end of the first double-stranded polynucleotide.

[0024] Some examples herein provide a method for generating fragments of a whole genome (WG). The method may include hybridizing a first set of CRISPR-associated protein guide RNA ribonucleoproteins (Cas-gRNA RNPs) to a first sequence in the WG that is spaced apart from each other by approximately a first number of base pairs in a first sample of the WG. The method may further include hybridizing a second set of Cas-gRNA RNPs to a second sequence in the WG that is spaced apart from each other by approximately a second number of base pairs in the first sample of the WG. The method may further include cleaving the first and second sequences with the first and second sets of Cas-gRNA RNPs in the first sample in the first sample of the WG, respectively, to generate a first set of WG fragments, each of which has approximately the same number of base pairs as each other.

[0025] In some examples, the first number of base pairs is approximately the same as the second number of base pairs. In some examples, the first number of base pairs is about 100 to about 2000 and the second number of base pairs is about 100 to about 2000. In some examples, the first number of base pairs is about 500 to about 700 and the second number of base pairs is about 500 to about 700. In some examples, the number of base pairs within the WG fragments of the first set of WG fragments differ by less than about 20%.

[0026] In some examples, the method further comprises hybridizing the first set of Cas-gRNA RNPs to the first sequence in the WG in the second sample of the WG. The method may further comprise hybridizing the second set of Cas-gRNA RNPs to the second sequence in the WG in the second sample of the WG. The method may further comprise hybridizing the third set of Cas-gRNA RNPs to a third sequence in the WG that is spaced apart from each other by approximately a third number of base pairs in the second sample of the WG. The method may further comprise cleaving the first, second, and third sequences with the first, second, and third sets of Cas-gRNA RNPs, respectively, in the second sample of the WG to generate a second set of WG fragments, each having approximately the same number of base pairs as each other.

[0027] In some examples, the third number of base pairs is different from the first number of base pairs. In some examples, the third number of base pairs is different from the second number of base pairs. In some examples, the third number of base pairs is about 100 to about 2000. In some examples, the third number of base pairs is about 200 to about 400. In some examples, the approximate number of base pairs in the WG fragments of the second set of WG fragments is different from the approximate number of base pairs in the WG fragments of the first set of WG fragments. In some examples, the number of base pairs in the WG fragments of the second set of WG fragments differs by less than about 20%.

[0028] In some examples, the method further comprises hybridizing, in a third sample of WG, the first, second, or third set of Cas-gRNA RNPs to the first, second, or third sequence in the WG, respectively. The method may further comprise cleaving the first, second, or third sequence with the first, second, or third set of Cas-gRNA RNPs, respectively, to generate a third set of WG fragments, each having approximately the same number of base pairs as each other.

[0029] In some examples, the approximate number of base pairs in the WG fragments of the third set of WG fragments differs from the approximate number of base pairs in the WG fragments of the first set of WG fragments. In some examples, the approximate number of base pairs in the WG fragments of the third set of WG fragments differs from the approximate number of base pairs in the WG fragments of the second set of WG fragments. In some examples, the number of base pairs in the WG fragments of the third set of WG fragments differs by less than about 20%.

[0030] In some examples, the method further comprises ligating an amplification adaptor to an end of the WG fragment of the third set of WG fragments. The method may further comprise generating an amplicon of the WG fragment of the third set of WG fragments to which the amplification adaptor has been ligated. The method may further comprise sequencing the amplicon of the WG fragment of the third set of WG fragments. In some examples, the amplicons of the WG fragments of the second and third sets of WG fragments are mixed together for sequencing. In some examples, the amplicons of the WG fragments of the first and third sets of WG fragments are mixed together for amplification and sequencing.

[0031] In some examples, the number of base pairs in the WG fragments of the third set of WG fragments is about 100 to about 1000. In some examples, the number of base pairs in the WG fragments of the third set of WG fragments is about 500 to about 700.

[0032] In some examples, the third set of Cas-gRNA RNPs comprises at least about 1,000,000 different Cas-gRNA RNPs.

[0033] In some examples, the method further comprises ligating an amplification adaptor to an end of the WG fragment of the second set of WG fragments. The method may further comprise generating an amplicon of the WG fragment of the second set of WG fragments to which the amplification adaptor is ligated. The method may further comprise sequencing the WG fragment amplicon of the second set of WG fragments.

[0034] In some examples, the WG fragment amplicons of the first and second sets of WG fragments are mixed together for amplification and sequencing.

[0035] In some examples, the number of base pairs in the WG fragments of the second set of WG fragments is about 100 to about 1000. In some examples, the number of base pairs in the WG fragments of the second set of WG fragments is about 100 to about 200.

[0036] In some examples, the method further comprises ligating an amplification adaptor to an end of the WG fragment of the first set of WG fragments. The method may further comprise generating an amplicon of the WG fragment of the first set of WG fragments to which the amplification adaptor is ligated. The method may further comprise sequencing the WG fragment amplicon of the first set of WG fragments.

[0037] In some examples, the amplification adapter comprises a unique molecular identifier (UMI).

[0038] In some examples, the number of base pairs in the WG fragments of the first set of WG fragments is about 100 to about 1000. In some examples, the number of base pairs in the WG fragments of the first set of WG fragments is about 200 to about 400.

[0039] In some examples, the first set of Cas-gRNA RNPs comprises at least about 1,000,000 different Cas-gRNA RNPs. In some examples, the second set of Cas-gRNA RNPs comprises at least about 1,000,000 different Cas-gRNA RNPs.

[0040] In some examples, the WG comprises double stranded DNA. In some examples, the Cas comprises Cas9.

[0041] Some examples herein provide a composition. The composition may include a sample of a whole genome (WG). The composition may include a first set of CRISPR-associated protein guide RNA ribonucleoprotein (Cas-gRNA RNP) hybridized to a first sequence in the WG that is approximately spaced apart from each other by a first number of base pairs. The composition may include a second set of Cas-gRNA RNP hybridized to a second sequence in the WG that is approximately spaced apart from each other by a second number of base pairs. The first and second sets of Cas-gRNA RNP may be for cleaving the first and second sequences in the sample, respectively, to generate WG fragments that each have approximately the same number of base pairs as each other.

[0042] In some examples, the first number of base pairs is approximately the same as the second number of base pairs. In some examples, the first number of base pairs is about 100 to about 2000, and the second number of base pairs is about 100 to about 2000. In some examples, the first number of base pairs is about 500 to about 700, and the second number of base pairs is about 500 to about 700.

[0043] In some examples, the number of base pairs in the WG fragment varies by less than about 20%. In some examples, the number of base pairs in the WG fragment is between about 100 base pairs and about 1000 base pairs. In some examples, the number of base pairs in the WG fragment is between about 200 base pairs and about 400 base pairs.

[0044] In some examples, the first set of Cas-gRNA RNPs comprises at least about 1,000,000 different Cas-gRNA RNPs. In some examples, the second set of Cas-gRNA RNPs comprises at least about 1,000,000 different Cas-gRNA RNPs.

[0045] In some examples, the WG comprises double stranded DNA. In some examples, the Cas comprises Cas9.

[0046] Some examples herein provide a composition. The composition may include a sample of a whole genome (WG). The composition may include a first set of CRISPR-associated protein guide RNA ribonucleoprotein (Cas-gRNA RNP) hybridized to a first sequence in the WG that is approximately spaced apart from each other by a first number of base pairs. The composition may include a second set of Cas-gRNA RNP hybridized to a second sequence in the WG that is approximately spaced apart from each other by a second number of base pairs. The composition may include a third set of Cas-gRNA RNP hybridized to a third sequence in the WG that is approximately spaced apart from each other by a third number of base pairs. The first, second, and third sets of Cas-gRNA RNP may be for cleaving the first, second, and third sequences in the sample, respectively, to generate WG fragments that each have approximately the same number of base pairs as each other.

[0047] In some examples, the first number of base pairs is approximately the same as the second number of base pairs. In some examples, the first number of base pairs is about 100 to about 2000, the second number of base pairs is about 100 to about 2000, and the third number of base pairs is about 100 to about 2000. In some examples, the first number of base pairs is about 500 to about 700, the second number of base pairs is about 500 to about 700, and the third number of base pairs is about 200 to about 400. In some examples, the third number of base pairs is different from the first number of base pairs. In some examples, the third number of base pairs is different from the second number of base pairs.

[0048] In some examples, the number of base pairs in the WG fragment varies by less than about 20%. In some examples, the number of base pairs in the WG fragment is from about 100 to about 1000. In some examples, the number of base pairs in the WG fragment is from about 100 to about 200.

[0049] In some examples, the first set of Cas-gRNA RNPs comprises at least about 1,000,000 different Cas-gRNA RNPs. In some examples, the second set of Cas-gRNA RNPs comprises at least about 1,000,000 different Cas-gRNA RNPs. In some examples, the third set of Cas-gRNA RNPs comprises at least about 1,000,000 different Cas-gRNA RNPs.

[0050] In some examples, the WG comprises double stranded DNA. In some examples, the Cas comprises Cas9.

[0051] Some examples herein provide a method for generating fragments of a whole genome (WG).The method may include hybridizing a set of CRISPR-associated protein guide RNA ribonucleoprotein (Cas-gRNA RNP) to a sequence in the WG that is approximately spaced apart from each other by a number of base pairs.The method may include respectively cutting the sequence with a set of Cas-gRNA RNP to generate a set of WG fragments that each have approximately the same number of base pairs as each other.

[0052] In some examples, the number of base pairs is about 100 to about 1000. In some examples, the number of base pairs is about 500 to about 700, or about 200 to about 400, or about 100 to about 200.

[0053] In some examples, the number of base pairs within the WG fragments of the set of WG fragments differs by less than about 20%. In some examples, the number of base pairs within the WG fragments of the set of WG fragments is about 100 to about 1000. In some examples, the number of base pairs within the WG fragments of the set of WG fragments is about 100 to about 200, or about 200 to about 400, or about 500 to about 700.

[0054] In some examples, the method further comprises ligating an amplification adaptor to an end of a WG fragment of the set of WG fragments. The method may further comprise generating an amplicon of the WG fragment of the set of WG fragments to which the amplification adaptor has been ligated. The method may further comprise sequencing the WG fragment amplicon of the set of WG fragments.

[0055] In some examples, the amplification adapter comprises a unique molecular identifier (UMI).

[0056] In some examples, the WG comprises double stranded DNA. In some examples, the Cas comprises Cas9.

[0057] Some examples herein provide compositions.The compositions can include a sample of whole genome (WG).The compositions can include a set of CRISPR-associated protein guide RNA ribonucleoproteins (Cas-gRNA RNPs) hybridized to sequences in the WG that are approximately spaced apart from each other by a number of base pairs.Each set of Cas-gRNA RNPs can be for cutting sequences in the sample to generate WG fragments that each have approximately the same number of base pairs as each other.

[0058] In some examples, the number of base pairs is about 100 to about 1000. In some examples, the number of base pairs is about 500 to about 700, or about 200 to about 400, or about 100 to about 200.

[0059] In some examples, the number of base pairs within the WG fragments of the set of WG fragments differs by less than about 20%. In some examples, the number of base pairs within the WG fragments of the set of WG fragments is about 100 to about 1000. In some examples, the number of base pairs within the WG fragments of the set of WG fragments is about 100 to about 200, or about 200 to about 400, or about 500 to about 700.

[0060] In some examples, the WG comprises double stranded DNA. In some examples, the Cas comprises Cas9.

[0061] Some examples herein provide compositions that can include a set of at least about 1,000,000 WG fragments, each having approximately the same number of base pairs as each other.

[0062] In some examples, the number of base pairs is about 100 to about 200. In some examples, the number of base pairs is about 200 to about 400. In some examples, the number of base pairs is about 500 to about 700.

[0063] In some instances, the WG comprises double-stranded DNA.

[0064] In some examples, the number of base pairs within the WG fragments of a set of WG fragments differs by less than about 20%. In some examples, the number of base pairs within the WG fragments of a set of WG fragments differs by less than about 10%. In some examples, the number of base pairs within the WG fragments of a set of WG fragments differs by less than about 5%.

[0065] Such compositions may be prepared according to the methods described above.

[0066] Some examples herein provide a method for cleaving a molecule of a target polynucleotide having a sequence. The method may include contacting a first and a second molecule of the target polynucleotide with a plurality of first and second CRISPR-associated protein guide RNA ribonucleoproteins (Cas-gRNA RNPs) in a fluid. The method may include hybridizing one of the first Cas-gRNA RNPs to a first subsequence in the first molecule. The method may include hybridizing one of the second Cas-gRNA RNPs to a second subsequence in the second molecule. The second subsequence may only partially overlap with the first subsequence. The method may include inhibiting hybridization of any of the second Cas-gRNA RNPs to the second subsequence in the first molecule by one of the first Cas-gRNA RNPs. The method may include inhibiting hybridization of any of the first Cas-gRNA RNPs to a first subsequence in the second molecule by one of the second Cas-gRNA RNPs. The method may include cleaving the first molecule at the first subsequence. The method may include cleaving the second molecule at the second subsequence.

[0067] In some examples, the cleavage site in the first molecule is at a different position in the sequence of the target polynucleotide than the cleavage site in the second molecule, In some examples, the cleavage site in the first molecule is offset from the cleavage site in the second molecule by about 2 base pairs to about 10 base pairs in the sequence of the target polynucleotide.

[0068] In some examples, the first molecule is cleaved using one of the first Cas-gRNA RNPs and the second molecule is cleaved using one of the second Cas-gRNA RNPs.

[0069] In some examples, the target polynucleotide comprises double-stranded DNA. In some examples, the Cas comprises Cas9 or dCas9.

[0070] In some examples, the method further comprises contacting the first and second molecules of the target polynucleotide with a plurality of third and fourth Cas-gRNA RNPs in the fluid. The method may further comprise hybridizing one of the third Cas-gRNA RNPs to a third subsequence in the first molecule. The method may further comprise inhibiting hybridization of any of the fourth Cas-gRNA RNPs to a fourth subsequence in the first molecule by one of the third Cas-gRNA RNPs. The fourth subsequence may only partially overlap with the third subsequence. The method may comprise cleaving the first molecule at the third subsequence using one of the third Cas-gRNA RNPs to generate a first fragment.

[0071] In some examples, the method further comprises contacting the first and second molecules of the target polynucleotide with a plurality of third and fourth Cas-gRNA RNPs in the fluid. The method may comprise hybridizing one of the fourth Cas-gRNA RNPs to a fourth subsequence in the first molecule. The method may comprise inhibiting hybridization of any of the third Cas-gRNA RNPs to the third subsequence in the first molecule by one of the fourth Cas-gRNA RNPs. The method may comprise cleaving the first molecule at the fourth subsequence using one of the fourth Cas-gRNA RNPs to generate a first fragment.

[0072] In some examples, the method further comprises hybridizing one of the third Cas-gRNA RNPs to a third subsequence in the second molecule. The method may further comprise inhibiting hybridization of any of the fourth Cas-gRNA RNPs to a fourth subsequence in the second molecule by one of the third Cas-gRNA RNPs. The method may further comprise cleaving the second molecule at the third subsequence using one of the third Cas-gRNA RNPs to generate a second fragment.

[0073] In some examples, the method further comprises hybridizing one of the fourth Cas-gRNA RNPs to a fourth subsequence in the second molecule. The method may further comprise inhibiting hybridization of any of the third Cas-gRNA RNPs to a third subsequence in the second molecule by one of the fourth Cas-gRNA RNPs. The method may further comprise cleaving the second molecule at the fourth subsequence using one of the fourth Cas-gRNA RNPs to generate a second fragment.

[0074] In some examples, the method further comprises degrading any portion of the first molecule that is not between one of the first Cas-gRNA RNPs and one of the third or fourth Cas-gRNA RNPs while the one of the first Cas-gRNA RNPs and one of the third or fourth Cas-gRNA RNPs are hybridized to the first molecule.

[0075] In some examples, the method further comprises degrading any portion of the second molecule that is not between one of the second Cas-gRNA RNPs and one of the third or fourth Cas-gRNA RNPs while one of the second Cas-gRNA RNPs and one of the third or fourth Cas-gRNA RNPs are hybridized to the second molecule. In some examples, the degradation is performed using Exonuclease III or Exonuclease VII.

[0076] In some examples, the first molecule is cleaved using one of the third or fourth Cas-gRNA RNPs, and the second molecule is cleaved using one of the third or fourth Cas-gRNA RNPs.

[0077] In some examples, the first and second fragments include different numbers of base pairs from each other. In some examples, the first fragment has a length of about 100 base pairs to about 1000 base pairs, and the second fragment has a length of about 100 base pairs to about 1000 base pairs. In some examples, the first fragment has a length of about 500 base pairs to about 700 base pairs, and the second fragment has a length of about 500 base pairs to about 700 base pairs. In some examples, the first fragment has a length of about 200 base pairs to about 400 base pairs, and the second fragment has a length of about 200 base pairs to about 400 base pairs. In some examples, the first fragment has a length of about 100 base pairs to about 200 base pairs, and the second fragment has a length of about 100 base pairs to about 200 base pairs.

[0078] Some examples herein provide a method for sequencing a target polynucleotide. The method may include generating a first and a second fragment of the target polynucleotide using the above method. The method may further include ligating an amplification adaptor to the ends of the first and the second fragment. The method may further include generating amplicons of the first and the second fragment, respectively, to which the amplification adaptor is ligated. The method may further include sequencing the amplicons of the first and the second fragment.

[0079] In some examples, the method further includes using the first, second, third, and fourth subsequences and identifying the first fragment amplicon as derived from the first molecule and identifying the second fragment amplicon as derived from the second molecule.

[0080] In some examples, the method further includes ligating a unique molecular identifier (UMI) to the ends of the first and second fragments prior to generating the amplicons. The method may further include using the UMI and identifying the amplicon of the first fragment as originating from the first molecule and identifying the amplicon of the second fragment as originating from the second molecule. In some examples, the UMI is coupled and ligated to the ends of the first and second fragments in the same operation as the amplification adaptors.

[0081] Some examples herein provide a composition. The composition may include a first and a second molecule of a target polynucleotide having a sequence. The composition may include a plurality of first and second CRISPR-associated protein guide RNA ribonucleoproteins (Cas-gRNA RNPs). One of the first Cas-gRNA RNPs may hybridize to a first subsequence in the first molecule and inhibit hybridization of any of the second Cas-gRNA RNPs to a second subsequence in the first molecule. The second subsequence may only partially overlap with the first subsequence. One of the second Cas-gRNA RNPs may hybridize to a second subsequence in the second molecule and inhibit hybridization of any of the first Cas-gRNA RNPs to the first subsequence in the second molecule.

[0082] In some examples, the cleavage site in the first molecule is at a different position in the sequence of the target polynucleotide than the cleavage site in the second molecule, In some examples, the cleavage site in the first molecule is offset from the cleavage site in the second molecule by about 2 base pairs to about 10 base pairs in the sequence of the target polynucleotide.

[0083] In some examples, one of the first Cas-gRNA RNPs is for cleaving a first molecule and one of the second Cas-gRNA RNPs is for cleaving a second molecule.

[0084] In some examples, the target polynucleotide comprises double-stranded DNA. In some examples, the Cas comprises Cas9 or dCas9.

[0085] In some examples, the composition further comprises a plurality of third and fourth Cas-gRNA RNPs. One of the third Cas-gRNA RNPs may hybridize to a third subsequence in the first molecule, inhibit any hybridization of the fourth Cas-gRNA RNP to a fourth subsequence in the first molecule, and may be for cleaving the first molecule at the third subsequence to generate a first fragment. The fourth subsequence may only partially overlap with the third subsequence.

[0086] In some examples, the composition further comprises a plurality of third and fourth Cas-gRNA RNPs. One of the fourth Cas-gRNA RNPs may hybridize to a fourth subsequence in the first molecule, inhibit hybridization of any of the third Cas-gRNA RNPs to the third subsequence in the first molecule, and may be for cleaving the first molecule at the fourth subsequence to generate a first fragment. The fourth subsequence may only partially overlap with the third subsequence.

[0087] In some examples, one of the third Cas-gRNA RNPs may hybridize to a third subsequence in the second molecule, may inhibit hybridization of any of the fourth Cas-gRNA RNPs to a fourth subsequence in the second molecule, and may be for cleaving the second molecule at the third subsequence to generate a second fragment.

[0088] In some examples, one of the fourth Cas-gRNA RNPs may hybridize to a fourth subsequence in the second molecule, may inhibit hybridization of any of the third Cas-gRNA RNPs to a third subsequence in the second molecule, and may be for cleaving the second molecule at the fourth subsequence to generate a second fragment.

[0089] In some examples, the composition further comprises an exonuclease to degrade any portion of the first molecule that is not between one of the first Cas-gRNA RNPs and one of the third or fourth Cas-gRNA RNPs.

[0090] In some examples, the composition further comprises an exonuclease to degrade any portion of the second molecule that is not between one of the second Cas-gRNA RNPs and one of the third or fourth Cas-gRNA RNPs.

[0091] In some examples, the exonuclease comprises exonuclease III or exonuclease VII.

[0092] In some examples, one of the third or fourth Cas-gRNA RNPs is for cleaving a first molecule, and one of the third or fourth Cas-gRNA RNPs is for cleaving a second molecule.

[0093] In some examples, the first and second fragments include different numbers of base pairs from each other. In some examples, the first fragment has a length of about 100 base pairs to about 1000 base pairs, and the second fragment has a length of about 100 base pairs to about 1000 base pairs. In some examples, the first fragment has a length of about 500 base pairs to about 700 base pairs, and the second fragment has a length of about 500 base pairs to about 700 base pairs. In some examples, the first fragment has a length of about 200 base pairs to about 400 base pairs, and the second fragment has a length of about 200 base pairs to about 400 base pairs. In some examples, the first fragment has a length of about 100 base pairs to about 200 base pairs, and the second fragment has a length of about 100 base pairs to about 200 base pairs.

[0094] Some examples herein provide a composition. The composition may include a first and a second molecule of a target polynucleotide having a sequence. The first molecule may have a first end at a first subsequence. The second molecule may have a first end at a second subsequence. The first subsequence may only partially overlap with the second subsequence.

[0095] In some examples, the first end of the first molecule is at a different position in the sequence of the target polynucleotide than the first end of the second molecule, in some examples, the first end of the first molecule is offset from the first end of the second molecule by about 2 base pairs to about 10 base pairs in the sequence of the target polynucleotide.

[0096] In some examples, the first molecule further has a second end at the third subsequence. The second molecule may further have a second end at the third subsequence or the fourth subsequence. The third subsequence may only partially overlap with the fourth subsequence. In some examples, the second end of the first molecule is at a different position in the sequence of the target polynucleotide than the second end of the second molecule. In some examples, the second end of the first molecule is offset from the second end of the second molecule by about 2 base pairs to about 10 base pairs in the sequence of the target polynucleotide.

[0097] In some instances, the target polynucleotide comprises double-stranded DNA.

[0098] In some examples, the first and second molecules comprise a different number of base pairs from each other. In some examples, the first molecule has a length of about 100 base pairs to about 1000 base pairs, and the second molecule has a length of about 100 base pairs to about 1000 base pairs. In some examples, the first fragment has a length of about 500 base pairs to about 700 base pairs, and the second fragment has a length of about 500 base pairs to about 700 base pairs. In some examples, the first fragment has a length of about 200 base pairs to about 400 base pairs, and the second fragment has a length of about 200 base pairs to about 400 base pairs. In some examples, the first fragment has a length of about 100 base pairs to about 200 base pairs, and the second fragment has a length of about 100 base pairs to about 200 base pairs.

[0099] Some examples herein provide a method for generating a fragment of a target polynucleotide having a sequence. The method may include contacting the target polynucleotide with a first and a second fusion protein in a fluid. The first fusion protein may include a first CRISPR-associated protein guide RNA ribonucleoprotein (Cas-gRNA RNP) coupled to a first transposase to which a first amplification adaptor is coupled. The second fusion protein may include a second Cas-gRNA RNP coupled to a second transposase to which a second amplification adaptor is coupled. The method may include hybridizing the first Cas-gRNA RNP to a first subsequence in the target polynucleotide and hybridizing the second Cas-gRNA RNP to a second subsequence in the target polynucleotide while promoting the activity of the first and second Cas-gRNA RNPs and inhibiting the activity of the first and second transposases. The method can then include adding a first amplification adapter to a first position in the target polynucleotide using a first transposase, and adding a second amplification adapter to a second position in the target polynucleotide using a second transposase, while promoting activity of the first and second transposases.

[0100] In some examples, the first condition of the fluid is used to promote the activity of the Cas-gRNA RNP and inhibit the activity of the transposase. In some examples, the first condition of the fluid includes the presence of calcium ions, manganese ions, or both calcium ions and manganese ions in an amount sufficient for the activity of the Cas-gRNA RNP. In some examples, the first condition of the fluid includes the absence of magnesium ions in an amount sufficient for the activity of the transposase.

[0101] In some examples, the second fluid condition is used to promote activity of the transposase. In some examples, the second fluid condition comprises the presence of magnesium ions in an amount sufficient for activity of the transposase.

[0102] In some examples, the method further comprises degrading any portion of the target polynucleotide that is not between the Cas-gRNA RNP of the first fusion protein and the Cas-gRNA RNP of the second fusion protein while the Cas-gRNA RNP of the first fusion protein hybridizes to the first subsequence and the Cas-gRNA RNP of the second fusion protein hybridizes to the second subsequence. In some examples, the degradation is performed using Exonuclease III or Exonuclease VII.

[0103] In some examples, the method further includes releasing the target polynucleotide from the first and second fusion proteins to provide a fragment of the target polynucleotide having a first amplification adaptor at one end and a second amplification adaptor at the other end, hi some examples, the releasing is performed using proteinase K, sodium dodecyl sulfate (SDS), or both proteinase K and SDS.

[0104] In some examples, the fragment has a length of about 100 base pairs to about 1000 base pairs. In some examples, the fragment has a length of about 500 base pairs to about 700 base pairs. In some examples, the fragment has a length of about 200 base pairs to about 400 base pairs. In some examples, the fragment has a length of about 100 base pairs to about 200 base pairs.

[0105] In some examples, the Cas comprises dCas9. In some examples, the transposase comprises Tn5.

[0106] In some examples, the first amplification adaptor comprises a P5 adaptor and the second amplification adaptor comprises a P7 adaptor.

[0107] In some examples, the first amplification adaptor comprises a first unique molecular identifier (UMI) and the second amplification adaptor comprises a second UMI.

[0108] In some instances, the first position is within about 10 bases of the first subsequence and the second position is within about 10 bases of the second subsequence.

[0109] In some examples, in each of the first and second fusion proteins, the Cas-gRNA RNP is coupled to the transposase via a covalent bond.

[0110] In some examples, in each of the first and second fusion proteins, the Cas-gRNA RNP is non-covalently coupled to the transposase. In some examples, the Cas-gRNA RNP is covalently coupled to an antibody and the transposase is covalently coupled to an antigen to which the antibody is non-covalently coupled, or the Cas-gRNA RNP is covalently coupled to an antigen and the transposase is covalently coupled to an antibody to which the antigen is non-covalently coupled. In some examples, the Cas-gRNA is non-covalently coupled to the transposase via hybridization between the gRNA and the first or second amplification adapter. In some examples, the Cas-gRNA is non-covalently coupled to the transposase via hybridization between the gRNA and an oligonucleotide within the transposase.

[0111] In some examples, in the first fusion protein, the portion of the gRNA that hybridizes to the first partial sequence has a length of about 15 to about 18 nucleotides, and in the second fusion protein, the portion of the gRNA that hybridizes to the second partial sequence has a length of about 15 to about 18 nucleotides.

[0112] In some instances, the first and second fusion proteins are in approximately stoichiometric ratio to the target polynucleotide.

[0113] In some instances, the target polynucleotide comprises double-stranded DNA.

[0114] In some examples, the first tag is coupled to the first Cas-gRNA RNP and the second tag is coupled to the second Cas-gRNA RNP. In some examples, the method includes coupling the first tag to a first tag partner coupled to the substrate and coupling the second tag to a second tag partner coupled to the substrate. In some examples, the coupling is performed after the first and second Cas-gRNA RNPs are hybridized to the first and second subsequences, respectively. In some examples, the first and amplification adaptors are added after the first and second tags are added to the first and second tag partners, respectively.

[0115] In some examples, the first and second tags comprise biotin. In some examples, the first and second tag partners comprise streptavidin. In some examples, the substrate comprises beads. In some examples, the Cas-gRNA RNP comprises Cas12k. In some examples, the transposase comprises a Tn5 or Tn7-like transposase.

[0116] Some examples herein provide a method for sequencing a target polynucleotide.The method may include: generating a fragment of the target polynucleotide using one of the above-mentioned methods; generating an amplicon of the fragment; and sequencing the amplicon.

[0117] Some examples herein provide a composition. The composition may include a target polynucleotide having a sequence. The composition may include a first fusion protein comprising a first CRISPR-associated protein guide RNA ribonucleoprotein (Cas-gRNA RNP) coupled to a first transposase to which a first amplification adaptor is coupled. The first Cas-gRNA RNP may hybridize to a first subsequence in the target polynucleotide.

[0118] In some examples, the composition may include a second fusion protein comprising a second Cas-gRNA RNP coupled to a second transposase to which a second amplification adaptor is coupled. The second Cas-gRNA RNP may hybridize to a second subsequence in the target polynucleotide.

[0119] In some examples, the composition further comprises a fluid having conditions that promote the activity of the first Cas-gRNA RNP and inhibit the activity of the first transposase. In some examples, the fluid conditions include the presence of calcium ions, manganese ions, or both calcium ions and manganese ions in an amount sufficient for the activity of the first Cas-gRNA RNP. In some examples, the fluid conditions include the absence of magnesium ions in an amount sufficient for the activity of the first transposase.

[0120] In some examples, the composition further comprises a fluid having conditions that promote activity of a first transposase, where the first transposase adds a first amplification adapter to a first position in the target polynucleotide. In some examples, the fluid conditions include the presence of magnesium ions in an amount sufficient for activity of the first transposase.

[0121] In some examples, the composition further comprises an agent for releasing the target polynucleotide from the first and second fusion proteins to provide a fragment of the target polynucleotide having a first amplification adaptor at one end and a second amplification adaptor at the other end, hi some examples, the agent comprises proteinase K, sodium dodecyl sulfate (SDS), or both proteinase K and SDS.

[0122] In some examples, the fragment has a length of about 100 base pairs to about 1000 base pairs. In some examples, the fragment has a length of about 500 base pairs to about 700 base pairs. In some examples, the fragment has a length of about 200 base pairs to about 400 base pairs. In some examples, the fragment has a length of about 100 base pairs to about 200 base pairs.

[0123] In some examples, the composition further comprises an exonuclease to degrade any portion of the target polynucleotide that is not between the first and second Cas-gRNA RNPs. In some examples, the exonuclease comprises Exonuclease III or Exonuclease VII.

[0124] In some examples, the Cas comprises dCas9. In some examples, the transposase comprises Tn5.

[0125] In some examples, the first adaptor comprises a P5 adaptor and the second adaptor comprises a P7 adaptor.

[0126] In some examples, the first amplification adaptor comprises a first unique molecular identifier (UMI) and the second amplification adaptor comprises a second UMI.

[0127] In some instances, the first position is within about 10 bases of the first subsequence and the second position is within about 10 bases of the second subsequence.

[0128] In some examples, the first Cas-gRNA RNP is coupled to the first transposase via a covalent bond.

[0129] In some examples, the first Cas-gRNA RNP is non-covalently coupled to the first transposase. In some examples, the first Cas-gRNA RNP is covalently coupled to an antibody and the first transposase is covalently coupled to an antigen to which the antibody is non-covalently coupled, or the Cas-gRNA RNP is covalently coupled to an antigen and the first transposase is covalently coupled to an antibody to which the antigen is non-covalently coupled. In some examples, the first Cas-gRNA is non-covalently coupled to the transposase via hybridization between the gRNA and the first or second amplification adapter. In some examples, the first Cas-gRNA is non-covalently coupled to the transposase via hybridization between the gRNA and an oligonucleotide within the transposase.

[0130] In some examples, in a first fusion protein, the portion of the gRNA that hybridizes to the first partial sequence has a length of about 15 to about 18 nucleotides. In examples involving a second fusion protein, the portion of the gRNA that hybridizes to the second partial sequence has a length of about 15 to about 18 nucleotides.

[0131] In some instances, the first fusion protein is in approximately stoichiometric ratio to the target polynucleotide.

[0132] In some instances, the target polynucleotide comprises double-stranded DNA.

[0133] Some examples further include a first tag coupled to the first Cas-gRNA RNP. Some examples further include a substrate and a first tag partner coupled to the substrate and the first tag.

[0134] Some examples further include a second tag coupled to the second Cas-gRNA RNP.Some examples further include a substrate, a first tag partner coupled to the substrate and the first tag, and a second tag partner coupled to the substrate and the second tag.

[0135] In some examples, the first and second tags comprise biotin. In some examples, the first and second tag partners comprise streptavidin. In some examples, the substrate comprises beads. In some examples, the Cas-gRNA RNP comprises Cas12k. In some examples, the transposase comprises a Tn5 or Tn7-like transposase.

[0136] Some examples herein provide a method for characterizing a protein coupled to each locus of a target polynucleotide. The method may include contacting the target polynucleotide with a first and a second CRISPR-associated protein guide RNA ribonucleoprotein (Cas-gRNA RNP). The method may include hybridizing the first and the second Cas-gRNA RNP to a first and a second subsequence in the target polynucleotide, respectively, and the protein may be coupled to each locus of the target polynucleotide between the first and the second subsequence. The method may include cleaving the target polynucleotide at the first subsequence using the first Cas-gRNA RNP and cleaving the target polynucleotide at the second subsequence using the second Cas-gRNA RNP to form fragments. The protein may be coupled to each locus of the fragments. The method may include labeling each of the proteins coupled to each locus of the fragments using a corresponding oligonucleotide. The method may include sequencing the corresponding oligonucleotide.

[0137] In some examples, the method includes enriching the fragments before labeling each of the proteins coupled to the respective loci of the fragments using corresponding oligonucleotides. In some examples, the first and second Cas-gRNA RNPs are each coupled to a tag such that the fragments are coupled to the tag via the first and second Cas-gRNA RNPs. Enriching may include contacting the fragments coupled to the tag via the first and second Cas-gRNA RNPs with a substrate coupled to a tag partner. Enriching may include coupling the fragments to the substrate by coupling the tag to the tag partner. Enriching may include removing any portion of the target polynucleotide that is not coupled to the substrate.

[0138] In some examples, the methods include identifying the protein using a corresponding oligonucleotide.

[0139] In some examples, the method includes identifying the locus using a corresponding oligonucleotide.

[0140] In some examples, the method includes quantifying the protein using a corresponding oligonucleotide.

[0141] In some examples, labeling each of the proteins with a corresponding oligonucleotide respectively includes contacting the fragments with a mixture of antibodies specific for different proteins. Each of the antibodies can be coupled to a corresponding oligonucleotide. For any antibodies in the mixture that are specific for a protein coupled to each locus of the fragment, those antibodies and corresponding oligonucleotides can be coupled to those proteins. In some examples, multiple proteins are coupled to each one of the loci and multiple antibodies in the mixture are coupled to a protein at that locus.

[0142] In some examples, sequencing the corresponding oligonucleotides comprises hybridizing the corresponding oligonucleotides to a bead array. In some examples, sequencing the corresponding oligonucleotides comprises performing sequencing by synthesis on the corresponding oligonucleotides.

[0143] In some instances, the corresponding oligonucleotide comprises a unique molecular identifier (UMI).

[0144] In some examples, the method includes identifying the protein using the presence of each of the corresponding oligonucleotides.

[0145] In some examples, the method includes quantifying the protein using the amount of each of the corresponding oligonucleotides.

[0146] In some examples, labeling each of the proteins with a corresponding oligonucleotide comprises contacting the fragment with a plurality of transposases, each of which is coupled to a corresponding oligonucleotide, inhibiting activity of the transposase at the locus with the proteins coupled to each locus of the fragment, and adding, at a location other than the locus, the oligonucleotide corresponding to the fragment using the transposase.

[0147] In some instances, sequencing the corresponding oligonucleotides includes performing sequencing by synthesis on the fragment to which the corresponding oligonucleotides are attached.

[0148] In some instances, each position within the corresponding oligonucleotide fragment is used to identify each locus of the protein.

[0149] In some instances, the transposase splits the fragment into subfragments and sequencing by synthesis is performed on the subfragments.

[0150] In some examples, the corresponding oligonucleotides comprise amplification adaptors. In some examples, the amplification adaptors comprise P5 and P7 adaptors.

[0151] In some examples, the amplification adapter comprises a unique molecular identifier (UMI).

[0152] In some examples, the Cas comprises Cas9.

[0153] In some examples, the fragment has a length of about 100 base pairs to about 1000 base pairs. In some examples, the fragment has a length of about 500 base pairs to about 700 base pairs. In some examples, the fragment has a length of about 200 base pairs to about 400 base pairs. In some examples, the fragment has a length of about 100 base pairs to about 200 base pairs.

[0154] In some instances, the target polynucleotide comprises double-stranded DNA.

[0155] Some examples herein provide compositions. The compositions may include fragments of a target polynucleotide. Proteins may be coupled to the loci of each of the fragments. The compositions may include a mixture of antibodies specific for different proteins. Each of the antibodies may be coupled to a corresponding oligonucleotide. For any antibodies in the mixture that are specific for proteins coupled to the loci of each of the fragments, those antibodies and corresponding oligonucleotides are coupled to those proteins.

[0156] In some instances, multiple proteins are coupled to each one of the loci and multiple antibodies in the mixture are coupled to the protein at that locus.

[0157] In some instances, the corresponding oligonucleotide comprises a unique molecular identifier (UMI).

[0158] In some instances, the presence of each of the corresponding oligonucleotides can be used to identify the protein.

[0159] In some instances, the amount of each of the corresponding oligonucleotides can be used to quantify the protein.

[0160] In some examples, the fragment has a length of about 100 base pairs to about 1000 base pairs. In some examples, the fragment has a length of about 500 base pairs to about 700 base pairs. In some examples, the fragment has a length of about 200 base pairs to about 400 base pairs. In some examples, the fragment has a length of about 100 base pairs to about 200 base pairs.

[0161] In some instances, the target polynucleotide comprises double-stranded DNA.

[0162] Some examples herein provide compositions. The compositions may include fragments of a target polynucleotide. A protein may be coupled to each locus of the fragment. The compositions may include multiple transposases. Each of the transposases may be coupled to a corresponding oligonucleotide. The protein coupled to each locus of the fragment may inhibit the activity of the transposase at that locus. The transposase may add an oligonucleotide corresponding to the fragment at a position other than the locus.

[0163] In some instances, each position within the corresponding oligonucleotide fragment can be used to identify each locus of the protein.

[0164] In some instances, the transposase splits the fragment into subfragments.

[0165] In some examples, the corresponding oligonucleotide comprises an amplification adaptor. In some examples, the amplification adaptor comprises a P5 and a P7 adaptor. In some examples, the amplification adaptor comprises a unique molecular identifier (UMI).

[0166] In some examples, the transposase comprises Tn5.

[0167] In some examples, the fragment has a length of about 100 base pairs to about 1000 base pairs. In some examples, the fragment has a length of about 500 base pairs to about 700 base pairs. In some examples, the fragment has a length of about 200 base pairs to about 400 base pairs. In some examples, the fragment has a length of about 100 base pairs to about 200 base pairs.

[0168] In some instances, the target polynucleotide comprises double-stranded DNA.

[0169] Some examples herein provide a composition comprising a target polynucleotide having a plurality of subsequences.The composition can comprise a plurality of complexes, each of which comprises ShCAST (Scytonema hofmanni CRISPR-associated transposase) coupled to a guide RNA (gRNA).The ShCAST can have an amplification adaptor coupled thereto.Each of the complexes can be hybridized to a corresponding one of the subsequences in the target polynucleotide.

[0170] In some embodiments, the composition further comprises a fluid having conditions that promote hybridization of the complex to the subsequence and inhibit activity of the transposase, hi some examples, the fluid conditions include the absence of magnesium ions in an amount sufficient for activity of the transposase.

[0171] In some examples, the composition further comprises a fluid having conditions that promote activity of a transposase, where the transposase adds an amplification adapter to a location in the target polynucleotide, hi some examples, the fluid conditions include the presence of magnesium ions in an amount sufficient for activity of the transposase.

[0172] In some examples, the ShCAST comprises Cas12k. In some examples, the transposase comprises a Tn5 or Tn7-like transposase. In some embodiments, the adaptor comprises at least one of a P5 adaptor and a P7 adaptor. In some examples, the target polynucleotide comprises double-stranded DNA.

[0173] In some examples, at least one of the gRNA and the transposase is biotinylated. The composition may further comprise streptavidin-coated beads to which the at least one of the biotinylated gRNA and the transposase is coupled.

[0174] Some examples herein provide a method of generating fragments of a double-stranded polynucleotide. The method may include coupling the double-stranded polynucleotide to a substrate. The method may include hybridizing a first and a second CRISPR-associated protein guide RNA ribonucleoprotein (Cas-gRNA RNP) nickase to a first and a second subsequence in the double-stranded polynucleotide, respectively. The first subsequence may be 3' to the target sequence along the first strand of the double-stranded polynucleotide. The second subsequence may be 3' to the target sequence along the second strand of the double-stranded polynucleotide. The method may include cleaving the first strand at the first subsequence using the first Cas-gRNA RNP nickase. The method may include cleaving the second strand at the second subsequence using the second Cas-gRNA RNP nickase. The method may include extending the first and second strands from their respective cuts using a polymerase and eluting the target sequence from the substrate. The method may include sequencing the eluted target sequences.

[0175] In some examples, the substrate comprises beads, for example paramagnetic beads.

[0176] In some examples, the 3' end of the double-stranded polynucleotide is coupled to a tag, the substrate is coupled to a tag partner, and the coupling comprises coupling the tag to the tag partner. In some examples, the tag comprises biotin and the tag partner comprises streptavidin.

[0177] In some examples, the first and second Cas-gRNA RNP nickase comprise Cas9.

[0178] In some examples, the polymerase comprises a strand displacing polymerase, hi some examples, the polymerase comprises Vent or Bsu.

[0179] In some examples, the polymerase has 5' exonuclease activity. In some examples, the polymerase comprises Taq, Bst, or DNA polymerase I.

[0180] Some examples provide a composition. The composition may include a double-stranded polynucleotide coupled to a substrate. The composition may include a first and a second CRISPR-associated protein guide RNA ribonucleoprotein (Cas-gRNA RNP) nickase hybridized to a first and a second subsequence in the double-stranded polynucleotide, respectively. The first subsequence may be 3' of the target sequence along the first strand of the double-stranded polynucleotide. The second subsequence may be 3' of the target sequence along the second strand of the double-stranded polynucleotide.

[0181] In some examples, the substrate comprises beads, for example paramagnetic beads.

[0182] In some examples, the 3' end of the double-stranded polynucleotide is coupled to a tag and the substrate is coupled to a tag partner that is coupled to the tag. In some examples, the tag comprises biotin and the tag partner comprises streptavidin.

[0183] In some examples, the first and second Cas-gRNA RNP nickase comprise Cas9.

[0184] Some examples provide a method of generating fragments of a double-stranded polynucleotide. The method may include hybridizing a first and a second complex to a first and a second subsequence in the double-stranded polynucleotide, respectively. Each of the first and the second complex may include a CRISPR-associated protein guide RNA ribonucleoprotein (Cas-gRNA RNP) coupled to an amplification adaptor. The method may include ligating the amplification adaptor of the hybridized first and the second complex to a first and a second end of the double-stranded polynucleotide, respectively. The method may include removing the Cas-gRNA RNP of the first and the second complex from the double-stranded polynucleotide. The method may include sequencing the double-stranded polynucleotide to which the amplification adaptor is ligated.

[0185] In some examples, the first subsequence is 3' to the target sequence along the first strand of the double-stranded polynucleotide, and the second subsequence is 3' to the target sequence along the second strand of the double-stranded polynucleotide.

[0186] In some instances, the amplification adapter is Y-shaped.

[0187] In some examples, each complex further comprises a linker that couples the Cas-gRNA RNP to an amplification adaptor. In some examples, the linker is coupled to the Cas of the Cas-gRNA RNP. In some examples, the linker is coupled to the gRNA. In some examples, the linker comprises a protein, a polynucleotide, or a polymer. In some examples, the linker remains coupled to the amplification adaptor even when the Cas-gRNA RNP is removed.

[0188] In some examples, ligation includes using a ligase. In some examples, the ligase is present during hybridization. In some examples, the ligase is inactive during hybridization and is activated for ligation using ATP. In some examples, the ligase is added after hybridization.

[0189] In some examples, the method includes A-tailing the double stranded polynucleotide prior to hybridization, and the amplification adaptor includes an unpaired T for hybridizing to the A-tail. Alternatively, the amplification adaptor may be ligated to a blunt end.

[0190] In some examples, the amplification adaptor comprises a unique molecular identifier. For example, the amplification adaptor can comprise a duplex unique molecular identifier.

[0191] In some examples, the Cas-gRNA RNP comprises dCas9.

[0192] Some examples provide a composition. The composition may comprise a fragment of a double-stranded polynucleotide. The composition may comprise a first and a second complex hybridized to a first and a second subsequence in the double-stranded polynucleotide. Each of the first and the second complex may comprise a CRISPR-associated protein guide RNA ribonucleoprotein (Cas-gRNA RNP) coupled to an amplification adaptor.

[0193] In some examples, the first subsequence is 3' to the target sequence along the first strand of the double-stranded polynucleotide, and the second subsequence is 3' to the target sequence along the second strand of the double-stranded polynucleotide.

[0194] In some instances, the amplification adapter is Y-shaped.

[0195] In some examples, each complex further comprises a linker that couples the Cas-gRNA RNP to an amplification adaptor. In some examples, the linker is coupled to the Cas of the Cas-gRNA RNP. In some examples, the linker is coupled to the gRNA. In some examples, the linker comprises a protein, a polynucleotide, or a polymer.

[0196] In some examples, the double-stranded polynucleotide comprises an A-tail and the amplification adaptor comprises an unpaired T to hybridize with the A-tail. Alternatively, the amplification adaptor may be ligated to a blunt end.

[0197] In some examples, the amplification adaptor comprises a unique molecular identifier. For example, the amplification adaptor can comprise a duplex unique molecular identifier.

[0198] In some examples, the Cas-gRNA RNP comprises dCas9.

[0199] Some examples herein provide a method of generating fragments of a polynucleotide. The method may include hybridizing a first CRISPR-associated protein guide RNA ribonucleoprotein (Cas-gRNA RNP) to a first sequence in the polynucleotide. The method may include hybridizing a second Cas-gRNA RNP to a second sequence in the polynucleotide that is spaced from the first sequence by at least a target sequence. The method may include cleaving the first and second sequences with the first and second Cas-gRNA RNPs to generate fragments that include a first and second end and a target sequence therebetween. The first end may have a first 5' overhang of at least one base. The second end may have a second 5' overhang of at least one base.

[0200] In some examples, the first and second 5' overhangs are each about 2-5 bases in length. In some examples, the first and second 5' overhangs are each about 5 bases in length.

[0201] In some examples, the first and second 5' overhangs have different sequences from each other.

[0202] Some examples further include ligating a first amplification adaptor to a first end of the fragment and ligating a second amplification adaptor to a second end of the fragment. The first amplification adaptor may have a third 5' overhang that is complementary to the first 5' overhang. The second amplification adaptor may have a fourth 5' overhang that is complementary to the second 5' overhang. The third and fourth 5' overhangs may have different sequences from each other. Some examples further include generating an amplicon of the fragment to which the first and second amplification adaptors are ligated, sequencing the amplicon, and identifying the target polynucleotide based on the sequencing. In some examples, the amplification adaptor includes a unique molecular identifier (UMI).

[0203] In some examples, the Cas comprises Cas12a.

[0204] Some examples herein provide a composition. The composition may include a polynucleotide. The composition may include a first CRISPR-associated protein guide RNA ribonucleoprotein (Cas-gRNA RNP) hybridized to a first sequence in the polynucleotide. The composition may include a second Cas-gRNA RNP hybridized to a second sequence in the polynucleotide that is spaced from the first sequence by at least the target sequence. The first and second Cas-gRNA RNPs may be for cleaving the first and second sequences of the polynucleotide, respectively, to generate fragments having first and second ends with the target sequence between them. The first end may have a first 5' overhang of at least one base. The second end may have a second 5' overhang of at least one base.

[0205] In some examples, the first and second 5' overhangs are each about 2-5 bases in length. In some examples, the first and second 5' overhangs are each about 5 bases in length.

[0206] In some examples, the first and second 5' overhangs have different sequences from each other.

[0207] In some examples, the Cas comprises Cas12a.

[0208] Some examples herein provide a composition. The composition may include a polynucleotide fragment having a first and a second end, each end having a target sequence therebetween. The first end may have a first 5' overhang of at least one base. The second end may have a second 5' overhang of at least one base. The first and second 5' overhangs may have different sequences from each other. The composition may also include a first amplification adaptor having a third 5' overhang that is complementary to the first 5' overhang and is not complementary to the second 5' overhang. The composition may also include a second amplification adaptor having a fourth 5' overhang that is complementary to the second 5' overhang and is not complementary to the first 5' overhang.

[0209] Some examples further include at least one ligase for ligating a first amplification adaptor to the first end and a second amplification adaptor to the second end.

[0210] In some examples, the first and second 5' overhangs are each about 2-5 bases in length. In some examples, the first and second 5' overhangs are each about 5 bases in length.

[0211] In some examples, the first and second amplification adapters comprise a unique molecular identifier (UMI).

[0212] In some examples, the ligase comprises T4 DNA ligase.

[0213] Some examples herein provide a plurality of polynucleotide fragments each having a first and second end with a target sequence therebetween. The first end can have a first 5' overhang of at least one base. The second end can have a second 5' overhang of at least one base. The first and second 5' overhangs can have different sequences from each other and from the first and second 5' overhangs of other fragments.

[0214] Some examples further include a plurality of first amplification adaptors. Each of the first amplification adaptors may have a third 5' overhang that is complementary to the first 5' overhang of the corresponding fragment and is not complementary to the second 5' overhang of the fragment and is not complementary to the first or second 5' overhang of the other fragment. Some examples herein further include a plurality of second amplification adaptors. Each of the second amplification adaptors may have a fourth 5' overhang that is complementary to the second 5' overhang of the corresponding fragment and is not complementary to the first 5' overhang of the fragment and is not complementary to the first or second 5' overhang of the other fragment.

[0215] Some examples further include a ligase for ligating a first amplification adaptor to a first end where the first and third 5' overhangs are complementary and for ligating a second amplification adaptor to a second end where the second and fourth 5' overhangs are complementary, hi some examples, the ligase comprises T4 DNA ligase.

[0216] In some examples, the first and second amplification adapters comprise a unique molecular identifier (UMI). In some examples, the first and second 5' overhangs are each about 2-5 bases in length. In some examples, the first and second 5' overhangs are each about 5 bases in length.

[0217] Some examples herein provide compositions. The compositions may include a plurality of polynucleotides. The compositions may include a plurality of first CRISPR-associated protein guide RNA ribonucleoproteins (Cas-gRNA RNPs) hybridized to respective first sequences in the polynucleotide. The compositions may include a plurality of second Cas-gRNA RNPs hybridized to respective second sequences in the polynucleotide that are spaced from the respective first sequences by at least the respective target sequences. The first and second plurality of Cas-gRNA RNPs may each be for cleaving the first and second sequences of the respective polynucleotides to generate fragments having respective first and second ends with the respective target sequences therebetween. The first end may have a first 5' overhang of at least one base. The second end may have a second 5' overhang of at least one base.

[0218] In some examples, the first and second 5' overhangs are each about 2-5 bases in length. In some examples, the first and second 5' overhangs are each about 5 bases in length.

[0219] In some examples, the first and second 5' overhangs have different sequences from each other.

[0220] In some examples, the Cas comprises Cas12a.

[0221] Some examples herein provide guide RNAs. The guide RNA may include a primer binding site, an amplification adapter site, and a CRISPR protospacer.

[0222] In some examples, the primer binding site is approximately complementary to at least a portion of the CRISPR protospacer.

[0223] In some examples, the amplification adapter site is located between the primer binding site and the CRISPR protospacer.

[0224] In some examples, the guide RNA comprises at least one loop. In some examples, the first loop is located between the amplification adaptor site and the CRISPR protospacer. In some examples, the second loop is located between the amplification adaptor site and the CRISPR protospacer.

[0225] Some examples herein provide CRISPR-associated protein guide RNA ribonucleoprotein (Cas-gRNA RNP). The Cas-gRNA RNP can include any one of the above-mentioned gRNAs and a Cas protein that binds to the CRISPR protospacer.

[0226] In some instances, the Cas protein is configured to perform double-stranded polynucleotide cleavage, hi some instances, the Cas protein comprises Cas9, Cas 12a, or Cas12f.

[0227] In some instances, the primer binding site and the amplification adapter site extend outside the Cas protein.

[0228] Some examples herein provide a complex. The complex may include a polynucleotide comprising a first and a second strand. The complex may include a first CRISPR-associated protein guide RNA ribonucleoprotein (Cas-gRNA RNP). The first Cas-gRNA RNP may include a first guide RNA comprising a first primer binding site, a first amplification adaptor site, and a first CRISPR protospacer, and a first Cas protein that binds to the first CRISPR protospacer. The first CRISPR protospacer may hybridize to the first strand, and the first primer binding site may hybridize to the second strand.

[0229] In some examples, the first and second strands are cleaved by the first Cas-gRNA RNP at respective positions based on the sequence of the first CRISPR protospacer. In some examples, the first Cas protein comprises Cas9, Cas 12a, or Cas12f.

[0230] In some examples, the complex further comprises a first reverse transcriptase for generating an amplified adapter site amplicon at the second strand break caused by the first Cas protein. In some examples, the first reverse transcriptase is coupled to the first Cas protein. In some examples, the first reverse transcriptase and the first Cas protein are components of a first fusion protein.

[0231] In some examples, the first primer binding site is approximately complementary to at least a portion of the first CRISPR protospacer.

[0232] In some examples, the first amplification adapter site is located between the first primer binding site and the first CRISPR protospacer.

[0233] In some examples, the first gRNA further comprises at least one loop. In some examples, the first loop is located between the first amplification adaptor site and the first CRISPR protospacer. In some examples, the second loop is located between the first amplification adaptor site and the first CRISPR protospacer.

[0234] Some examples further include a second Cas-gRNA RNP. The second Cas-gRNA RNP may include a second guide RNA that includes a second primer binding site, a second amplification adapter site, and a second CRISPR protospacer. The second Cas-gRNA RNP may include a second Cas protein that binds to the second CRISPR protospacer. The second CRISPR protospacer may hybridize to the first strand, and the second primer binding site may hybridize to the second strand.

[0235] In some examples, the first and second strands are cleaved by the second Cas-gRNA RNP at their respective positions based on the sequence of the second CRISPR protospacer. In some examples, the cleavage of the first and second strands by the second Cas-gRNA RNP is spaced apart from the cleavage of the first and second strands by the first Cas-gRNA RNP by at least the target sequence. In some examples, the second Cas protein comprises Cas9, Cas 12a, or Cas12f.

[0236] In some examples, the complex further comprises a second reverse transcriptase to generate an amplified adapter site amplicon at the second strand break caused by the second Cas protein. In some examples, the second reverse transcriptase is coupled to the second Cas protein. In some examples, the second reverse transcriptase and the second Cas protein are components of a second fusion protein.

[0237] In some examples, the second primer binding site is approximately complementary to at least a portion of the second CRISPR protospacer.

[0238] In some examples, the second amplification adapter site is located between the second primer binding site and the second CRISPR protospacer.

[0239] Some examples herein provide a partially double-stranded polynucleotide fragment. The fragment may comprise a first end including a first 3' overhang; a second end; and a target sequence located between the first and second ends.

[0240] In some examples, the first 3' overhang comprises a first amplification adaptor.

[0241] In some instances, the second end comprises a second 3' overhang.

[0242] In some examples, the second 3' overhang comprises a second amplification adaptor.

[0243] Some examples herein provide a method. The method may include contacting a first CRISPR-associated protein guide RNA ribonucleoprotein (Cas-gRNA RNP) with a polynucleotide comprising a first and a second strand. The first Cas-gRNA may include a first guide RNA comprising a first primer binding site, a first amplification adaptor site, and a first CRISPR protospacer, and a first Cas protein that binds to the first CRISPR protospacer. The method may include hybridizing the first CRISPR protospacer to the first strand. The method may include hybridizing the first primer binding site to the second strand.

[0244] In some examples, the method further comprises cleaving the first and second strands with a first Cas-gRNA RNP at respective positions based on the sequence of the first CRISPR protospacer. In some examples, the first Cas protein comprises Cas9, Cas 12a, or Cas12f.

[0245] In some examples, the method further includes using a first reverse transcriptase to generate an amplified adapter site amplicon at the second strand break caused by the first Cas protein. In some examples, the first reverse transcriptase is coupled to the first Cas protein. In some examples, the first reverse transcriptase and the first Cas protein are components of a first fusion protein.

[0246] In some examples, the first primer binding site is approximately complementary to at least a portion of the first CRISPR protospacer.

[0247] In some examples, the first amplification adapter site is located between the first primer binding site and the first CRISPR protospacer.

[0248] In some examples, the first gRNA further comprises at least one loop. In some examples, the first loop is located between the first amplification adaptor site and the first CRISPR protospacer. In some examples, the second loop is located between the first amplification adaptor site and the first CRISPR protospacer.

[0249] In some examples, the method further comprises contacting the polynucleotide with a second Cas-gRNA RNP. The second Cas-gRNA RNP may comprise a second guide RNA comprising a second primer binding site, a second amplification adaptor site, and a second CRISPR protospacer; and a second Cas protein that binds to the second CRISPR protospacer. The method may comprise hybridizing the second CRISPR protospacer to the first strand. The method may comprise hybridizing the second primer binding site to the second strand.

[0250] In some examples, the method may include cleaving the first and second strands by a second Cas-gRNA RNP at respective positions based on the sequence of the second CRISPR protospacer. In some examples, the cleavage of the first and second strands by the second Cas-gRNA RNP is spaced from the cleavage of the first and second strands by the first Cas-gRNA RNP by at least the target sequence. In some examples, the second Cas protein includes Cas9, Cas 12a, or Cas12f.

[0251] In some examples, the method may further include using a second reverse transcriptase to generate an amplified adapter site amplicon at the second strand break caused by the second Cas protein. In some examples, the second reverse transcriptase is coupled to the second Cas protein. In some examples, the second reverse transcriptase and the second Cas protein are components of a second fusion protein.

[0252] In some examples, the second primer binding site is approximately complementary to at least a portion of the second CRISPR protospacer.

[0253] In some examples, the second amplification adapter site is located between the second primer binding site and the second CRISPR protospacer.

[0254] In some examples, the first and second Cas-gRNA RNPs and the first and second reverse transcriptases generate a partial double-stranded polynucleotide fragment having a first end and a second end. The first end may include a first 3' overhang. The second end may include a second 3' overhang. The target sequence may be located between the first end and the second end. In some examples, the first 3' overhang includes an amplicon of the first amplification adaptor site. In some examples, the second 3' overhang includes an amplicon of the second amplification adaptor site. In some examples, the method further includes ligating a third amplification adaptor to the 5' group of the first end, ligating a fourth amplification adaptor to the 5' group of the second end, amplifying the fragment using the first, second, third, and fourth amplification adaptors, and sequencing the amplified fragment.

[0255] It should be understood that any respective feature / example of each of the aspects of the present disclosure described herein may be implemented together in any suitable combination, and any feature / example from any one or more of these aspects may be implemented together in any suitable combination with any of the features of the other aspects described herein, in order to achieve the benefits described herein. [Brief description of the drawings]

[0256] [Figure 1A] Schematic diagram of exemplary compositions and process flow operations for Cas-gRNA RNP-mediated dehosting. [Figure 1B]Schematic diagram of exemplary compositions and process flow operations for Cas-gRNA RNP-mediated dehosting. [Figure 1C] Schematic diagram of exemplary compositions and process flow operations for Cas-gRNA RNP-mediated dehosting. [Figure 1D] Schematic diagram of exemplary compositions and process flow operations for Cas-gRNA RNP-mediated dehosting. [Figure 1E] Schematic diagram of exemplary compositions and process flow operations for Cas-gRNA RNP-mediated dehosting. [Figure 1F] Schematic diagram of exemplary compositions and process flow operations for Cas-gRNA RNP-mediated dehosting. [Figure 1G] Schematic diagram of exemplary compositions and process flow operations for Cas-gRNA RNP-mediated dehosting. [Figure 1H] Schematic diagram of exemplary compositions and process flow operations for Cas-gRNA RNP-mediated dehosting. [Figure 1I] Schematic diagram of exemplary compositions and process flow operations for Cas-gRNA RNP-mediated dehosting. [Figure 1J] Schematic diagram of exemplary compositions and process flow operations for Cas-gRNA RNP-mediated dehosting. [Figure 1K] Schematic diagram of exemplary compositions and process flow operations for Cas-gRNA RNP-mediated dehosting.

[0257] [Figure 2A] 1 illustrates schematic diagrams of exemplary compositions and process flow operations for WG fragmentation into different defined fragment sizes. [Figure 2B] 1 illustrates schematic diagrams of exemplary compositions and process flow operations for WG fragmentation into different defined fragment sizes. [Figure 2C]1 illustrates schematic diagrams of exemplary compositions and process flow operations for WG fragmentation into different defined fragment sizes. [Figure 2D] 1 illustrates schematic diagrams of exemplary compositions and process flow operations for WG fragmentation into different defined fragment sizes. [Figure 2E] 1 illustrates schematic diagrams of exemplary compositions and process flow operations for WG fragmentation into different defined fragment sizes. [Figure 2F] 1 illustrates schematic diagrams of exemplary compositions and process flow operations for WG fragmentation into different defined fragment sizes. [Figure 2G] 1 illustrates schematic diagrams of exemplary compositions and process flow operations for WG fragmentation into different defined fragment sizes. [Figure 2H] 1 illustrates schematic diagrams of exemplary compositions and process flow operations for WG fragmentation into different defined fragment sizes. [Figure 2I] 1 illustrates schematic diagrams of exemplary compositions and process flow operations for WG fragmentation into different defined fragment sizes. [Figure 2J] 1 illustrates schematic diagrams of exemplary compositions and process flow operations for WG fragmentation into different defined fragment sizes. [Figure 2K] 1 illustrates schematic diagrams of exemplary compositions and process flow operations for WG fragmentation into different defined fragment sizes.

[0258] [Figure 3A] 1 illustrates generally exemplary compositions and operations in a process flow for labeling polynucleotides using cleavage moieties. [Figure 3B] 1 illustrates generally exemplary compositions and operations in a process flow for labeling polynucleotides using cleavage moieties. [Figure 3C] 1 illustrates generally exemplary compositions and operations in a process flow for labeling polynucleotides using cleavage moieties. [Figure 3D]1 illustrates generally exemplary compositions and operations in a process flow for labeling polynucleotides using cleavage moieties. [Figure 3E] 1 illustrates generally exemplary compositions and operations in a process flow for labeling polynucleotides using cleavage moieties.

[0259] [Figure 4A] 1A-1C generally depict exemplary compositions and operations in a process flow for coupling amplification adaptors to polynucleotides. [Figure 4B] 1A-1C generally depict exemplary compositions and operations in a process flow for coupling amplification adaptors to polynucleotides. [Figure 4C] 1A-1C generally depict exemplary compositions and operations in a process flow for coupling amplification adaptors to polynucleotides. [Figure 4D] 1A-1C generally depict exemplary compositions and operations in a process flow for coupling amplification adaptors to polynucleotides. [Figure 4E] 1A-1C generally depict exemplary compositions and operations in a process flow for coupling amplification adaptors to polynucleotides. [Figure 4F] 1A-1C generally depict exemplary compositions and operations in a process flow for coupling amplification adaptors to polynucleotides. [Figure 4G] 1A-1C generally depict exemplary compositions and operations in a process flow for coupling amplification adaptors to polynucleotides. [Figure 4H] 1A-1C generally depict exemplary compositions and operations in a process flow for coupling amplification adaptors to polynucleotides. [Figure 4I] 1A-1C generally depict exemplary compositions and operations in a process flow for coupling amplification adaptors to polynucleotides. [Figure 4J]1A-1C generally depict exemplary compositions and operations in a process flow for coupling amplification adaptors to polynucleotides.

[0260] [Figure 5A] 1 illustrates generally exemplary compositions and operations in a process flow for targeted epigenetic assays. [Figure 5B] 1 illustrates generally exemplary compositions and operations in a process flow for targeted epigenetic assays. [Figure 5C] 1 illustrates generally exemplary compositions and operations in a process flow for targeted epigenetic assays. [Figure 5D] 1 illustrates generally exemplary compositions and operations in a process flow for targeted epigenetic assays. [Figure 5E] 1 illustrates generally exemplary compositions and operations in a process flow for targeted epigenetic assays. [Figure 5F] 1 illustrates generally exemplary compositions and operations in a process flow for targeted epigenetic assays. [Figure 5G] 1 illustrates generally exemplary compositions and operations in a process flow for targeted epigenetic assays. [Figure 5H] 1 illustrates generally exemplary compositions and operations in a process flow for targeted epigenetic assays. [Figure 5I] 1 illustrates generally exemplary compositions and operations in a process flow for targeted epigenetic assays. [Figure 5J] 1 illustrates generally exemplary compositions and operations in a process flow for targeted epigenetic assays. [Figure 5K] 1 illustrates generally exemplary compositions and operations in a process flow for targeted epigenetic assays.

[0261] [Figure 6A]Schematic depiction of exemplary compositions and operations in a process flow for ShCAST (Scytonema hofmanni CRISPR-associated transposase) targeted library preparation and enrichment. [Figure 6B] Schematic depiction of exemplary compositions and operations in a process flow for ShCAST (Scytonema hofmanni CRISPR-associated transposase) targeted library preparation and enrichment.

[0262] [Figure 7A] 1A-1C generally depict exemplary compositions and operations in a process flow for coupling amplification adaptors to polynucleotides. [Figure 7B] 1A-1C generally depict exemplary compositions and operations in a process flow for coupling amplification adaptors to polynucleotides. [Figure 7C] 1A-1C generally depict exemplary compositions and operations in a process flow for coupling amplification adaptors to polynucleotides. [Figure 7D] 1A-1C generally depict exemplary compositions and operations in a process flow for coupling amplification adaptors to polynucleotides. [Figure 7E] 1A-1C generally depict exemplary compositions and operations in a process flow for coupling amplification adaptors to polynucleotides. [Figure 7F] 1A-1C generally depict exemplary compositions and operations in a process flow for coupling amplification adaptors to polynucleotides. [Figure 7G] 1A-1C generally depict exemplary compositions and operations in a process flow for coupling amplification adaptors to polynucleotides. [Figure 7H] 1A-1C generally depict exemplary compositions and operations in a process flow for coupling amplification adaptors to polynucleotides.

[0263] [Figure 8A]FIG. 13 shows schematic diagrams of exemplary compositions and process flow operations for enriching selected polynucleotide fragments using Cas-gRNA RNP nickase. [Figure 8B] FIG. 13 shows schematic diagrams of exemplary compositions and process flow operations for enriching selected polynucleotide fragments using Cas-gRNA RNP nickase. [Figure 8C] FIG. 13 shows schematic diagrams of exemplary compositions and process flow operations for enriching selected polynucleotide fragments using Cas-gRNA RNP nickase. [Figure 8D] FIG. 13 shows schematic diagrams of exemplary compositions and process flow operations for enriching selected polynucleotide fragments using Cas-gRNA RNP nickase. [Figure 8E] FIG. 13 shows schematic diagrams of exemplary compositions and process flow operations for enriching selected polynucleotide fragments using Cas-gRNA RNP nickase. [Figure 8F] FIG. 13 shows schematic diagrams of exemplary compositions and process flow operations for enriching selected polynucleotide fragments using Cas-gRNA RNP nickase. [Figure 8G] FIG. 13 shows schematic diagrams of exemplary compositions and process flow operations for enriching selected polynucleotide fragments using Cas-gRNA RNP nickase. [Figure 8H] FIG. 13 shows schematic diagrams of exemplary compositions and process flow operations for enriching selected polynucleotide fragments using Cas-gRNA RNP nickase.

[0264] [Figure 9A] 1A-1C show schematic diagrams of exemplary compositions and process flow operations previously known for ligating amplification adaptors to fragments of a dsDNA library.

[0265] [Figure 9B]Schematic diagram of an exemplary composition and process flow for ligating amplification adapters to selected polynucleotide fragments using Cas-gRNA RNPs. [Figure 9C] Schematic diagram of an exemplary composition and process flow for ligating amplification adapters to selected polynucleotide fragments using Cas-gRNA RNPs. [Figure 9D] Schematic diagram of an exemplary composition and process flow for ligating amplification adapters to selected polynucleotide fragments using Cas-gRNA RNPs. [Figure 9E] Schematic diagram of an exemplary composition and process flow for ligating amplification adapters to selected polynucleotide fragments using Cas-gRNA RNPs. [Figure 9F] Schematic diagram of an exemplary composition and process flow for ligating amplification adapters to selected polynucleotide fragments using Cas-gRNA RNPs.

[0266] [Figure 10A] Schematic diagram of an exemplary composition and process flow for generating fragments using Cas-gRNA RNPs and coupling adapters thereto. [Figure 10B] Schematic diagram of an exemplary composition and process flow for generating fragments using Cas-gRNA RNPs and coupling adapters thereto. [Figure 10C] Schematic diagram of an exemplary composition and process flow for generating fragments using Cas-gRNA RNPs and coupling adapters thereto.

[0267] [Figure 11A] Schematic diagram of an exemplary composition and process flow for generating fragments using Cas-gRNA RNPs and coupling adapters thereto. [Figure 11B] Schematic diagram of an exemplary composition and process flow for generating fragments using Cas-gRNA RNPs and coupling adapters thereto. [Figure 11C] Schematic diagram of an exemplary composition and process flow for generating fragments using Cas-gRNA RNPs and coupling adapters thereto. [Figure 11D] Schematic diagram of an exemplary composition and process flow for generating fragments using Cas-gRNA RNPs and coupling adapters thereto. [Figure 11E] Schematic diagram of an exemplary composition and process flow for generating fragments using Cas-gRNA RNPs and coupling adapters thereto. [Figure 11F] Schematic diagram of an exemplary composition and process flow for generating fragments using Cas-gRNA RNPs and coupling adapters thereto. [Figure 11G] Schematic diagram of an exemplary composition and process flow for generating fragments using Cas-gRNA RNPs and coupling adapters thereto.

[0268] [Figure 12] Schematic representation of target DNA fragments after tagmentation, arrest and TWB wash.

[0269] [Figure 13] Schematic representation of the target DNA fragment after gap filling and ligation with an ELM.

[0270] [Figure 14] Schematic of Cas9 nickase (D10A) cleaving opposite the PAM site.

[0271] [Figure 15] Schematic representation of target DNA containing a 3' nick.

[0272] [Figure 16] Schematic showing polymerase extension of the 3′ end resulting in elution of the target fragment.

[0273] [Figure 17] An example of an IGV trace showing enrichment of four lambda targets is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0274] Provided herein are genomic library preparation and targeted epigenetic assays using Cas-gRNA ribonucleoproteins (RNPs).

[0275] With respect to genomic library preparation, some examples herein relate to Cas-gRNA RNP-mediated dehosting. Some examples herein relate to fragmentation of the whole genome (WG) into different defined fragment sizes. Some examples herein relate to cleaving polynucleotides, and some examples herein relate to coupling polynucleotides to amplification adaptors. It will be understood that one or more aspects of any such examples relating to genomic library preparation can be used in combination with one or more aspects of any other such examples relating to genomic library preparation.

[0276] With regard to targeted epigenetic assays, some examples herein relate to using Cas-gRNA RNPs to enrich DNA regions (small or large) that retain epigenetic features (e.g., chromatin), which are then processed in epigenetic-NGS assays. This approach allows for ultra-deep epigenetic assays, improving the resolution of fine epigenetic changes (e.g., compared to ATAC-seq or ChIP-seq) and complex networks (e.g., locus-associated proteomics), which may facilitate a better understanding of epigenetic mechanisms that may be of interest in research or clinical development. It will be understood that one or more aspects of any such examples relating to targeted epigenetic assays may be used in combination with one or more aspects of any examples relating to genomic library preparation, and vice versa.

[0277] We first provide a brief description of some of the terms used herein, then we describe some exemplary compositions and exemplary methods for genomic library preparation and targeted epigenetic assays using Cas-RNPs.

[0278] term Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. The use of the term "including" and other forms such as "include", "includes" and "included" is not limiting. The use of the term "having" and other forms such as "have", "has" and "had" is not limiting. As used herein, whether in a transitional phrase or in the body of a claim, the terms "comprise" and "comprising" should be interpreted as having an open-ended meaning. That is, the above terms should be interpreted as synonymous with the phrase "having at least" or "comprising at least". For example, when used in the context of a process, the term "comprising" means that the process includes at least the recited steps, but may include additional steps. When used in the context of a compound, composition, or device, the term "comprising" means that the compound, composition, or device includes at least the recited features or components, but may include additional features or components.

[0279] As used herein, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0280] As used throughout this specification, the terms "substantially," "approximately," and "about" are used to describe and take into account small variations due to processing variations, etc. For example, they can refer to ±10% or less, such as ±5% or less, such as ±2% or less, such as ±1% or less, such as ±0.5% or less, such as ±0.2% or less, such as ±0.1% or less, such as ±0.05% or less.

[0281] As used herein, terms such as "hybridize" and "hybridization" are intended to mean non-covalent association of polynucleotides with each other along the length of the polynucleotides to form a double stranded "duplex", a triple stranded "triple strand", or a higher order structure. For example, two DNA polynucleotide strands may associate to form a duplex through complementary base pairing. Primary interactions between polynucleotide strands may typically be nucleotide base specific, e.g., A:T, A:U, and G:C. Base stacking and hydrophobic interactions may also contribute to duplex stability. Hybridization conditions may include salt concentrations of less than about 1 M, more usually less than about 500 mM, or less than about 200 mM. Hybridization buffers may include buffered salt solutions such as 5% SSPE, or other suitable buffers known in the art. Hybridization temperatures may be as low as 5°C, but are typically greater than 22°C, more typically greater than about 30°C, and typically greater than 37°C. The strength of the association between a first and a second polynucleotide increases with the complementarity between the nucleotide sequences within those polynucleotides. The strength of hybridization between polynucleotides can be characterized by the melting temperature (Tm) at which 50% of the polynucleotide strands of a duplex separate.

[0282] As used herein, the term "nucleotide" is intended to mean a molecule that includes a sugar and at least one phosphate group, and in some instances also includes a nucleobase. A nucleotide that lacks a nucleobase may be referred to as "abasic." Nucleotides include deoxyribonucleotides, modified deoxyribonucleotides, ribonucleotides, modified ribonucleotides, peptide nucleotides, modified peptide nucleotides, modified phosphate sugar backbone nucleotides, and mixtures thereof. Examples of nucleotides include adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (GT ... deoxyadenosine monophosphate (UTP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), and deoxythymidine monophosphate (DTMP).These include deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxycytidine diphosphate (dCDP), deoxycytidine triphosphate (dCTP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), and deoxyuridine triphosphate (dUTP).

[0283] As used herein, the term "nucleotide" is also intended to encompass any nucleotide analog, which is a type of nucleotide that contains a modified nucleobase, sugar, backbone and / or phosphate moiety compared to naturally occurring nucleotides. Nucleotide analogs may also be referred to as "modified nucleic acids." Exemplary modified nucleobases include inosine, xanthate, hypoxanthate, isocytosine, isoguanine, 2-aminopurine, 5-methylcytosine, 5-hydroxymethylcytosine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 2-propylguanine, 2-propyladenine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 15-halouracil, 15-halocytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil ... These include cytosine, 6-azothymine, 5-uracil, 4-thiouracil, 8-halo adenine or guanine, 8-amino adenine or guanine, 8-thiol adenine or guanine, 8-thioalkyl adenine or guanine, 8-hydroxyl adenine or guanine, 5-halo substituted uracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, and the like. As is known in the art, certain nucleotide analogs cannot become incorporated into polynucleotides, such as nucleotide analogs such as adenosine 5'-phosphosulfate. A nucleotide can include any suitable number of phosphates, such as 3, 4, 5, 6, or more than 6 phosphates. Nucleotide analogs also include locked nucleic acids (LNA), peptide nucleic acids (PNA), and 5-hydroxybutynyl-2'-deoxyuridine ("Super T").

[0284] As used herein, the term "polynucleotide" refers to a molecule that comprises a sequence of nucleotides that are linked together. A polynucleotide is a non-limiting example of a polymer. Examples of polynucleotides include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and their analogs, such as locked nucleic acid (LNA) and peptide nucleic acid (PNA). A polynucleotide can be a single-stranded sequence of nucleotides, such as RNA or single-stranded DNA, a double-stranded sequence of nucleotides, such as double-stranded DNA, or a mixture of single-stranded and double-stranded sequences of nucleotides. Double-stranded DNA (dsDNA) includes genomic DNA, and PCR and amplification products. Single-stranded DNA (ssDNA) can be converted to dsDNA and vice versa. A polynucleotide can include non-naturally occurring DNA, such as enantiomeric DNA, LNA, or PNA. The exact sequence of nucleotides in a polynucleotide can be known or unknown. The following are examples of polynucleotides: a gene or gene fragment (e.g., a probe, primer, expressed sequence tag (EST), or serial analysis of gene expression (SAGE) tag), genomic DNA, genomic DNA fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, synthetic polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, primers, or amplified copies of any of the foregoing.

[0285] As used herein, "polymerase" is intended to mean an enzyme having an active site that assembles a polynucleotide by polymerizing nucleotides into a polynucleotide. A polymerase can bind to a primed single-stranded target polynucleotide and grow it by successively adding nucleotides to the primer to form a "complementary copy" polynucleotide having a sequence complementary to that of the target polynucleotide. Another polymerase, or the same polymerase, can then form a copy of the target nucleotide by forming a complementary copy of the complementary copy polynucleotide. Any such copy may be referred to herein as an "amplicon." A DNA polymerase can bind to a target polynucleotide and then move downstream of the target polynucleotide while it grows it by successively adding nucleotides to the free hydroxyl group at the 3' end of the polynucleotide chain (amplicon growth). A DNA polymerase can synthesize a complementary DNA molecule from a DNA template, and an RNA polymerase can synthesize an RNA molecule from a DNA template (transcription). Polymerases can use short RNA or DNA strands (primers) to initiate strand growth. Some polymerases can displace the strand upstream of the site where they add a base to the strand. Such polymerases can also be said to be strand displacing, which means they have the activity of removing the complementary strand from the template strand that is read by the polymerase.

[0286] Examples of polymerases include Bst DNA polymerase, 9°Nm DNA polymerase, Phi29 DNA polymerase, DNA polymerase I (E. coli), DNA polymerase I (Large), (Klenow) fragment, Klenow fragment (3'-5' exo-), T4 DNA polymerase, T7 DNA polymerase, Deep VentR™ (exo-) DNA polymerase, Deep VentR™ DNA polymerase, DyNAzyme™ EXT DNA, DyNAzyme™ II Hot Start DNA polymerase, Phusion™ High-Fidelity DNA polymerase, Therminator™ DNA polymerase, Therminator™ II DNA polymerase, VentR™ DNA polymerase, VentR™ (exo-) DNA polymerase, RepliPHI™ Phi29 DNA polymerase, rBst Examples of suitable polymerases include DNA polymerase, rBst DNA polymerase (Large), Fragment (IsoTherm™ DNA polymerase), MasterAmp™ AmpliTherm™ DNA polymerase, Taq DNA polymerase, Tth DNA polymerase, Tfl DNA polymerase, Tgo DNA polymerase, SP6 DNA polymerase, Tbr DNA polymerase, DNA polymerase beta, and ThermoPhi DNA polymerase. In certain non-limiting examples, the polymerase is selected from the group consisting of Bst, Bsu, and Phi29. Since the polymerase extends the hybridized strand, it may be beneficial to include a single-stranded binding protein (SSB). The SSB may stabilize the displaced (non-template) strand. Exemplary polymerases with strand displacement activity include, but are not limited to, Vent polymerase, Bsu polymerase, Bst (Bacillus stearothermophilus) polymerase, exo-Klenow polymerase, or a large fragment of sequencing grade T7 exo-polymerase. Some polymerases cleave their preceding strand and effectively replace it with the growing strand (5' exonuclease activity).Examples of polymerases with 5' exonuclease activity include Taq, Bst, and DNA polymerase I. Some polymerases have the activity of degrading their trailing strand (3' exonuclease activity). Some useful polymerases have been mutated or otherwise modified to reduce or eliminate 3' and / or 5' exonuclease activity. Polymerases can include reverse transcriptase (RT). Non-limiting examples of RT include MMLV and its variants, such as those described in Anzalone et al., "Search-and-replace genome editing without double-strand breaks or donor DNA," Nature 576:149-157 (2019), the entire contents of which are incorporated herein by reference.

[0287] As used herein, the term "primer" is defined as a polynucleotide to which a nucleotide can be added via a free 3'OH group. A primer may include a 3' block that prevents polymerization until the block is removed. A primer may include a modification at the 5' end to allow a coupling reaction or to allow the primer to be coupled to another moiety. A primer may include one or more moieties, such as 8-oxo-G, that can be cleaved under appropriate conditions, such as UV light, chemicals, enzymes, etc. The length of a primer may be any suitable number of bases long and may include a suitable combination of natural and / or non-natural nucleotides. A target polynucleotide may include an "amplification adapter" or more simply an "adapter" that hybridizes to the primer (having a sequence complementary to the primer) and can be amplified to generate a complementary copy polynucleotide by adding a nucleotide to the free 3'OH group of the primer. A "capture primer" is intended to mean a primer that can bind to a substrate and hybridize to a first adapter of a target polynucleotide, and an "orthogonal capture primer" is intended to mean a primer that can bind to a substrate and hybridize to a second adapter of that target polynucleotide. The first adaptor may have a sequence complementary to that of the capture primer, and the second adaptor may have a sequence complementary to that of the orthogonal capture primer. The capture primer and the orthogonal capture primer may have independent sequences that are different from each other. Furthermore, the capture primer and the orthogonal capture primer may differ from each other in at least one other characteristic. For example, the capture primer and the orthogonal capture primer may have different lengths from each other, either the capture primer or the orthogonal capture primer may include a non-nucleic acid portion (such as a blocking group or a removal portion) that the other of the capture primer or the orthogonal capture primer does not have, or may include any suitable combination of such characteristics. The modified capture primer may further include a plurality of naturally occurring nucleic acids, such as, but not limited to, DNA.

[0288] In some examples, the capture primer is a P5 or P7 primer, commercially available from Illumina, Inc. P5 and P7 primers are non-limiting examples of primers that are orthogonal to each other. The P5 and P7 primer sequences, in some examples, can have the following sequences: Paired lead set: P5: 5'-AATGATACGGCGACCACCGAGAUCTACAC-3' (SEQ ID NO: 1) P7: 5'-CAAGCAGAAGACGGCATACGAG * AT-3' (SEQ ID NO:2) Single Lead Set: P5: 5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO: 3) P7: 5'-CAAGCAGAAGACGGCATACGA3' (SEQ ID NO: 4) In the formula, G * is G or 8-oxoguanine.

[0289] As used herein, the term "plurality" is intended to mean a population of two or more distinct members. A plurality can range in size from small, medium, large, to very large. A small size plurality can range, for example, from a few members to tens of members. A medium size plurality can range, for example, from tens of members to about 100 members or hundreds of members. A large plurality can range, for example, from about hundreds of members to about 1000 members, thousands of members, and tens of thousands of members. A very large plurality can range, for example, from tens of thousands of members to about hundreds of thousands, millions, tens of millions, or hundreds of millions or more members. Thus, a plurality can range in size from 2 to well over 100 million members, as well as between all sizes measured by number of members and larger than the exemplary ranges listed above. Exemplary polynucleotides include, for example, about 1×10 5 That's it, 5 x 10 5 or more or 1×10 6The term includes a population of more than one different polynucleotides. Thus, the definition of this term is intended to include all integer values ​​greater than 2. The upper limit of the multiple value can be set, for example, by the theoretical diversity of polynucleotide sequences in a sample.

[0290] As used herein, the term "double-stranded" when used in reference to a polynucleotide is intended to mean that all or substantially all of the nucleotides in a polynucleotide are hydrogen bonded to their respective nucleotides in a complementary polynucleotide. A double-stranded polynucleotide may also be referred to as a "duplex." As used herein, the term "single-stranded" when used in reference to a polynucleotide means that none of the nucleotides in a polynucleotide are hydrogen bonded to their respective nucleotides in a complementary polynucleotide.

[0291] As used herein, the term "target polynucleotide" is intended to mean a polynucleotide that is the subject of analysis or action, and may also be referred to using terms such as "library polynucleotide", "template polynucleotide", or "library template". Analysis or action may include subjecting a polynucleotide to capture, amplification, sequencing, and / or other procedures. A target polynucleotide may include additional nucleotide sequences to the target sequence being analyzed. For example, a target polynucleotide may include one or more adapters, including amplification adapters that function as primer binding sites, that flank the target polynucleotide sequence being analyzed. A target polynucleotide hybridized to a capture primer may include nucleotides that extend beyond the 5' or 3' end of the capture oligonucleotide such that not all of the target polynucleotide is suitable for extension. In certain instances, multiple target polynucleotides may have first and second adapters that are the same as each other, although they may have different sequences from each other. Two adapters that may flank a particular target polynucleotide sequence may have the same sequence as each other, or complementary sequences to each other, or the two adapters may have different sequences. Thus, a species in a plurality of target polynucleotides may include, for example, a region of known sequence flanked by regions of unknown sequence that are evaluated by sequencing (e.g., SBS). In some examples, the target polynucleotide carries an amplification adapter at a single end, and such an adapter may be located at either the 3' or 5' end of the target polynucleotide. The target polynucleotide may be used without an adapter, in which case the primer binding sequence may directly use a sequence present in the target polynucleotide.

[0292] The terms "polynucleotide" and "oligonucleotide" are used interchangeably herein. The difference in the terminology is not intended to indicate any particular difference in size, sequence, or other properties, unless otherwise specified. For clarity of explanation, when describing a particular method or composition that includes several polynucleotide species, different terms may be used to distinguish one species of polynucleotide from another species.

[0293] The terms "sequence" and "subsequence" may in some cases be used interchangeably herein. For example, a sequence may include one or more subsequences therein. Each such subsequence may also be referred to as a sequence.

[0294] As used herein, the term "amplicon" when used in reference to a polynucleotide refers to a product of the replication of a polynucleotide, which product has a nucleotide sequence that is substantially the same as or substantially complementary to at least a portion of the nucleotide sequence of the polynucleotide. "Amplification" and "amplifying" refer to the process of creating an amplicon of a polynucleotide. A first amplicon of a target polynucleotide can typically be a complementary copy. Additional amplicons are copies made from the target polynucleotide or the first amplicon after the generation of the first amplicon. Subsequent amplicons can have a sequence that is substantially complementary to or substantially identical to the target polynucleotide. It will be understood that a small number of mutations of a polynucleotide (e.g., due to amplification artifacts) may occur when generating an amplicon of the polynucleotide.

[0295] As used herein, the term "protection element", when used in reference to the 5' or 3' end of a polynucleotide, is intended to mean an element that inhibits modification of the end of a polynucleotide. Illustratively, a protection element can inhibit the action of one or more enzymes at the end of a polynucleotide, such as the action of a 5' or 3' exonuclease. Non-limiting examples of protection elements include hairpin sequences ligated to the 5' and 3' strands of a double-stranded polynucleotide end, modified bases (e.g., containing phosphorothioate bonds or 3' phosphates), or dephosphorylated bases.

[0296] As used herein, terms such as "CRISPR-Cas system", "Cas-gRNA ribonucleoprotein", and Cas-gRNA RNP refer to an enzymatic system that includes a guide RNA (gRNA) sequence that includes an oligonucleotide sequence that is complementary or substantially complementary to a sequence in a target polynucleotide, and a Cas protein. CRISPR-Cas systems can generally be classified into three main types based on core element content and sequence, which are further subdivided into 10 subtypes; see, for example, Makarova et al., "Evolution and classification of the CRISPR-Cas systems," Nat Rev Microbiol. 9(6):467-477 (2011). Cas proteins can have various activities, such as nuclease activity. Thus, CRISPR-Cas systems provide a mechanism for targeting specific sequences (e.g., via gRNA) as well as certain enzymatic activities against the sequences (e.g., via Cas proteins).

[0297] Type I CRISPR-Cas systems can contain a Cas3 protein that has separate helicase and DNase activities. For example, in type 1-E systems, the crRNA is incorporated into a multisubunit effector complex called Cascade (CRISPR-associated complex for antiviral defense), which binds to target DNA and triggers its degradation by the Cas3 protein; see, e.g., Brouns et al., "Small CRISPR RNAs guide antiviral defense in prokaryotes", Science 321(5891):960-964(2008); Sinkunas et al., "Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR-Cas immune system", EMBO J 30:1335-1342(2011); and Beloglazova et al., "Structure and activity of the Cas3 HD nuclease MJ0384, an effector enzyme of the CRISPR interference", EMBO J 30:4616-4627(2011). Type II CRISPR-Cas systems contain a signature Cas9 protein, a single protein (approximately 160 KDa) that can generate crRNA and cleave target DNA. Cas9 proteins typically contain two nuclease domains: a RuvC-like nuclease domain near the amino terminus and an HNH (or McrA-like) nuclease domain near the center of the protein. Each nuclease domain of the Cas9 protein is specialized to cleave one strand of the double helix; see, e.g., Jinek et al., "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity," Science 337(6096):816-821 (2012). Type III CRISPR-Cas systems contain a polymerase and a RAMP module.Type III systems can be further divided into subtypes III-A and III-B. Type III-A CRISPR-Cas systems have been shown to target plasmids, and the polymerase-like protein of the type III-A system is responsible for cleaving the target DNA; see, e.g., Marraffini et al., "CRISPR interference limits horizontal gene transfer in Staphylococci by targeting DNA," Science 322(5909):1843-1845 (2008). Type III-B CRISPR-Cas systems have also been shown to target RNA; see, e.g., Hale et al., "RNA-guided RNA cleavage by a CRISPR-RNA-Cas protein complex," Cell 139(5):945-956 (2009). CRISPR-Cas systems include engineered and / or programmed nuclease systems derived from naturally occurring CRISPR-Cas systems. The CRISPR-Cas system may include engineered and / or mutated Cas proteins. The CRISPR-Cas system may include engineered and / or programmed guide RNAs.

[0298] In some embodiments, the Cas protein in one of the Cas-gRNA RNPs of the invention can comprise Cas9, or other suitable Cas capable of cleaving a target polynucleotide at a sequence to which the gRNA is complementary, in a manner as described in the following references, the entire contents of each of which are incorporated herein by reference: Nachmanson et al., "Targeted genome fragmentation with CRISPR / Cas9 enables fast and efficient enrichment of small genomic regions and ultra-accurate sequencing with low DNA input (CRISPR-DS)", Genome Res. 28(10):1589-1599 (2018); Vakulskas et al., "A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells", Nature Medicine 24:1216-1224 (2018); Chatterjee et al., "Minimal PAM specificity of a highly similar SpCas9 ortholog”, Science Advances 4(10):eaau0766,1-10 (2018); Lee et al., “CRISPR-Cap: multiplexed double-stranded DNA enrichment based on the CRISPR system”, Nucleic Acids Research 47(1):1-13 (2019). The Cas9-crRNA complex isolated from the S. thermophilus CRISPR-Cas system, as well as a complex assembled in vitro from separate components, demonstrate that it binds to both synthetic oligodeoxynucleotides and plasmid DNA bearing nucleotide sequences complementary to the crRNA.Cas9 has two nuclease domains, RuvC and HNH active site / nuclease domain, which have been shown to be involved in cleaving opposite DNA strands. In some instances, the Cas9 protein is derived from the Cas9 protein of the S. thermophilus CRISPR-Cas system. In some instances, the Cas9 protein is a multi-domain protein with approximately 1,409 amino acid residues.

[0299] In other examples, the Cas may be engineered so that it does not cleave the target polynucleotide at a sequence to which the gRNA is complementary, e.g., in a manner as described in the following references, the entire contents of each of which are incorporated herein by reference: Guilinger et al., "Fusion of catalytically inactive Cas9 to Fokl nuclease improves the specificity of genome modification," Nature Biotechnology 32:577-582 (2014); Bhatt et al., "Targeted DNA transposition using a dCas9-transposase fusion protein," https: / / doi.org / 10.1101 / 571653, pages 1-89 (2019); Xu et al., "CRISPR-assisted targeted enrichment-sequencing (CATE-seq)," available at URL www.biorxiv.org / content / 10.1101 / 672816v1, 1-30 (2019); and Tijan et al. al., "dCas9-targeted locus-specific protein isolation method identifies histone gene regulators," PNAS 115(12):E2734-E2741 (2018). A Cas that lacks nuclease activity may be referred to as an inactivated Cas (dCas). In some examples, dCas may include a nuclease-deficient variant of the Cas9 protein in which both the RuvC and HNH active site / nuclease domains are mutated. The nuclease-deficient mutant of the Cas9 protein (dCas9) binds to double-stranded DNA but does not cleave the DNA. Another variant of the Cas9 protein has two inactivated nuclease domains, with a first mutation in the domain that cleaves the strand complementary to the crRNA and a second mutation in the domain that cleaves the strand non-complementary to the crRNA.In some instances, the Cas9 protein has a first mutation, D10A, and a second mutation, H840A.

[0300] In yet another example, the Cas protein comprises a Cascade protein. The Cascade complex of E. coli recognizes double-stranded DNA (dsDNA) targets in a sequence-specific manner. The Cascade complex of E. coli is a 405 kDa complex that contains five functionally essential CRISPR-associated (Cas) proteins (CasA1B2C6D1E1, also called Cascade proteins) and a 61-nucleotide crRNA. The crRNA guides the Cascade complex to the dsDNA target sequence by forming base pairs with the complementary DNA strand while displacing the non-complementary strand to form an R-loop. Cascade recognizes target DNA without consuming ATP, suggesting that continuous invader DNA surveillance occurs without energy investment; see, for example, Matthijs et al., "Structural basis for CRISPR RNA-guided DNA recognition by Cascade," Nature Structural & Molecular Biology 18(5):529-536 (2011). In yet another example, the Cas protein includes a Cas3 protein. Illustratively, E. coli Cas3 can catalyze ATP-independent annealing of RNA and DNA to form R-loops and RNA base-pair hybrids into double-stranded DNA. Cas3 protein can use longer gRNAs than Cas9; see, for example, Howard et al., "Helicase disassociation and annealing of RNA-DNA hybrids by Escherichia coli Cas3 protein," Biochem J. 439(1):85-95 (2011). Such longer gRNAs may allow other elements to more easily access the target DNA, such as the primer that is extended by the polymerase. Another feature provided by the Cas3 protein is that it does not require a PAM sequence like Cas9, thus providing more flexibility to target the desired sequence.R-loop formation by Cas3 can utilize magnesium as a cofactor; see, e.g., Howard et al., "Helicase disassociation and annealing of RNA-DNA hybrids by Escherichia coli Cas3 protein," Biochem J. 439(1):85-95 (2011). Cas9 variants that reduce or avoid the need for PAM sequences have also been developed; see, e.g., Walton et al., "Unconstrained genome targeting with near PAMless engineered CRISPR-Cas9 variants," Science 368(6488):290-296 (2020), the entire contents of which are incorporated herein by reference. It will be understood that any suitable cofactor, such as a cation, can be used with the Cas proteins used in the compositions and methods of the invention.

[0301] It should also be understood that any CRISPR-Cas system capable of disrupting a double-stranded polynucleotide and creating a loop structure can be used. For example, Cas proteins can include, but are not limited to, Cas proteins as described in the following references, the entire contents of each of which are incorporated herein by reference: Haft et al., "A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR / Cas subtypes exist in prokaryotic genomes", PLoS Comput Biol. 1(6):e60,1-10 (2005); Zhang et al., "Expanding the catalog of cas genes with metagenomes", Nucl. Acids Res. 42(4):2448-2459 (2013); and Strecker et al., "RNA-guided DNA insertion with CRISPR-associated transposases", Science 365(6448):48-53 (2019) (Cas proteins can include Cas12k). Some of these CRISPR-Cas systems may utilize specific sequences to recognize and bind to target sequences, for example, Cas9 may utilize the presence of a 5'-NGG protospacer adjacent motif (PAM).

[0302] In some examples, the Cas protein may be selected to leave a single-stranded DNA overhang region after dsDNA cleavage, for example, of one or more bases, illustratively 2-5 bases. For example, CRISPR-Cas12a (Cpf1) is commercially available from Integrated DNA Technologies, Inc. (Coralville, Iowa). According to the manufacturer, CRISPR-Cas12a (Cpf1) generates staggered cuts with 5' overhangs and may target different sites than CRISPR-Cas9. In some examples, the 5' overhang may be 5 bases long. Some of these CRISPR-Cas systems may utilize PAM. For example, Cas12a (Cpf1 or C2c1) or FnCas12a may use a PAM of TTTN upstream of the cleavage site, but emerging Cas12a orthologs may have reduced PAM requirements (e.g., YTN), as described in Teng et al., "Enhanced mammalian genome editing by new Cas12a orthologs with optimized crRNA scaffolds," Genome Biology 20:15 (2019), the entire contents of which are incorporated herein by reference. Cas12 may be derived from organisms such as Francisella novicida, Acidaminococcus sp., Lachnospiraceae sp., and Prevotella sp. For further details regarding Cas12a, see Covsky et al., “CRISPR-Cas12a exploits R-loop asymmetry to form double-strand breaks,” eLife, 9:e55143 (2020), the entire contents of which are incorporated herein by reference.

[0303] The CRISPR-Cas system may also include an engineered and / or programmed guide RNA (gRNA). As used herein, the terms "guide RNA" and "gRNA" (sometimes referred to in the art as single guide RNA or sgRNA) are intended to mean an RNA that includes a sequence that is complementary or substantially complementary to a region of a target DNA sequence and that guides a Cas protein to that region. A guide RNA may include nucleotide sequences in addition to the nucleotide sequence that is complementary or substantially complementary to a region of a target DNA sequence.Methods for designing gRNAs are well known in the art, and non-limiting examples are provided in the following references, the entire contents of each of which are incorporated herein by reference: Stevens et al., "A novel CRISPR / Cas9 associated technology for sequence-specific nucleic acid enrichment," PLoS ONE 14(4):e0215441, pages 1-7 (2019); Fu et al., "Improving CRISPR-Cas nuclease specificity using truncated guide RNAs, Nature Biotechnology 32(3):279-284 (2014); Kocak et al., "Increasing the specificity of CRISPR systems with engineered RNA secondary structures," Nature Biotechnology 37:657-666 (2019); Lee et al., "CRISPR-Cap: multiplexed double-stranded DNA enrichment based on the CRISPR system," Nucleic Acids Research 47(1):e1,1-13 (2019); Quan et al., “FLASH: a next-generation CRISPR diagnostic for multiplexed detection of antimicrobial resistance sequences”, Nucleic Acids Research 47(14):e83,1-9 (2019); and Xu et al., “CRISPR-assisted targeted enrichment-sequencing (CATE-seq)”, https: / / doi.org / 10.1101 / 672816,1-30 (2019).

[0304] In some examples, the gRNA comprises a chimera, e.g., a CRISPR RNA (crRNA) fused to a transactivating CRISPR RNA (tracrRNA). Such chimeric single guide RNAs (sgRNAs) are described in Jinek et al., "A programmable dual-RNA-guided endonuclease in adaptive bacterial immunity," Science 337(6096):816-821 (2012). The Cas protein can be directed by the chimeric sgRNA to any genomic locus followed by a 5'-NGG protospacer adjacent motif (PAM). In one non-limiting example, the crRNA and tracrRNA can be synthesized by in vitro transcription using a synthetic double-stranded DNA template containing a T7 promoter. The tracrRNA may have a fixed sequence, but the target sequence may determine part of the sequence of the crRNA. Equimolar concentrations of the crRNA and tracrRNA may be mixed and heated at 55°C for 30 seconds. Cas9 can be added at the same molar concentration and incubated with the RNA mix for 10 min at 37 °C. A 10-20 fold molar excess of the resulting Cas9-gRNA RNP can then be added to the target DNA. The binding reaction can occur within 15 min. Other suitable reaction conditions can easily be used.

[0305] As used herein, the terms "fusion protein" and "chimeric protein" are intended to mean an element comprising two or more polypeptide domains having different functional properties from each other (e.g., different enzymatic activity). The domains may be covalently or non-covalently coupled to each other. A fusion protein may optionally comprise a third, fourth or fifth polypeptide domain or other polypeptide domains operably linked to one or more other polypeptide domains. A fusion protein may comprise multiple copies of the same polypeptide domain. A fusion protein may also, or alternatively, comprise one or more mutations in one or more polypeptides. A fusion protein may comprise one or more non-protein elements, such as a polynucleotide (exemplarily, a gRNA) and / or a linker that couples the domains to each other. For non-limiting examples of fusion proteins, see the following references, the entire contents of which are incorporated herein by reference: Guilinger et al., "Fusion of catalytically inactive Cas9 to Fokl nuclease improves the specificity of genome modification", Nature Biotechnology 32:577-582 (2014); Bhatt et al., "Targeted DNA transposition using a dCas9-transposase fusion protein", https: / / doi.org / 10.1101 / 571653, pages 1-89 (2019); and Strecker et al., "RNA-guided DNA insertion with CRISPR-associated transposases", Science 365(6448):48-53 (2019). Another exemplary fusion protein is ShCAST (Scytonema hofmanni CRISPR-associated transposase), which comprises Cas12k and a Tn7-like transposase.For further details regarding ShCAST, which contains Cas12k and Tn7, see Strecker et al., "RNA-Guided DNA insertion with CRISPR-associated transposases," Science 365(6448):48-53 (2019), the entire contents of which are incorporated herein by reference.

[0306] As used herein, the term "transposase" is intended to mean an enzyme capable of coupling an oligonucleotide to a polynucleotide. In some examples, the oligonucleotide may include an amplification adapter and, optionally, a unique molecular identifier (UMI). The transposase may cleave the polynucleotide while adding the oligonucleotide. A non-limiting example of a transposase is Tn5. In yet another example, the transposase may include an integrase from a retrotransposon or retrovirus. Transposases, transposomes, and transposome complexes are generally known to those of skill in the art, as exemplified by the disclosure of U.S. Patent Application Publication No. 2010 / 0120098, the entire contents of which are incorporated herein by reference.

[0307] For further non-limiting examples of transposases that may be used in the methods provided herein, see the following references, the entire contents of each of which are incorporated herein by reference: Strecker et al., "RNA-guided DNA insertion with CRISPR-associated transposases", Science 365(6448):48-53(2019); Klompe et al., "Transposon-encoded CRISPR-Cas systems direct RNA-guided DNA integration", Nature 571:219-225(2019); Bhatt et al., "Targeted DNA transposition using a dCas9-transposase fusion protein", https: / / doi.org / 10.1101 / 571653, pages 1-89(2019).Other examples of known transposition systems that can be used in the provided methods include Staphylococcus aureus Tn552, Tyl, transposons Tn7, Tn / O and IS10, mariner transposase, Tel, P elements, Tn3, bacterial insertion sequences, retroviruses, and yeast retrotransposons (see, e.g., Colegio et al. 2001, J. Bacteriol. 183:2384-8; Kirby et al., 2002, Mol. Microbiol. 43:173-86; Devine and Boeke, 1994, Nucleic Acids Res., 22:3765-72; International Patent Application No. 95 / 23875; Craig, 1996, Science 271:1512; Craig, 1996, Review in: Curr Top Microbiol. Immunol.204:27-48;Kleckner et al.,1996,Curr Top Microbiol Immunol.204:49-82;Lampe et al.,1996,EMBO J.15:5470-9;Plasterk,1996,Curr Top Microbiol Immunol 204:125-43;Gloor,2004,Methods Mol.Biol.260:97-114;Ichikawa and Ohtsubo,1990,J Biol.Chem.265:18829-32;Ohtsubo and Sekine,1996,Curr.Top.Microbiol.Immunol.204:1-26;Brown et al.,1989,Proc Natl Acad Sci USA86:2525-9;and Boeke and Corces, 1989, Annu Rev Microbiol. 43:403-34).As another example, ShCAST (Scytonema hofmanni CRISPR-associated transposase) comprises a Tn7-like transposase; for further details, see Strecker et al., "RNA-Guided DNA insertion with CRISPR-associated transposases," Science 365(6448):48-53 (2019), the entire contents of which are incorporated herein by reference.

[0308] In some instances, the transposase may carry out a process that may be referred to as "tagmentation" or "transposition" that results in fragmentation of a target polynucleotide and ligation of adapters to the 5' ends, or to the 5' and 3' ends, of both strands of a double-stranded DNA fragment, e.g., in a manner such as described in U.S. Patent Application Publication No. 2010 / 0120098 or WO 2010 / 04860, the entire contents of each of which are incorporated herein by reference.

[0309] The transposase may form a "transposition complex" comprising the transposase, a transposon end-containing composition, and a double-stranded polynucleotide, and may catalyze the insertion or transposition of the transposon end-containing composition into a double-stranded target polynucleotide. Examples of transposition complexes include, but are not limited to, those formed by hyperactive Tn5 transposase and Tn5-type transposon ends, or by MuA transposase and Mu transposon ends containing R1 and R2 end sequences; see, for example, the following references, the entire contents of each of which are incorporated herein by reference: Goryshin et al., "Tn5 in vitro transposition", J. Biol. Chem. 273:7367-7394(1998); Mizuuchi, "In vitro transposition of bacteriophage Mu: a biochemical approach to a novel replication reaction", Cell 35(3 pt 2):785-794(1983); and Savilahti et al., "The phage Mu transposomes core: DNA requirements for assembly and function", EMBO J. 14(19):4893-4903(1995). The combination of a transposase and a transposon end may be referred to as a "transposome".

[0310] Further examples of transposases and other suitable transposition systems include Staphylococcus aureus Tn552 (see, e.g., Colegio et al., "In vitro transposition system for efficient generation of random mutants of Campylobacter jejuni", J Bacteriol. 183:2384-2388 (2001) and Kirby et al., "Cryptic plasmids of Mycobacterium avium: Tn552 to the rescue", Mol Microbiol., 43(1):173-186 (2002)); TyI (Devine et al., "Efficient integration of artificial transposons into plasmid targets in vitro: a useful tool for DNA mapping, sequencing and genetic analysis", Nucleic Acids Res. 22(18):3765-3772 (1994) and International Patent Application No. 95 / 23875); transposon Tn7 (Craig, "V(D)J recombination and transposition: Closer than expected", Science 271(5255):1512 (1996) and Craig, Review in: Curr Top Microbiol Immunol, 204:27-48 (1996)); TnIO and ISLO (Kleckner et al., Curr Top Microbiol Immunol, 204:49-82 (1996)); mariner transposase (Lampe et al., "A purified mariner transposase is sufficient to mediate transposition in vitro", EMBO J.15(19):5470-5479(1996));Tci(Plasterk,Curr Top Microbiol Immunol,204:125-143(1996)),P Element(Gloor,"Gene targeting in Drosophila",Methods Mol Biol 260:97-114(2004));TnJ(Ichikawa et al. al., "In vitro transposition of transposon Tn3", J Biol Chem. 265(31):18829-18832 (1990)); integration:Structure of the initial covalent product and its precursor,and a role for the viral IN protein," Proc Natl Acad Sci USA, 86:2525-2529 (1989)); and yeast retrotransposons (see Boeke et al., "Transcription and reverse transcription of retrotransposons," Annu Rev Microbiol. 43:403-434 (1989).

[0311] As used herein, the term "nuclease" is intended to mean an enzyme capable of cleaving phosphodiester bonds between nucleotide subunits of a polynucleotide. The term "endonuclease" refers to an enzyme capable of cleaving phosphodiester bonds within a polynucleotide chain.

[0312] As used herein, the term "nickases" refers to endonucleases that cleave only one strand of a DNA duplex. Some CRISPR-Cas systems can cleave only one strand of a double-stranded polynucleotide, and therefore can be referred to as CRISPR nickases or Cas-gRNA RNP nickases. For example, the term "Cas9 nickases" refers to nickases derived from Cas9 proteins, typically by inactivating one nuclease domain of the Cas9 protein. A non-limiting example of a CRISPR nickase includes S.pyogenes Cas9 with a first mutation D10A and a second mutation H840A.

[0313] In the context of a polypeptide, the terms "variant" and "derivative" as used herein refer to a polypeptide comprising the amino acid sequence of a polypeptide or a fragment of a polypeptide that has been altered by the introduction of substitutions, deletions, or additions of amino acid residues. A variant or derivative of a polypeptide may be a fusion protein that comprises a portion of the amino acid sequence of a polynucleotide. The terms "variant" or "derivative" as used herein also refer to a polypeptide or a fragment thereof that has been chemically modified, for example, by the covalent attachment of any type of molecule to the polypeptide. For example, but not limited to, a polypeptide or a fragment of a polypeptide can be chemically modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, attachment to a cellular ligand or other protein, etc. A variant or derivative is modified either in the type or location of the attached molecule that is naturally present or in a different manner than the starting peptide or polypeptide. A variant or derivative further includes the deletion of one or more chemical groups that are naturally present on the peptide or polypeptide. A variant or derivative of a polypeptide or a fragment of a polypeptide can be chemically modified by chemical modification using techniques known to those skilled in the art, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. Additionally, a variant or derivative of a polypeptide or a fragment of a polypeptide can contain one or more non-classical amino acids. A polypeptide variant or derivative can have a similar or identical function as a polypeptide or a fragment of a polypeptide described herein. A polypeptide variant or derivative can have additional or different functions compared to a polypeptide or a fragment of a polypeptide described herein.

[0314] As used herein, the term "sequencing" is intended to mean determining the sequence of a polynucleotide. Sequencing may include one or more of sequencing by synthesis, bridge PCR, chain termination sequencing, sequencing by hybridization, nanopore sequencing, and sequencing by ligation.

[0315] As used herein, the term "dehosting" is intended to mean the selective inactivation or degradation of polynucleotides of one species relative to the polynucleotides of another species. For example, a first species, such as a mammal (e.g., a human), can act as a host for many other species, such as bacteria, fungi, and viruses. It may be desirable to selectively inactivate or degrade the polynucleotides of the first species so that the polynucleotides of one or more other species can be amplified and sequenced.

[0316] "Selective" for a target is intended to mean binding to that target and not binding to a different target. For example, a Cas-gRNA RNP selective for a species-specific repeat element may bind to that species-specific repeat element and may not bind to a different species-specific repeat element.

[0317] As used herein, the term "species-specific repetitive element" is intended to mean a repetitive sequence that is present in a polynucleotide of a given species and may not be present in a polynucleotide of another species. Species with multiple chromosomes (e.g., mammals, e.g., humans) may contain different species-specific elements on each chromosome, or may contain the same species-specific elements on each chromosome, or a mixture of the same and different species-specific elements on each chromosome. One example of a species-specific repetitive element is a photospacer adjacent motif or PAM sequence (e.g., NGG). The gRNA of the Cas-gRNA RNP may have a sequence that hybridizes to the species-specific repetitive element.

[0318] As used herein, the terms "unique molecular identifier" and "UMI" are intended to mean an oligonucleotide that can be coupled to a polynucleotide through which the polynucleotide can be identified. For example, a set of different UMIs can be attached to multiple different polynucleotides, each of which can be identified using the particular UMI coupled to that polynucleotide.

[0319] As used herein, the term "whole genome" or "WG" of a species is intended to mean a set of one or more polynucleotides that together provide a majority of the polynucleotides used by the cellular processes of the species. The whole genome of a species may include any suitable combination of chromosomal DNA and / or mitochondrial DNA of the species, and in the case of plant species, may include DNA contained in chloroplasts. The set of one or more polynucleotides together may provide at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99% of the polynucleotides used by the cellular processes of the species.

[0320] As used herein, the term "fragment" is intended to mean a portion of a polynucleotide. For example, a polynucleotide may be a total number of bases in length, and a fragment of that polynucleotide may be less than the total number of bases in length.

[0321] As used herein, the term "sample" is intended to mean a volume of fluid that contains one or more polynucleotides. The polynucleotides in a sample may include an entire genome or may include only a portion of an entire genome. A sample may contain polynucleotides from a single species or multiple species.

[0322] The term "antibody" as used herein includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity of binding to a target antigen site and its isoform of interest. The term "antibody fragment" includes a portion of a full-length antibody, generally the antigen-binding or variable region thereof. The term "antibody" as used herein includes any antibody from any species and source, including, but not limited to, human antibodies, rat antibodies, mouse antibodies, rabbit antibodies, etc., and may be synthetically produced or naturally occurring.

[0323] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies; that is, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically contain different antibodies against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. "Monoclonal antibodies" may also be isolated from phage antibody libraries using techniques known in the art. Monoclonal antibodies, as the term is used herein, may include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chains are identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chains are identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

[0324] As used herein, terms such as "target specific" and "selective" when used in reference to a guide RNA or other polynucleotide are intended to mean a polynucleotide that includes a sequence that is specific (substantially complementary to and capable of hybridizing to) a sequence in another polynucleotide.

[0325] As used herein, the terms "complementary" and "substantially complementary," when used in reference to a polynucleotide, are intended to mean that the polynucleotide comprises a sequence that can selectively hybridize to a sequence in another polynucleotide under specified conditions.

[0326] As used herein, terms such as "amplification" and "amplifying" refer to the use of any suitable amplification method to generate polynucleotide amplicons. Polymerase chain reaction (PCR) is one non-limiting amplification method. Other suitable amplification methods known in the art include, but are not limited to, rolling circle amplification; riboprimer amplification (e.g., as described in U.S. Pat. No. 7,413,857); ICAN; UCAN; ribospia; end tagging; (e.g., as described in U.S. Pat. App. Pub. No. 2005 / 0153333) and Eberwine-type aRNA amplification or strand displacement amplification. Further non-limiting examples of amplification methods include those described in WO 02 / 16639; WO 00 / 56877; Australian Patent Application Publication No. 00 / 29742; U.S. Patent No. 5,523,204; U.S. Patent No. 5,536,649; U.S. Patent No. 5,624,825; U.S. Patent No. 5,631,147; U.S. Patent No. 5,648,211; U.S. Patent No. 5,733,752; U.S. Patent No. 5,744,311; U.S. Patent No. 5,756,702; U.S. Patent No. 5,916,779; U.S. Patent No. 6,238,868; U.S. Patent No. 6,309,833; U.S. Patent No. 6,326,173; U.S. Patent No. 5,849,54 No. 7; U.S. Pat. No. 5,874,260; U.S. Pat. No. 6,218,151; U.S. Pat. No. 5,786,183; U.S. Pat. No. 6,087,133; U.S. Pat. No. 6,214,587; U.S. Pat. No. 6,063,604; U.S. Pat. No. 6,251,639; U.S. Pat. No. 6,410,278; WO 00 / 28082; U.S. Pat. No. 5,591,609; U.S. Pat. No. 5,614,389; U.S. Pat. No. 5,773,733; U.S. Pat. No. 5,834,202; U.S. Pat. No. 6,448,017; U.S. Pat. No. 6,124,120; and U.S. Pat. No. 6,280,949.

[0327] The terms "polymerase chain reaction" and "PCR" as used herein refer to a procedure in which small amounts of polynucleotides, such as RNA and / or DNA, are amplified. Generally, amplification primers are coupled to polynucleotides for use during PCR. See, for example, the following references, each of which is incorporated herein by reference in its entirety: Mullis, U.S. Pat. No. 4,683,195; Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51:263 (1987); and Erlich, ed., PCR Technology, (Stockton Press, NY, 1989). As known to those skilled in the art, a wide variety of enzymes and kits are available for performing PCR. For example, in some instances, PCR amplification is performed using either the FAILSAFE™ PCR System or the MASTERAMP™ Extra-Long PCR System from EPICENTRE Biotechnologies (Madison, Wis.), as described by the manufacturer.

[0328] As used herein, terms such as "ligation" and "ligating" are intended to mean forming a covalent bond or linkage between the ends of two or more polynucleotides. The nature of the bond or linkage can vary widely, and ligation can be performed enzymatically or chemically. Ligation is typically performed enzymatically, forming a phosphodiester bond between the 5' carbon terminal nucleotide of one oligonucleotide and the 3' carbon of another nucleotide. Template-driven ligation reactions are described in the following references, the entire contents of each of which are incorporated herein by reference: U.S. Pat. No. 4,883,750; U.S. Pat. No. 5,476,930; U.S. Pat. No. 5,593,826; and U.S. Pat. No. 5,871,921. Ligation may also be performed using non-enzymatic formation of phosphodiester bonds, or formation of non-phosphodiester covalent bonds between the ends of polynucleotides, such as phosphorothioate bonds, disulfide bonds, etc.

[0329] As used herein, the term "substrate" refers to a material used as a support for the compositions described herein. Exemplary substrate materials can include glass, silica, plastic, quartz, metal, metal oxide, organo-silicates (e.g., polyhedral organic silsesquioxanes (POSS)), polyacrylates, tantalum oxide, complementary metal oxide semiconductor (CMOS), or combinations thereof. An example of a POSS can be that described in Kehagias et al., Microelectronic Engineering 86 (2009), pp. 776-778, which is incorporated herein by reference in its entirety. In some examples, the substrate used in this application includes a silica-based substrate, such as glass, fused silica, or other silica-containing materials. In some examples, the silica-based substrate can include silicon, silicon dioxide, silicon nitride, or hydrogenated silicone. In some examples, the substrates used in this application include plastic materials or components such as polyethylene, polystyrene, poly(vinyl chloride), polypropylene, nylon, polyester, polycarbonate, and poly(methyl methacrylate). Examples of plastic materials include poly(methyl methacrylate), polystyrene, and cyclic olefin polymer substrates. In some examples, the substrate is or includes a silica-based material or a plastic material, or a combination thereof. In certain examples, the substrate has at least one surface that includes glass or a silicon-based polymer. In some examples, the substrate can include a metal. In some such examples, the metal is gold. In some examples, the substrate has at least one surface that includes a metal oxide. In one example, the surface includes tantalum oxide or tin oxide. Acrylamides, enones, or acrylates can also be utilized as substrate materials or components. Other substrate materials include, but are not limited to, gallium arsenide, indium phosphide, aluminum, ceramics, polyimides, quartz, resins, polymers, and copolymers.In some examples, the substrate and / or substrate surface can be or include quartz. In some other examples, the substrate and / or substrate surface can be or include a semiconductor, such as GaAs or ITO. The above list is intended to illustrate, but not limit, the present application. The substrate can include a single material or multiple different materials. The substrate can be a composite or laminate. In some examples, the substrate includes an organosilicate material.

[0330] The substrate can be horizontal, circular, spherical, rod-shaped, or any other suitable shape. The substrate can be rigid or flexible. In some examples, the substrate is a bead or a flow cell.

[0331] The substrate may be unpatterned, textured, or patterned on one or more surfaces of the substrate. In some examples, the substrate is patterned. Such patterns may include posts, pads, wells, ridges, channels, or other three-dimensional concave or convex structures. The pattern may be regular or irregular across the surface of the substrate. The pattern may be formed, for example, by nanoimprint lithography or by using, for example, metal pads to form features on a non-metallic surface.

[0332] In some examples, the substrates described herein form at least a portion of a flow cell, are located within a flow cell, or are coupled to a flow cell. A flow cell may include a flow chamber that is divided into multiple lanes or multiple sectors. Examples of flow cells that can be used in the methods and compositions described herein, as well as substrates for manufacturing flow cells, include, but are not limited to, those commercially available from Illumina, Inc. (San Diego, CA).

[0333] Compositions and methods for Cas-gRNA RNP-mediated dehosting Some examples herein relate to Cas-gRNA RNP-mediated dehosting. For example, Figures 1A-1K show schematic diagrams of exemplary compositions and process flow operations for Cas-gRNA RNP-mediated dehosting.

[0334] A more complex species, illustratively a mammal, can host multiple other simpler species, such as bacteria, fungi, and viruses. It may be desirable to sequence the polynucleotides (such as DNA) of the host species, but it may be difficult to adequately separate such polynucleotides from the polynucleotides of the host species. For example, a sample of polynucleotides purified from a fluid or tissue derived from a host may contain primarily polynucleotides from the host (e.g., about 99% or more) and relatively small amounts of polynucleotides from other species (e.g., about 1% or less). Thus, sequencing of the sample may yield primarily the host sequence, with relatively little information on the sequences of the other species. As provided herein, polynucleotides of a given species (such as the host) may be removed from the sample in a manner that enhances the ability to sequence the polynucleotides of one or more other species within the sample.

[0335] For example, as shown in FIG. 1A, a sample obtained from a first species may include a mixture of a first double-stranded polynucleotide derived from the first species and a second double-stranded polynucleotide derived from one or more second species. Illustratively, the first species (S1) may be a mammal (e.g., human) that may act as a host for a number of other species, such as bacteria, fungi, and viruses (S2, S3, etc.). In the non-limiting example shown in FIG. 1A, the composition 101 includes a mixture of polynucleotides S1-1, S1-2, S1-3 derived from the first species; a polynucleotide S2-1 derived from the second species; and a polynucleotide S3-1 derived from a third species. Each of the polynucleotides S1-1, S1-2, S1-3 derived from the first species may include a species-specific repeat element 140 as shown in FIG. 1A. For example, if the first species is a mammal, the polynucleotide derived from that species may include a mammal-specific repeat element. For example, if the first species is human, each of the human-derived polynucleotides may contain one or more human-specific repetitive elements 140.

[0336] It will be understood that the concentration, number, and type of polynucleotides from each given species may vary for each particular sample. For example, if a first species is a host for a second and a third species, the sample may contain a significantly higher concentration of polynucleotides from the first species than the second and third species. Furthermore, the first species may have greater genetic complexity, e.g., a genome with multiple polynucleotides, such as the 23 relatively long chromosomes S1-1, S1-2, S1-3, S1-23 in humans, while the second and / or third species may be genetically simpler, e.g., a genome with only a single relatively short polynucleotide. Furthermore, the polynucleotides of one or more species in the mixture may be fragmented ex vivo into shorter fragments than those species normally use in vivo during physiological processes. Furthermore, the polynucleotides of one or more species in the mixture may be circular (such as S3-1) and therefore may not have any ends.

[0337] As shown in FIG. 1A, each polynucleotide in the mixture may be double-stranded. For example, polynucleotide S1-1 may include a first strand 111 and a complementary second strand 111'; polynucleotide S1-2 may include a first strand 112 and a complementary second strand 112'; polynucleotide S1-3 may include a first strand 113 and a complementary second strand 113'; polynucleotide S2-1 may include a first strand 121 and a complementary second strand 121'; polynucleotide S3-1 may include a first strand 131 and a complementary second strand 131'. In some examples, the double-stranded polynucleotides derived from the first, second, and / or third species may include double-stranded DNA.

[0338] The ends of the first double-stranded polynucleotide and, if present, the ends of the second double-stranded polynucleotide may be protected. For example, as shown in FIG. 1B, the composition 102 includes a protection element 150 that protects any ends of the double-stranded polynucleotides in the mixture. Illustratively, the protection element 150 is coupled to and protects the ends of the first species of polynucleotides S1-1, S1-2, and S1-3, and the second species of polynucleotide S2-1. Because the third species of polynucleotide S3-1 is circular, such polynucleotide may not have an end to which the protection element 150 can be coupled. The protection element 150 may include any suitable chemical moiety that inhibits the action of one or more enzymes (such as exonucleases) on the end of the double-stranded polynucleotide to which such protection element is coupled. For example, as shown in the inset of FIG. 1B, the protection element 150 may include a modified base 151, a hairpin adaptor 152 ligated to the end, or a 5' dephosphorylated end. The modified bases 151 may be added using a terminal transferase, which may include, for example, a phosphorothioate bond or a 3' phosphate. The hairpin adaptor 152 may comprise an oligonucleotide that includes a stem sequence that hybridizes to one another and a loop sequence that extends between the stem sequences, and may be added by methods known in the art, such as, for example, by performing end repair to fill in any overhangs, then adding an A overhang ("A tail") (e.g., using an exonuclease such as Klenow Fragment exo-), and then ligating the hairpin adaptor 152 to the end. The 5' end of the double-stranded polynucleotide may be dephosphorylated using an appropriate phosphatase enzyme.

[0339] After protecting the ends of the first and second double-stranded polynucleotides, a free end in the first double-stranded polynucleotide can be selectively generated. For example, FIG. 1C shows a composition 103 in which a Cas-gRNA RNP 160 hybridizes to a sequence, e.g., a species-specific repeat element 140, that is present in the first double-stranded polynucleotide and not present in the second double-stranded polynucleotide. The sequence can then be cleaved with a Cas-gRNA RNP to generate free ends in a manner as shown in Figure ID, including composition 104 in which free ends 141, 141' are generated on the strand of polynucleotide S1-1, free ends 143, 142' are generated on the strand of polynucleotide S1-2, and free ends 142, 143' are generated on the strand of polynucleotide S1-3, but no free ends are generated on polynucleotides S2-1 and S3-1 because those polynucleotides do not contain the species-specific repeat element 150 to which the Cas-gRNA RNP 160 selectively hybridizes. The Cas can include, for example, Cas9.

[0340] The first double-stranded polynucleotide can then be degraded from the free end generated by the Cas-gRNA RNP 160 toward the protected end. For example, the composition 105 illustrated in FIG. 1E includes an exonuclease 170 for degrading the first double-stranded polynucleotides S1-1, S1-2, S1-3. Any suitable exonuclease 170 can be used. Illustratively, the free end can include a 3' end in a manner as shown in the upper part of the inset in FIG. 1E, and the first double-stranded polynucleotides S1-1, S1-2, S1-3 can be degraded using exonuclease III. As another purely illustrative example, the free end can include a 5' end in a manner as shown in the lower part of the inset in FIG. 1E, and one strand of each of the first double-stranded polynucleotides S1-1, S1-2, S1-3 can be degraded using lambda exonuclease. Depending on the particular type of protection element 150 used, the use of an exonuclease may result in a composition 106 illustrated in FIG. 1F in which both strands of each of the polynucleotides S1-1, S1-2, S1-3 are degraded, or a composition 107 illustrated in FIG. 1G in which the polynucleotides S1-1, S1-2, S1-3 are rendered single-stranded. Illustratively, if the protection element 150 includes a hairpin oligonucleotide, after degrading one strand, the exonuclease may degrade the other strand following the hairpin, resulting in degradation of both strands. As another example, if the protection element 150 includes modified bases or 5' dephosphorylated bases, after the exonuclease degrades one strand, the protection element may inhibit the exonuclease from degrading the other strand. Regardless of the particular exonuclease used and whether the first species polynucleotide is fully degraded or rendered single stranded, polynucleotides S2-1 and S3-1 cannot be degraded by that exonuclease because the end of polynucleotide S2-1 is protected by protection element 150 and polynucleotide S3-1 lacks an end.

[0341] After degradation of the first species of polynucleotides, amplification adaptors can be ligated to the ends of any remaining double-stranded polynucleotides in the mixture. For example, FIG. 1H shows a composition 108 in which polynucleotides S1-1, S1-2, and S1-3 are degraded (e.g., both strands are degraded as shown in FIG. 1F, or the polynucleotides are rendered single-stranded as shown in FIG. 1G and 1H), and protecting groups 150 are removed from any remaining double-stranded polynucleotides in the mixture, such as polynucleotide S2-1. Any remaining protecting groups 150 coupled to any remaining portions of the first species of polynucleotides can be removed as well. As illustrated in FIG. 1I, any circular polynucleotides (e.g., S3-1 of the third species) can be opened using, for example, tagmentation, shearing, or other suitable fragmentation techniques that can fragment any remaining double-stranded polynucleotides in the mixture (e.g., S2-1). The amplification adaptors can then be ligated to the remaining double-stranded polynucleotides, e.g., those of the second and third species, or the remaining double-stranded polynucleotides can be tagged to obtain the composition 109 shown in FIG. 1J. The composition 109 comprises substantially only single-stranded polynucleotides S1-1, S1-2, S1-3 from the first species; substantially only double-stranded polynucleotides S1-2, S1-3 from the second and / or third species; and amplification adaptors 180 ligated to the ends of fragments of the second double-stranded polynucleotides S2-1, S3-1, and substantially not ligated to any ends of the first double-stranded polynucleotides S1-1, S1-2, S1-3. It will be appreciated that if the polynucleotides of the first species are completely degraded in a manner as described with reference to FIG. 1F, the composition 109 may instead not comprise any polynucleotides derived from the first species.In the method as shown in FIG. 1J, the amplification adaptor 180 may be Y-shaped and may include a unique molecular identifier (UMI) as described in the following references, the entire contents of each of which are incorporated herein by reference: Kennedy et al., "Detecting ultralow-frequency mutations by Duplex Sequencing," Nat Protoc. 9:2586-2606 (2014); and Kivioja et al., "Counting absolute numbers of molecules using unique molecular identifiers," Nature Methods 9:72-42 (2012). The double-stranded polynucleotides S2-1 and S3-1 may then be amplified (e.g., using PCR) and sequenced without substantially sequencing any of the polynucleotides derived from the first species. Thus, sequences of polynucleotides S2-1 and S3-1 may be obtained with relatively low or substantially no background signal from the first species, which may host the second and third species.

[0342] It should be noted that the first species polynucleotides S1-1, S1-2, and S1-3 do not necessarily have to be completely degraded to render these polynucleotides unavailable for amplification and sequencing. For example, the amplification adaptor 180 may be configured to selectively ligate to any double-stranded polynucleotides and not substantially ligate to any single-stranded polynucleotides. Thus, any double-stranded polynucleotides in the mixture to which the amplification adaptor is ligated can be amplified and then sequenced, but any single-stranded polynucleotides cannot be amplified due to lack of a suitable amplification adaptor. Illustratively, tagmentation may only add adaptors to dsDNA and not to ssDNA. As another example, T4 DNA ligase may act only on dsDNA. In this regard, it should be noted that the amplification adaptor 180 may be blunt-ended or A-tailed in any of such approaches.

[0343] FIG. 1K shows an exemplary flow of operations in a method for processing a mixture of a first double-stranded polynucleotide derived from a first species and a second double-stranded polynucleotide derived from a second species. The method 1000 illustrated in FIG. 1K may include protecting the ends of the first double-stranded polynucleotide and any ends of the second double-stranded polynucleotide in the mixture (operation 1001). For example, in a method as described with reference to FIG. 1B, a protection element 150 may be added to the ends of the first double-stranded polynucleotides S1-1, S1-2, and S1-3 and the second double-stranded polynucleotide S2-1, while the double-stranded polynucleotide S3-1 lacks an end and therefore cannot be coupled to the protection element 150.

[0344] The method 1000 illustrated in FIG. 1K may also include selectively generating free ends in the first double-stranded polynucleotide after protecting the ends of the first and second double-stranded polynucleotides (operation 1002). For example, in a manner as described with reference to FIG. 1C, the Cas-gRNA RNP 160 may selectively hybridize with sequences, such as species-specific repeat elements, that are in the first species polynucleotides S1-1, S1-2, and S1-3 and are not in the second species polynucleotide S2-1 (or the third species polynucleotide S3-1). The Cas-gRNA RNP 160 may cleave the first species polynucleotides S1-1, S1-2, and S1-3 to generate free ends as described with reference to FIG. 1D. The method 1000 illustrated in FIG. 1K may also include degrading the first double-stranded polynucleotide from the free ends toward the protected ends (operation 1003). For example, an exonuclease can be used to degrade the first species polynucleotides S1-1, S1-2, and S1-3 from their respective free ends 141, 141', 142, 142', and 143, 143', in a manner as described with reference to Figures 1E-1G. An amplification adaptor can then be coupled to the second species polynucleotide S2-1 (optionally including fragmentation prior to adding the amplification adaptor), in a manner as described with reference to Figures 1I-1J, and the polynucleotide then amplified and sequenced.

[0345] Thus, as provided herein, Cas-gRNA RNPs can be used to selectively generate free ends in polynucleotides of a desired species, which are degraded in such a way as to render them substantially unavailable for amplification or sequencing, for one or more other species of polynucleotides that can then be amplified and sequenced.

[0346] Fragmentation of the whole genome (WG) into different defined fragment sizes Some examples herein relate to fragmentation of a whole genome (WG) into different defined fragment sizes. For example, Figures 2A-2K show schematic diagrams of exemplary compositions and process flow operations for fragmenting a WG into different defined fragment sizes.

[0347] Depending on the species, the WG of that species contains a distinct number of chromosomes. Although the general sequence of each human chromosome has been well characterized, the sequence of each individual's chromosome contains genetic variations specific to that individual. Furthermore, the sequence of one or more chromosomes may even vary within an individual, for example, if the individual has a tumor with a different genetic variation than that individual's normal tissue; the tumor may even have different genetic variations at different locations. These and other types of genetic variations make it desirable to perform WG sequencing. Typically, WG sequencing begins by obtaining an aliquot of blood or other fluid or tissue from an individual, purifying the DNA in the aliquot, and then fragmenting the DNA into smaller fragments of a size suitable for sequencing. Depending on the particular instrument used to sequence the DNA, only fragments in a certain size range (e.g., about 100 to about 1000 base pairs) can be properly sequenced. However, previously known methods of fragmenting DNA using mechanical processes such as sonication or enzymatic fragmentation produce a relatively wide distribution of different fragment sizes. Only a small portion of the fragments within that distribution (e.g., about 20%) may have a size range suitable for sequencing, and the remainder of the WG (e.g., about 80%) may be discarded. As provided herein, a WG - or any other suitable polynucleotide or collection of polynucleotides - can be fragmented into any desired number of different fragment sizes, each of which can be relatively well controlled.

[0348] For example, as shown in FIG. 2A, a first purified sample 201 of WGs can be obtained that includes some or even all of the chromosomes of a given species. In the non-limiting example shown in FIG. 2A, the sample 201 includes human WGs and thus includes 23 DNA chromosomes C1, C2, ... C23. It will be understood that a given sample that can be processed as provided herein can include any suitable number of any suitable type of polynucleotides. The chromosomes C1, C2, ... C23 in the sample 201 include different sequences 210, 220 along their length, and different portions of these sequences can be used as predetermined targets for Cas-gRNA RNPs that are used to cut the chromosomes at approximately uniformly spaced locations to form fragments of approximately uniform size. Illustratively, the first sequences 210 can be spaced apart from each other by approximately a first number of base pairs, and the second sequences 220 can be spaced apart from each other by approximately a second number of base pairs. It should be noted that sequence 210 need not contain the same specific sequence at each individual location, and similarly sequence 220 need not contain the same specific sequence at each individual location. Instead, sequence 210 may represent a first set of selected locations in different chromosomes used as predetermined targets for a first set of Cas-gRNA RNPs, each of which may be targeted to a particular one of sequence 210, and sequence 220 may represent a second set of selected locations in different chromosomes used as predetermined targets for a second set of Cas-gRNA RNPs, each of which may be targeted to a particular one of sequence 220.

[0349] The composition 202 shown in FIG. 2B includes a first set 251 of Cas-gRNA RNPs hybridized to a first sequence 210 and a second set 252 of Cas-gRNA RNPs hybridized to a second sequence 220. The first set 251 and the second set 252 of Cas-gRNA RNPs can be for cleaving the first and second sequences in a sample, respectively, to generate WG fragments, each having approximately the same number of base pairs as each other. The Cas can include Cas9. The first set 251 and the second set 252 of Cas-gRNA RNPs can each include any suitable number of Cas-gRNA RNPs. Each given one of the RNPs of the first set 251 may be the same as one or more other RNPs in the first or second set, in which case such RNPs may target the same specific sequence 210 or 220 as each other, or may be different from multiple other RNPs in the first or second set, in which case such RNPs may target a different specific sequence from such other RNPs. Similarly, each given one of the RNPs of the second set 252 may be the same as one or more other RNPs in the first or second set, in which case such RNPs may target the same specific sequence 210 or 220 as each other, or may be different from multiple other RNPs in the first or second set, in which case such RNPs may target a different specific sequence from such other RNPs.

[0350] The number of RNPs in each of the first and second sets of Cas-gRNA RNPs 251, 252 can be appropriately selected to fragment a desired polynucleotide (e.g., one or more double-stranded DNA chromosomes, or the entire set of double-stranded DNA chromosomes). Illustratively, the first set of Cas-gRNA RNPs 251 can include at least about 50,000 different Cas-gRNA RNPs, or at least about 100,000 different Cas-gRNA RNPs, or at least about 1,000,000 different Cas-gRNA RNPs, or at least about 10,000,000 different Cas-gRNA RNPs, or at least about 20,000,000 different Cas-gRNA RNPs. Illustratively, the second set of Cas-gRNA RNPs 252 may include at least about 50,000 different Cas-gRNA RNPs, or at least about 100,000 different Cas-gRNA RNPs, or at least about 1,000,000 different Cas-gRNA RNPs, or at least about 10,000,000 different Cas-gRNA RNPs, or at least about 20,000,000 different Cas-gRNA RNPs.

[0351] The composition 203 shown in FIG. 2C includes or essentially consists of a set of fragments 260 resulting from such cleavage by the first set 251 and the second set 252 of Cas-gRNA RNPs, each comprising approximately X base pairs. Thus, substantially the entire WG (or any suitable polynucleotide) in the first sample 201 can be fragmented into fragments 260 of a defined size. It will be understood that the specific locations of the sequences 210, 220 along the chromosomes C1, C2, ... C23 targeted by the first and second sets 251, 252 of Cas-gRNA RNPs, respectively, can be selected to provide fragments 260 of any suitable length. In this particular example, the first number of base pairs by which the sequences 210 are spaced is approximately the same as the second number of base pairs by which the sequences 220 are spaced, such that the sequences 210 and 220 are substantially alternating along the length of each chromosome. Exemplarily, the first number of base pairs can be about 100 to about 2000 (e.g., about 500 to about 700) and the second number of base pairs can be about 100 to about 2000 (e.g., about 500 to about 700), or the first number of base pairs can be about 1000 base pairs to about 3000 base pairs (exemplarily, about 2000 base pairs) and the second number of base pairs can be about 1000 base pairs to about 3000 base pairs (exemplarily, about 2000 base pairs).

[0352] Because sequences 210 and 220 collectively are appropriately predefined and relatively equally spaced, the number of base pairs in each of fragments 260 may have a relatively tight distribution. For example, the number of base pairs in WG fragments 260 may vary by less than about 20%, or less than about 10%, or less than about 5%, or less than about 2%, or even less than about 1%. The number of base pairs (X) in each of WG fragments 260 may illustratively be about 100 base pairs to about 1000 base pairs, for example, about 200 base pairs to about 400 base pairs (exemplarily, about 300 base pairs), or about 1000 base pairs to about 3000 base pairs (exemplarily, about 2000 base pairs).

[0353] It should be noted that the first and / or second set of Cas-gRNA RNPs may be used to generate WG fragments with other lengths. Indeed, for a given WG, it may be desirable to generate fragments with different defined lengths and then compare the sequences obtained using each of such different defined lengths. As provided herein, different fragment lengths may each be generated in a different sample of WG (or different samples of other polynucleotides). For example, as shown in FIG. 2D, a second purified sample 204 of WG may be obtained, which includes 23 DNA chromosomes C1, C2, ... C23, with a first sequence 210 spaced apart from each other by approximately a first number of base pairs, and a second sequence 220 spaced apart from each other by approximately a second number of base pairs, as in sample 201 shown in FIG. 2A. Although not specifically shown in FIG. 2A, chromosomes C1, C2, ... C23 may include other sequences that may represent other sets of selected positions within different chromosomes that may be used as predetermined targets for the first set of Cas-gRNA RNPs. For example, sequences 230 shown in FIG. 2D represent a third set of selected locations in different chromosomes to be used as predetermined targets for a third set of Cas-gRNA RNPs, each of which can be targeted to a particular one of sequences 230.

[0354] The composition 205 shown in FIG. 2E includes a first set 251 of Cas-gRNA RNPs hybridized to a first sequence 210, a second set 252 of Cas-gRNA RNPs hybridized to a second sequence 220, and a third set 253 of Cas-gRNA RNPs hybridized to a third sequence 230. In a manner similar to that described with reference to FIG. 2B, the first set 251, the second set 252, and the third set 253 of Cas-gRNA RNPs may be for cleaving the first, second, and third sequences in the sample, respectively, to generate WG fragments, each having approximately the same number of base pairs as each other. The Cas may include Cas9. In a manner similar to that described with reference to FIG. 2B, the first set 251, the second set 252, and the third set 253 of Cas-gRNA RNPs may each include any suitable number of Cas-gRNA RNPs. Each given one of the RNPs of the first set 251 may be the same as one or more other RNPs in the first set, second set, or third set, in which case such RNPs may target the same specific sequence 210, 220, or 230 as each other, or may be different from multiple other RNPs in the first set, second set, or third set, in which case such RNPs may target a different specific sequence from such other RNPs. Similarly, each given one of the RNPs of the second set 252 may be the same as one or more other RNPs in the first set, second set, or third set, in which case such RNPs may target the same specific sequence 210, 220, or 230 as each other, or may be different from multiple other RNPs in the first set, second set, or third set, in which case such RNPs may target a different specific sequence from such other RNPs.Similarly, each given one of the RNPs of the third set 253 may be the same as one or more other RNPs in the first set, second set, or third set, in which case such RNPs may target the same particular sequence 210, 220, or 230 as each other, or may be different from multiple other RNPs in the first set, second set, or third set, in which case such RNP may target a different particular sequence from such other RNPs.

[0355] The number of RNPs in each of the first, second, and third sets of Cas-gRNA RNPs 251, 252, 253 can be appropriately selected to fragment a desired polynucleotide (e.g., one or more double-stranded DNA chromosomes, or the entire set of double-stranded DNA chromosomes). Illustratively, the first set of Cas-gRNA RNPs 251 can include at least about 50,000 different Cas-gRNA RNPs, or at least about 100,000 different Cas-gRNA RNPs, or at least about 1,000,000 different Cas-gRNA RNPs, or at least about 10,000,000 different Cas-gRNA RNPs, or at least about 20,000,000 different Cas-gRNA RNPs. Illustratively, the second set of Cas-gRNA RNPs 252 may include at least about 50,000 different Cas-gRNA RNPs, or at least about 100,000 different Cas-gRNA RNPs, or at least about 1,000,000 different Cas-gRNA RNPs, or at least about 10,000,000 different Cas-gRNA RNPs, or at least about 20,000,000 different Cas-gRNA RNPs. Illustratively, the third set of Cas-gRNA RNPs 253 may include at least about 50,000 different Cas-gRNA RNPs, or at least about 100,000 different Cas-gRNA RNPs, or at least about 1,000,000 different Cas-gRNA RNPs, or at least about 10,000,000 different Cas-gRNA RNPs, or at least about 20,000,000 different Cas-gRNA RNPs.

[0356] The composition 206 shown in FIG. 2F comprises or consists essentially of a set of fragments 270 resulting from such cleavage by the first set 251, the second set 252, and the third set 253 of Cas-gRNA RNPs, each comprising approximately Y base pairs (X≠Y). Thus, substantially the entire WG (or any suitable polynucleotide) in the second sample 204 can be fragmented into fragments 270 of defined size. It will be appreciated that the particular locations of the sequences 210, 220, 230 along the chromosomes C1, C2, ... C23 targeted by the first, second, and third sets 251, 252, 253 of Cas-gRNA RNPs, respectively, can be selected to provide fragments 270 of any suitable length. In this particular example, the first number of base pairs by which sequence 210 is spaced is approximately the same as the second number of base pairs by which sequence 220 is spaced, such that sequences 210 and 220 substantially alternate along the length of each chromosome, in a manner similar to that described with reference to Figures 2A-2C. However, the third number of base pairs by which sequence 230 is spaced may be different from the first and / or second number of base pairs. Thus, sequences 210 and 220 may substantially alternate along the length of each chromosome, but sequence 230 may be regularly intercalated between different ones of sequences 210 and 220, in a manner such as that shown in Figure 2E. Exemplarily, the first number of base pairs can be about 100 to about 2000 (e.g., about 500 to about 700), the second number of base pairs can be about 100 to about 2000 (e.g., about 500 to about 700), and the third number of base pairs can be about 100 to about 2000 (e.g., about 200 to about 400), or the first number of base pairs can be about 1000 to about 3000 (e.g., about 2000), the second number of base pairs can be about 1000 to about 3000 (e.g., about 2000), and the third number of base pairs can be about 500 to about 2000 (e.g., about 1000).

[0357] Because sequences 210, 220, 230 collectively are appropriately predefined and relatively equally spaced in position, the number of base pairs in each of fragments 270 may have a relatively tight distribution. For example, the number of base pairs in WG fragments 270 may vary by less than about 20%, or less than about 10%, or less than about 5%, or less than about 2%, or even less than about 1%. The number of base pairs (Y) in each of WG fragments 270 may illustratively be from about 100 base pairs to about 1000 base pairs, for example, from about 100 base pairs to about 200 base pairs (e.g., about 150 base pairs).

[0358] Comparing the process performed using sample 201 with the process performed using sample 204, it can be seen that the same set of Cas-gRNA RNPs can be used to generate WG fragments with different lengths. For example, the first and second sets of Cas-gRNA RNPs 251, 252 can be used to generate fragments 260 with length X and can also be used (in combination with the third set of Cas-gRNA RNPs 253) to generate fragments 270 with length Y (X≠Y). The first, second and / or third sets of Cas-gRNA RNPs can be similarly used to generate further fragments of defined lengths for other samples of WG without the need to provide further different sets of Cas-gRNA RNPs.

[0359] For example, as shown in FIG. 2G, a third purified sample 207 of WG can be obtained, which includes 23 DNA chromosomes C1, C2, ... C23 with a first sequence 210 spaced apart from each other by approximately a first number of base pairs, as in sample 201 shown in FIG. 2A and sample 204 shown in FIG. 2D. Although not specifically shown in FIG. 2G, chromosomes C1, C2, ... C23 can include other sequences that can represent other sets of selected locations in different chromosomes that can be used as predetermined targets for other sets of Cas-gRNA RNPs. For example, sequence 220 shown in FIG. 2A and sequence 230 shown in FIG. 2D represent other sets of selected locations in different chromosomes that can be used as predetermined targets for other sets of Cas-gRNA RNPs. Composition 208 shown in FIG. 2H includes a first set 251 of Cas-gRNA RNPs hybridized to the first sequence 210. In a similar manner to that described with reference to FIG. 2B, the first set of Cas-gRNA RNPs 251 may be for cleaving the first sequence 210 in the sample to generate WG fragments each having approximately the same number of base pairs as each other. The Cas may include Cas9. In a similar manner to that described with reference to FIG. 2B, the first set of Cas-gRNA RNPs 251 may each include any suitable number of Cas-gRNA RNPs. Each given one of the RNPs of the first set 251 may be the same as one or more other RNPs in the first set, in which case such RNPs may target the same specific sequence 210 as each other, or may be different from a plurality of other RNPs in the first set, in which case such RNPs may target a different specific sequence than such other RNPs. The number of RNPs in the first set of Cas-gRNA RNPs 251 may be suitably selected to fragment a desired polynucleotide (e.g., one or more double-stranded DNA chromosomes, or an entire set of double-stranded DNA chromosomes).Illustratively, the first set of Cas-gRNA RNPs 251 may include at least about 50,000 different Cas-gRNA RNPs, or at least about 100,000 different Cas-gRNA RNPs, or at least about 1,000,000 different Cas-gRNA RNPs, or at least about 10,000,000 different Cas-gRNA RNPs, or at least about 20,000,000 different Cas-gRNA RNPs.

[0360] The composition 209 shown in Figure 2I results from such cleavage by the first set of Cas-gRNA RNPs 251 (shown in Figure 2H) and comprises or consists essentially of a set of fragments 280, each comprising approximately Z base pairs (X ≠ Y ≠ Z). Thus, substantially the entire WG (or any suitable polynucleotide) in the third sample 207 can be fragmented into fragments 280 of defined size. It will be appreciated that the particular locations of the sequences 210 along the chromosomes C1, C2, ... C23 targeted by the first set of Cas-gRNA RNPs 251, respectively, can be selected to provide fragments 280 of any suitable length. Exemplarily, the first number of base pairs can be about 100 to about 2000 (e.g., about 500 to about 700, e.g., about 600, or about 200 to about 400, e.g., about 300), or can be about 1000 to about 3000 base pairs, e.g., about 2000. Because sequences 210 collectively are at appropriately predefined and relatively evenly spaced positions, the number of base pairs in each of fragments 280 can have a relatively tight distribution. For example, the number of base pairs in WG fragments 280 can vary by less than about 20%, or less than about 10%, or less than about 5%, or less than about 2%, or even less than about 1%. The number of base pairs (Z) in each of the WG fragments 280 can be, for example, about 100 base pairs to about 1000 base pairs, for example, about 500 to about 700 base pairs (for example, about 600), or about 200 to about 400 base pairs (for example, about 300), or about 1000 base pairs to about 3000 base pairs, for example, about 2000.

[0361] It will be appreciated that instead of using the first set 251 of Cas-gRNA RNPs with the third sample 207, either the second set 252 or the third set 253 can be used in place of the first set 251 to target the target sequence 220 or 230, which may provide fragments having other lengths instead. It will also be appreciated that any suitable number of samples (including one sample) of any suitable number of polynucleotides (including one polynucleotide) can be prepared using any suitable number of sets (including one set) of Cas-gRNA RNPs. For example, FIG. 2J shows a flow of operations in a method for generating fragments of WG. The method 2000 shown in FIG. 2J includes hybridizing a set of Cas-gRNA RNPs to sequences in a sample of WG that are spaced apart from each other by approximately a number of base pairs (operation 2001). The resulting composition may include a set of Cas-gRNA RNPs hybridized to sequences in a sample of WG that are spaced apart from each other by approximately a number of base pairs. The set of Cas-gRNA RNPs can each be for cleaving a sequence in a sample to generate WG fragments, each having approximately the same number of base pairs as each other. For example, method 2000 shown in FIG. 2J can include cleaving a sequence with a set of Cas-gRNA RNPs to generate a set of WG fragments, each having approximately the same number of base pairs as each other (operation 2002). The number of base pairs between the sequences can be about 100 to about 2000, e.g., about 500 to about 700 (e.g., about 600), or about 200 to about 400 (e.g., about 300), or about 100 to about 200 (e.g., about 150), or about 1000 base pairs to about 3000 base pairs, e.g., about 2000. In some examples, the number of base pairs in the WG fragments can be from about 100 to about 2000, e.g., from about 100 to about 200 (e.g., about 150), or from about 200 to about 400 (e.g., about 300), or from about 500 to about 700 (e.g., about 600), or from about 1000 base pairs to about 3000 base pairs, e.g., about 2000. The number of base pairs in the WG fragments of a set of WG fragments can vary by less than about 20%.

[0362] Additionally or alternatively, other samples may use one or more other sets of Cas-gRNA RNPs in combination with each other to generate fragments of WG. For example, FIG. 2K shows a flow of operations in another method of generating fragments of WG in a sample of WG. The method 2010 shown in FIG. 2K may include hybridizing a first set of Cas-gRNA RNPs to a first sequence in the WG that is spaced apart from each other by approximately a first number of base pairs (operation 2011). The method 2010 shown in FIG. 2K may also include hybridizing a second set of Cas-gRNA RNPs to a second sequence in the WG that is spaced apart from each other by approximately a second number of base pairs (operation 2012). Operations 2011 and 2012 may be performed simultaneously with each other, for example, by contacting a sample of WG with the first and second sets of Cas-gRNA RNPs. Alternatively, the sample may be contacted with a first set of Cas-gRNA RNPs followed by a second set of Cas-gRNA RNPs, or vice versa. The method 2010 shown in FIG. 2K may also further comprise cleaving the first and second sequences with the first and second sets of Cas-gRNA RNPs, respectively, in the first sample to generate a first set of WG fragments, each having approximately the same number of base pairs as each other. The first and second sequences may be cleaved simultaneously with each other. Alternatively, the first sequence may be cleaved with the first set of Cas-gRNA RNPs followed by cleavage with the second set of Cas-gRNA RNPs, or vice versa. It will be appreciated that FIG. 2K may be suitably modified to cleave additional sequences using one or more additional sets of Cas-gRNA RNPs, for example, in a manner as described with reference to FIG. 2D-FIG. 2F.

[0363] Regardless of the particular number of sets of Cas-gRNA RNPs used to cleave polynucleotides in a given sample, it will be understood that the resulting fragments may be amplified and sequenced. For example, amplification adapters may be ligated to the ends of the fragments in a manner similar to that described with reference to FIG. 1J, amplicons may be generated from the fragments having the amplification adapters ligated thereto, and the amplicons may be sequenced. For example, amplification adapters may be ligated to the ends of fragment 260, fragment 270, and / or fragment 280, and such fragments may then be amplified and sequenced. In some examples, the amplification adapters include unique molecular identifiers (UMIs). Different sets of fragments may be amplified and sequenced separately from each other, or may be mixed together for amplification and / or sequencing. Illustratively, amplicons of any suitable ones of fragments 260, 270, and / or 280 may be mixed together for amplification and / or sequencing.

[0364] Thus, provided herein is a composition comprising or consisting essentially of a set of at least about 1,000,000 WG fragments, each having approximately the same number of base pairs as each other. Illustratively, the number of base pairs can be about 100 to about 200 (e.g., about 150), or about 200 to about 400 (e.g., about 300), or about 500 to about 700 (e.g., about 600), or about 1000 to about 3000, e.g., about 2000. The composition can be derived from the entire genome of a species and can be amplified and sequenced to provide the sequence of the entire genome. The size of the WG fragments can be adjusted for use with the sequencing technique used, allowing substantially all WG in a given sample to be sequenced, as compared to mechanical fragmentation techniques, where a relatively low portion of the WG may be of a length that is usable for sequencing.

[0365] Labeling of polynucleotides using cleavage As described elsewhere herein, a unique molecular identifier (UMI) can be coupled to each polynucleotide as a way to label those polynucleotides for sequencing. Illustratively, any amplicon of a given polynucleotide molecule coupled to a given UMI can also include that UMI, thereby allowing those amplicons to be uniquely identified as originating from that polynucleotide molecule, compared to other polynucleotide molecules coupled to other UMIs. However, such UMIs can mutate during the amplification process, and such mutations can inhibit the ability to identify the polynucleotide molecule from which the amplicon originates. As provided herein, Cas-gRNA RNPs can be used to cleave polynucleotide molecules in such a way that they label those polynucleotide molecules and their amplicons for sequencing without the need for a UMI, although such a UMI can be optionally coupled to the polynucleotide to be cleaved in such a way as provided herein.

[0366] For example, Figures 3A-3E show schematics of exemplary compositions and process flow operations for labeling polynucleotides using cleavage moieties. Figure 3A shows a composition 301 including first and second molecules M1, M2 of a target polynucleotide, such as double-stranded DNA. Each of the molecules M1, M2 may have substantially the same sequence, and thus it is arbitrary which molecule is considered to be the "first" and which molecule is the "second." The sequence of the target polynucleotide may include different subsequences that can be used to cleave the polynucleotide molecules M1, M2 at one or more different positions relative to each other, and each such position of cleavage may be considered for labeling each polynucleotide molecule. For example, each of the polynucleotide molecules may comprise a first subsequence 311 to which a first Cas-gRNA RNP may be targeted (having a sequence complementary to the relevant portion of the gRNA), a second subsequence 312 to which a second Cas-gRNA RNP may be targeted, a third subsequence 313 to which a third Cas-gRNA RNP may be targeted, and a fourth subsequence 314 to which a fourth Cas-gRNA RNP may be targeted. The first and second subsequences 311, 312 may only partially overlap with each other, and the third and fourth subsequences 313, 314 may only partially overlap with each other.

[0367] In the composition 302 shown in FIG. 3B, the first and second molecules M1, M2 of the target polynucleotide are in contact with a plurality of each of the first and second Cas-gRNA RNPs 351, 352, and may also be in contact with a plurality of each of the third and fourth Cas-gRNA RNPs 353, 354, in a fluid. Depending on which of the RNPs first hybridize with the corresponding subsequence in each of the molecules M1, M2, other portions of the RNPs may be inhibited from hybridizing with other subsequences in these molecules in a manner as shown in FIG. 3B. In one non-limiting example, one of the first Cas-gRNA RNPs 351 may hybridize to a first subsequence 311 in the first molecule M1, and one of the second Cas-gRNA RNPs may hybridize to a second subsequence 312 in the second molecule M2. Since the first and second partial sequences 311, 312 only partially overlap each other, one of the first Cas-gRNA RNPs 351 hybridizing to the first molecule M1 may inhibit hybridization of any of the second Cas-gRNA RNPs 351 to the second partial sequence 311 in the first molecule M1, and one of the second Cas-gRNA RNPs 352 hybridizing to the second molecule M2 may inhibit hybridization of any of the first Cas-gRNA RNPs 351 to the first partial sequence 312 in the second molecule M2. That is, if one of the first Cas-gRNA RNPs 351 hybridizes to one of the molecules, the second Cas-gRNA RNP 352 may not hybridize to that molecule, and if one of the second Cas-gRNA RNPs 352 hybridizes to one of the molecules, the first Cas-gRNA RNP 351 may not hybridize to that molecule. The molecule may then be cleaved at the first or second subsequence 311, 312 to which the first or second Cas-gRNA RNP 351, 352 is hybridized, in a manner as described in more detail with reference to FIG. 3C. Thus, the cleavage sites may be at different positions from each other. Exemplarily, the cleavage site of the first molecule M1 may be at a different position in the sequence of the target polynucleotide than the cleavage site of the second molecule M2.It will be appreciated that in some circumstances, the same type of RNP may hybridize to both the first and second molecules M1, M2, in which case these molecules may be cleaved at the same location.

[0368] In a manner as illustrated in FIG. 3B, the third and fourth Cas-gRNA RNPs 353, 354 may similarly hybridize to the third or fourth subsequences 313, 314 and inhibit hybridization of other RNPs to those subsequences. For example, one of the third Cas-gRNA RNPs 353 may hybridize to the third subsequence 313 in the first molecule M1 and inhibit hybridization of any of the fourth Cas-gRNA RNPs to the fourth subsequence 314 in the first molecule M1. The first molecule M1 may then be cleaved at the third subsequence using one of the third Cas-gRNA RNPs 353 to generate fragments in a manner as described in more detail with reference to FIG. 3C. Alternatively, one of the fourth Cas-gRNA RNPs 354 may hybridize to the fourth subsequence 354 in the first molecule M1 and inhibit hybridization of any of the third Cas-gRNA RNPs to the third subsequence in the first molecule. The first molecule M1 may then be cleaved at the fourth subsequence using one of the fourth Cas-gRNA RNPs 354 to generate fragments, in a manner as described in more detail with reference to FIG. 3C. The RNPs may hybridize to different subsequences of the second molecule M2 in a similar manner. For example, one of the third Cas-gRNA RNPs 353 may hybridize to the third subsequence 313 in the second molecule M2 and inhibit hybridization of any of the fourth Cas-gRNA RNPs 354 to the fourth subsequence 314 in the second molecule M2. The second molecule M1 may then be cleaved at the third subsequence 313 using one of the third Cas-gRNA RNPs 354 to generate fragments, in a manner as described in more detail with reference to Figure 3C. Alternatively, one of the fourth Cas-gRNA RNPs 354 may hybridize to the fourth subsequence 314 in the second molecule M2 and inhibit hybridization of any of the third Cas-gRNA RNPs 353 to the third subsequence 313 in the second molecule M2.The second molecule M2 can then be cleaved at the fourth subsequence using one of the fourth Cas-gRNA RNPs 354 to generate fragments, in a manner as described in more detail with reference to Figure 3C. It will be understood that in some circumstances, the same type of RNP can hybridize to both the first and second molecules M1, M2, in which case these molecules can be cleaved at the same position. However, statistically, it is highly likely that at least one of the cleavages in the first and second molecules can be at different positions in the sequence of the target polynucleotide.

[0369] Referring now to FIG. 3C, the first and second molecules M1, M2 may be cleaved using Cas-gRNA RNPs to generate a composition 303. Exemplarily, the first molecule M1 may be cleaved at position 341 using one of the first Cas-gRNA RNPs 351 hybridized thereto, and the second molecule M2 may be cleaved at position 342 using one of the second Cas-gRNA RNPs. Similarly, the first molecule M1 may be cleaved at position 343 or 344 using one of the third or fourth Cas-gRNA RNPs 353, 354 hybridized thereto, and the second molecule M2 may be cleaved at position 343 or 344 using one of the third or fourth Cas-gRNA RNPs 353, 354 hybridized thereto. However, it should be understood that any molecule of the target polynucleotide may be cleaved at any suitable position, for example, at a position to which the Cas-gRNA RNP may hybridize. The cleavage at position 341 in one molecule may be offset from the cleavage at position 342 in another molecule, for example, by about 2 base pairs to about 40 base pairs (e.g., about 2-20 base pairs, or about 5-10 base pairs) in the sequence of the target polynucleotide. Similarly, the cleavage at position 343 in one molecule may be offset from the cleavage at position 344 in another molecule, for example, by about 2 base pairs to about 40 base pairs (e.g., about 2-20 base pairs, or about 5-10 base pairs) in the sequence of the target polynucleotide. Thus, as shown in FIG. 3C, fragments of different lengths and with different numbers of base pairs may be formed depending on the particular combination of cleavage sites 341 or 342 and 343 or 344 made in each of the first and second molecules M1, M2. For example, fragment 331 may have a length between the location of cut 341 and the location of cut 343. Fragment 332 may have a length between the location of cut 342 and the location of cut 344. Fragment 333 may have a length between the location of cut 341 and the location of cut 344, and fragment 334 may have a length between the location of cut 342 and the location of cut 343. It should be noted that fragments 331 and 332 may have approximately the same length as one another, but may be shorter than fragment 333 and longer than fragment 334 due to the particular locations of the cuts within the various fragments.Each of fragments 331, 332, 333, and 334 may have a length of about 100 base pairs to about 1000 base pairs, for example, about 500 base pairs to about 700 base pairs (e.g., about 600 base pairs), or about 200 base pairs to about 400 base pairs (e.g., about 300 base pairs), or about 100 base pairs to about 200 base pairs (e.g., about 150 base pairs), or about 1000 to about 3000 base pairs, for example, about 2000 base pairs.

[0370] Thus, the composition 303 shown in FIG. 3C may include a first and a second molecule M1, M2 of a target polynucleotide having a sequence. The first molecule (e.g., fragment 331 or fragment 333) may have a first end at a first subsequence 311, and the second molecule (e.g., fragment 332 or 334) may have a first end at a second subsequence 312. The first subsequence 311, 312 may only partially overlap with the second subsequence in a manner as described with reference to FIG. 4. The first end of the first molecule may be at a different position in the sequence of the target polynucleotide from the first end of the second molecule. The first end of the first molecule may be offset from the first end of the second molecule, for example, by about 2 base pairs to about 10 base pairs in the sequence of the target polynucleotide. The first molecule (e.g., fragment 331) may further have a second end at the third subsequence 313, and the second molecule (e.g., fragment 332 or 334) may further have a second end at the third subsequence 313 or the fourth subsequence 314. The third subsequence may only partially overlap with the fourth subsequence. The first end of the second molecule may be at a different position in the sequence of the target polynucleotide from the second end of the second molecule. The second end of the first molecule may be offset from the second end of the second molecule by about 2 base pairs to about 10 base pairs in the sequence of the target polynucleotide. The first and second molecules may include a different number of base pairs from each other, or may have the same number of base pairs as each other.

[0371] In some examples, the Cas comprises a Cas9 that cleaves the molecule to which each of the Cas-gRNA RNPs 351, 352, 353, and / or 354 is hybridized. In other examples, the Cas comprises an inactivated Cas9 (dCas9). In one non-limiting example, while one of the first Cas-gRNA RNPs 351 and one of the third or fourth Cas-gRNA RNPs 353, 354 are hybridized to the first molecule M1, any portion of the first molecule that is not between the first Cas-gRNA RNP and the third or fourth Cas-gRNA RNP can be degraded, for example, using exonuclease III or exonuclease VII. In another non-limiting example, while one of the second Cas-gRNA RNP 352 and one of the third or fourth Cas-gRNA RNP 353, 354 are hybridized to the second molecule M2, any part of the second molecule that is not between the second Cas-gRNA RNP and the third or fourth Cas-gRNA RNP can be degraded, for example, using exonuclease III or exonuclease VII. That is, a suitable exonuclease can be used to degrade the part of the molecule that is not located between the Cas-gRNA RNPs that hybridize to it. Thus, the Cas-gRNA RNPs can be considered to protect the part of the molecule between them.

[0372] The fragments generated using the methods of the present invention can be amplified and sequenced. For example, as shown in FIG. 3D, an amplification adapter 360 can be ligated to the ends of the fragments in a manner similar to that described with reference to FIG. 1J, amplicons can be generated from the fragments with the amplification adapter ligated thereto, and the amplicons can be sequenced. For example, an amplification adapter 360 can be ligated to the ends of fragments 331, 332, 333, 334, and such fragments can then be amplified and sequenced. In some examples, the amplification adapters include unique molecular identifiers (UMIs), although such UMIs are purely arbitrary. Any UMI can be coupled and ligated to the ends of the first and second fragments in the same operation as the amplification adapters.

[0373] Using the first subsequence 311, the second subsequence 312, the third subsequence 313, and the fourth subsequence 314, the different fragment amplicons can be identified as originating from different ones of the first and second molecules M1, M2. Illustratively, fragment 331 and its amplicon can have a first end at position 341 that falls within subsequence 311 and a second end at position 342 that falls within subsequence 313. Fragment 332 and its amplicon can have a first end at position 342 that falls within subsequence 312 and a second end at position 344 that falls within subsequence 314. Fragment 333 and its amplicon may have a first end at position 341 that falls within subsequence 311, and a second end at position 344 that falls within subsequence 314; fragment 334 and its amplicon may have a first end at position 342 that falls within subsequence 312, and a second end at position 332 that falls within subsequence 313. Thus, based on the location of each end of a given amplicon within subsequences 311, 312, 313, 314, it can be determined that such an amplicon originates from a particular one of molecules M1 or M2. Similarly, any UMI can be used to identify an amplicon as originating from a particular one of molecules M1 or M2. This ability to identify all of the reads originating from a particular molecule allows these reads to be collapsed to determine the true sequence of the original molecule. In practice, this can result in error correction and increased accuracy, allowing for the identification of true variants as opposed to errors that may be introduced during preparation and sequencing. This also provides a very efficient method of adding UMI. In comparison, UMIs ligated before amplification may suffer from low conversion efficiency. The method of the present invention can incorporate UMI identification into the cutting of the library, which is less susceptible to errors introduced during PCR and therefore may be more accurate.

[0374] FIG. 3E shows an exemplary flow of operations in a method for cleaving a polynucleotide. The method 3000 shown in FIG. 3E includes contacting a first and a second molecule of a target polynucleotide with a plurality of first and second Cas-gRNA RNPs in a fluid (operation 3001). The method 3000 shown in FIG. 3E includes hybridizing one of the first Cas-gRNA RNPs to a first subsequence in the first molecule (operation 3002). For example, one of the first Cas-gRNA RNPs 351 may hybridize to a first subsequence 311 in molecule M1 in a manner as described with reference to FIG. 3B. The method 3000 shown in FIG. 3E includes hybridizing one of the second Cas-gRNA RNPs to a second subsequence in a second molecule, where the second subsequence only partially overlaps with the first subsequence (operation 3003). For example, one of the second Cas-gRNA RNPs 352 may hybridize to the second subsequence 312 in the molecule M2 in a manner as described with reference to FIG. 3B. The method 3000 shown in FIG. 3E includes inhibiting hybridization of any of the second Cas-gRNA RNPs to the second subsequence in the first molecule by one of the first Cas-gRNA RNPs (operation 3004). For example, the first Cas-gRNA RNP 351 hybridized to the molecule M1 may inhibit the second Cas-gRNA RNP 352 from also hybridizing to the molecule. The method 3000 shown in FIG. 3E includes inhibiting hybridization of any of the first Cas-gRNA RNPs to the first subsequence in the second molecule by one of the second Cas-gRNA RNPs (operation 3005). For example, a second Cas-gRNA RNP 352 hybridized to a molecule M2 may inhibit the first Cas-gRNA RNP 351 from also hybridizing to that molecule. The method 3000 shown in Figure 3E includes cleaving a first molecule at a first subsequence (operation 3006) and cleaving a second molecule at a second subsequence (operation 3007). Exemplary operations for cleaving such molecules using Cas-gRNA RNPs are provided with reference to Figure 3C.

[0375] It can thus be seen that different molecules of a target polynucleotide can be cleaved at defined positions to generate termini at various positions, and following amplification and sequencing, the positions of such termini in the sequence of the target polynucleotide are used to identify the molecule from which the amplicon originated.

[0376] Coupling of amplification adaptors to polynucleotides Coupling of amplification adapters to polynucleotides facilitates their amplification and sequencing. As provided herein, amplification adapters can be coupled to polynucleotides using a fusion protein that includes both a Cas-gRNA RNP and a transposase. For example, Figures 4A-4J show schematic operations in an exemplary composition and process flow for introducing an amplification adapter into a polynucleotide. As shown in Figure 4A, the composition 401 can include a target polynucleotide P1 (such as a double-stranded DNA) that includes a first subsequence 410 that can be targeted using a first Cas-gRNA RNP (i.e., includes a sequence to which the gRNA of the Cas-gRNA RNP can hybridize). Optionally, the composition 401 can further include a second subsequence 420 that can be targeted using a second Cas-gRNA RNP. As shown in Figure 4B, the target polynucleotide P1 can be contacted with a first fusion protein 430 and an optional second fusion protein 440 in a fluid. The first fusion protein 430 (and, if present, the second fusion protein 440) can be in approximately stoichiometric ratio to the target polynucleotide P1 in the fluid.

[0377] The first fusion protein 430 may include a first Cas-gRNA RNP 431 coupled to a first transposase 432 to which a first amplification adaptor (shown in dashed lines) is coupled. The optional second fusion protein 440 may include a second Cas-gRNA RNP 441 coupled to a second transposase 442 to which a second amplification adaptor (shown in dotted lines) is coupled. Non-limiting examples for coupling Cas-gRNA RNPs to transposases are provided further below with reference to Figures 4F-4I. It will be understood that any suitable amplification adaptor may be coupled to a target polynucleotide using the transposases 432, 442. Illustratively, the first amplification adaptor may include a P5 adaptor and the second amplification adaptor may include a P7 adaptor. Optionally, the first amplification adaptor may also include a first unique molecular identifier (UMI) and the second amplification adaptor may include a second UMI. UMIs may be used during sequencing in the manner described elsewhere herein.

[0378] A composition 402 as shown in FIG. 4B may be provided in which the first Cas-gRNA RNP 431 hybridizes to a first subsequence 410 in the target polynucleotide P1, and the second Cas-gRNA RNP 441 hybridizes to a second subsequence 420 in the target polynucleotide, while promoting the activity of the first Cas-gRNA RNP 431 (and, if present, the second Cas-gRNA RNP 441) and inhibiting the activity of the first transposase 432 (and, if present, the second transposase 442). In some examples, using fluid conditions, the activity of the first and second Cas-gRNA RNPs 431, 441 may be promoted and the activity of the transposases 432, 442 may be inhibited. For example, it is well known that different enzymes may use certain ions to function. Illustratively, the Cas-gRNA RNP 431, 441 may function using calcium ions (Ca2+), manganese ions (Mn2+), or both calcium and manganese ions, e.g., to hybridize to sequences 420, 430, respectively. In comparison, the transposase 432, 442 may function using magnesium ions (Mg2+), e.g., to couple an amplification adapter to the target polynucleotide P. Thus, by contacting the target polynucleotide P1 with the first and second fusion proteins 430, 440 in a fluid having conditions including the presence of a sufficient amount of calcium ions, manganese ions, or both calcium and manganese ions for the activity of the Cas-gRNA RNP 432, 442, and the absence of a sufficient amount of magnesium ions for the activity of the transposase 431, 441, the Cas-gRNA RNP may function properly, but the transposase may not function properly.Additionally or alternatively, binding of the transposase to the target polynucleotide may be inhibited in any suitable manner, including, for example, reversibly blocking the binding site on the transposase, hybridizing the Cas-gRNA RNP using a temperature different from that used for the transposase, and / or delaying binding of the transposase adapter to the transposase until after the Cas-gRNA hybridizes to the target polynucleotide to delay the ability of the transposase to bind to the target polynucleotide, etc. Optionally, while the Cas-gRNA RNP 431 of the first fusion protein 430 hybridizes to the first subsequence 410 and the Cas-gRNA RNP 441 of the second fusion protein 440 hybridizes to the second subsequence 420, any portion of the target polynucleotide P1 that is not between the Cas-gRNA RNPs 431, 441 may be degraded, for example, using exonuclease III or exonuclease VII.

[0379] Subsequently, a first transposase can be used to add a first amplification adaptor to a first position in the target polynucleotide P1, and a second transposase can be used to add a second amplification adaptor to a second position in the target polynucleotide, while promoting the activity of the first and second transposases 432, 442. For example, the activity of the transposases 432, 442 can be promoted using a second condition of the fluid, such as the presence of magnesium ions in an amount sufficient for the activity of the transposase. Illustratively, the magnesium ions can be mixed into the fluid. Thus, a composition 403 as shown in FIG. 4C can be provided, in which the transposases 432, 442 act on the target polynucleotide P1 to couple the first and second amplification adaptors thereto. The target polynucleotide P1 may be released from the first and second fusion proteins 430, 440 to provide a composition 404, shown in FIG. 4D, comprising a fragment 450 of the target polynucleotide P1 having a first amplification adaptor at one end and a second amplification adaptor at the other end. Such release may be performed using proteinase K, sodium dodecyl sulfate (SDS), or both proteinase K and SDS. The amplification adaptor-coupled fragment 450 may be amplified and sequenced.

[0380] The length of the fragment 450 may be closely related to the first sequence 410 and the second sequence 420, e.g., the approximate distance between them. For example, as shown in FIG. 4C, a first Cas-gRNA RNP 431 of a fusion protein 440 may be coupled to a first transposase 432 via a linker 433, and a second Cas-gRNA RNP 441 of a fusion protein 430 may be coupled to a second transposase 442 via a linker 443. Non-limiting examples of linkers 433, 443 are provided in more detail below with reference to FIGS. 4F-4I. The linkers 433, 443 may have a defined length and thus provide a defined distance over which the transposase may move from the respective Cas-gRNA RNP. Thus, once the Cas-gRNA RNPs 431, 441 are hybridized to their respective sequences 410, 420 within the target polynucleotide P1 and the transposases 432, 442 are activated (e.g., using fluidic conditions), the transposases can each be coupled to a region of the target polynucleotide relatively close to the Cas-gRNA RNP at any position that may be permitted by the length of the linkers 433, 443. However, because the transposases may not be coupled to a specific sequence within the target polynucleotide P1 (as the Cas-gRNA RNPs are), there may be a range of positions to which the transposases can each bind. Illustratively, the first position at which the transposase 432 adds a first adaptor may be within about 10 bases of the first subsequence 410, and the second position at which the transposase 442 adds a second adaptor may be within about 10 bases of the second subsequence 420.

[0381] It will be understood that fragment 450 shown in Figure 4D can have any suitable length, for example, as approximately defined by the distance between sequences 410, 420 (illustrated in Figures 4A-4C). For example, fragment 450 can have a length of about 100 base pairs to about 1000 base pairs, for example, about 500 base pairs to about 700 base pairs (e.g., about 600 base pairs), or about 200 base pairs to about 400 base pairs (e.g., about 300 base pairs), or about 100 base pairs to about 200 base pairs (e.g., about 150 base pairs), or about 1000 base pairs to about 3000 base pairs (e.g., about 2000 base pairs).

[0382] As shown in FIG. 4E, each gRNA 434, 444 in the first and second fusion proteins 430, 440 may have any suitable length and sequence to facilitate its hybridization to the respective sequences 410, 420. For example, the 5' end of the gRNA 434, 444 that hybridizes to the first or second subsequence 410, 420 may be truncated relative to the 5' end of a gRNA more typically used in a Cas-gRNA RNP. Illustratively, as shown in FIG. 4E, a typical gRNA may have a 5' end of length x, where x may be about 20 nucleotides, while the gRNA 434, 444 may have a 5' end of length y, where y is less than x. In some examples, the portion y of gRNA 434 that hybridizes to the first subsequence 410 may have a length of about 15 to about 18 nucleotides, and the portion y of gRNA 444 that hybridizes to the second subsequence 420 may have a length of about 15 to about 18 nucleotides. For further details regarding truncated gRNAs, see Fu et al., "Improving CRISPR-Cas nuclease specificity using truncated guide RNAs," Nat. Biotechnol. 32(3):279-284 (2014), the entire contents of which are incorporated herein by reference.

[0383] It will be understood that any suitable Cas and any suitable transposase can be used in the fusion protein 430, 440. Exemplarily, the Cas may include dCas9 (e.g., to prevent the Cas from cleaving the target polynucleotide P1 before the transposase is activated), and the transposase may include Tn5 (e.g., so that the activity of the transposase can be well controlled through fluid conditions, such as adding a sufficient amount of magnesium ions). The Cas and the transposase can be coupled to each other via any suitable coupling, for example, via a covalent bond or via a non-covalent bond. The covalent bond can be formed by, exemplarily, a copper(I)-catalyzed click reaction, or a strain-promoted azide-alkyne cycloaddition. The non-covalent bond can be formed in any suitable manner. For example, in a manner as shown in FIG. 4F, the Cas-gRNA RNP can be covalently coupled to an antibody 461 and the transposase can be covalently coupled to an antigen 462 to which the antibody is non-covalently coupled, or in a manner as shown in FIG. 4G, the Cas-gRNA RNP can be covalently coupled to an antigen 461 and the transposase can be covalently coupled to an antibody 462 to which the antigen is non-covalently coupled. Alternatively, in a manner as shown in FIG. 4H, the Cas-gRNA can be non-covalently coupled to the transposase via hybridization between a portion 463 of the gRNA and a first or second amplification adapter. As yet another example, in a manner as shown in FIG. 4I, the Cas-gRNA can be non-covalently coupled to the transposase via hybridization between a portion 464 of the gRNA and an oligonucleotide 465 within the transposase.For further examples of how Cas can be coupled to another protein, see the following references, the entire contents of which are incorporated herein by reference: Guilinger et al., "Fusion of catalytically inactive Cas9 to Fokl nuclease improves the specificity of genome modification," Nature Biotechnology 32:577-582 (2014); and Bhatt et al., "Targeted DNA transposition in vitro using a dCas9-transposase fusion protein," Nucleic Acids Res. 47:8126-8135 (2019).

[0384] FIG. 4J shows an exemplary flow of operations in a method for generating a fragment of a target polynucleotide having a sequence. The method 4000 shown in FIG. 4J includes contacting a target polynucleotide in a fluid with a first and a second fusion protein (operation 4001), each of which includes a Cas-gRNA RNP coupled to a transposase coupled with an amplification adaptor. For example, the target polynucleotide P1 can be contacted with a first fusion protein 430 and a second fusion protein 440 in a manner as described with reference to FIG. 4B. The method 4000 shown in FIG. 4J includes (i) hybridizing a first Cas-gRNA RNP to a first subsequence in the target polynucleotide, and (ii) hybridizing a second Cas-gRNA RNP to a second subsequence in the target polynucleotide (operation 4002), while promoting the activity of the Cas-gRNA RNP and inhibiting the activity of the transposase. For example, the fluid may have a first condition that promotes such hybridization to a first subsequence 410 of the first Cas-gRNA RNP 431 and to a second subsequence 420 of the second Cas-gRNA RNP 442 while inhibiting activity of the transposases 432 and 442 in a manner as described with reference to FIG. 4B (illustratively, the presence of a sufficient amount of Ca2+ and / or Mn2+ and the absence of a sufficient amount of Mg2+). The method 4000 shown in FIG. 4J includes (i) adding a first amplification adaptor to a first position in the target polynucleotide using a first transposase, and (ii) adding a second amplification adaptor to a second position in the target polynucleotide using a second transposase (operation 4003), while promoting activity of the first and second transposases. For example, the fluid may have a second condition (illustratively, the presence of a sufficient amount of Mg2+) that promotes activity of the first and second transposases 432 and 442 in a manner as described with reference to FIG. 4C.

[0385] In some embodiments, ShCAST (Scytonema hofmanni CRISPR-associated transposase) targeted library preparation and enrichment may be used.

[0386] Targeted sequencing of specific genes using separate enrichment steps after library preparation can be time-consuming.For example, such separate enrichment steps can include hybridizing oligonucleotide probes to library DNA and isolating hybridized DNA on streptavidin-coated beads.Although efficiency and time required have improved significantly, such separate enrichment protocols can take about 2 hours and many reagents, making automation of such protocols difficult.

[0387] In comparison, some examples herein can be used to prepare and enrich libraries for targeted sequencing of specific genes using a single step for both preparation and enrichment.

[0388] For example, Figures 7A and 7H show schematics of exemplary compositions and operations in a process flow for coupling an amplification adaptor to a polynucleotide. Referring first to Figure 7A, a composition 701 may include a target polynucleotide P3 (such as double-stranded DNA) that includes a first subsequence 710 that may be targeted using a first Cas-gRNA RNP (i.e., includes a sequence to which a gRNA of the Cas-gRNA RNP may hybridize). Optionally, the composition 701 may further include a second subsequence 720 that may be targeted using a second Cas-gRNA RNP. The target polynucleotide P3 may include partially fragmented dsDNA, such as cell-free DNA, or DNA that has been fragmented in a manner as described elsewhere herein. Alternatively, the target polynucleotide P3 may include whole chromosomal DNA. As shown in Figure 7B, the target polynucleotide P3 may be contacted in a fluid with a first fusion protein 730 and an optional second fusion protein 740 in a manner similar to that described with reference to Figures 4A-4D. The first fusion protein 730 (and, if present, the second fusion protein 740) can be in approximately stoichiometric ratio to the target polynucleotide P3 in the fluid.

[0389] The first fusion protein 730 may include a first Cas-gRNA RNP 731, which includes a tag 733, coupled to a first transposase 732 to which a first amplification adaptor (shown by a dashed line) is coupled. The optional second fusion protein 740 may include a second Cas-gRNA RNP 741, which includes a tag 733, coupled to a second transposase 742 to which a second amplification adaptor (shown by a dotted line) is coupled. The tag 733 may be coupled to any suitable portion of any gRNA RNP of the respective Cas-gRNA RNP. Non-limiting examples for coupling the Cas-gRNA RNP to the transposase are further provided above with reference to Figures 4F-4I. It will be understood that any suitable amplification adaptor may be coupled to the target polynucleotide using the transposase 732, 742. Illustratively, the first amplification adaptor may include a P5 adaptor and the second amplification adaptor may include a P7 adaptor. Optionally, the first amplification adapter may also include a first unique molecular identifier (UMI) and the second amplification adapter may include a second UMI, which may be used during sequencing in a manner as described elsewhere herein.

[0390] A composition 702 as shown in FIG. 7B may be provided in which the first Cas-gRNA RNP 731 hybridizes to a first subsequence 710 in the target polynucleotide P3 and the second Cas-gRNA RNP 741 hybridizes to a second subsequence 720 in the target polynucleotide, while promoting the activity of the first Cas-gRNA RNP 731 (and, if present, the second Cas-gRNA RNP 741) and inhibiting the activity of the first transposase 732 (and, if present, the second transposase 742). In some examples, the activity of the first and second Cas-gRNA RNPs 731, 741 may be promoted and the activity of the transposases 732, 742 may be inhibited using fluid conditions in a manner as described with reference to FIGS. 4A-4D.

[0391] The target polynucleotide P3 may be enriched using the tag 733. For example, in the composition 703 shown in FIG. 7C, a target polynucleotide having hybridized first and second Cas-gRNA RNPs 731, 732 (coupled to a tag 733 and a transposase 732, 742, respectively) may be contacted with a substrate 750 coupled to a tag partner 751 via a respective linker. The tag partner 751 may be selected to be covalently or non-covalently coupled to the tag 733, forming a composition 704 as shown in FIG. 7D, in which the target polynucleotide P3 is coupled to the substrate 750 via the tag 733 and the tag partner 751. Any other polynucleotides not coupled to the substrate 750 may be washed away.

[0392] Subsequently, a first transposase can be used to add a first amplification adaptor to a first position in the target polynucleotide P3, and a second transposase can be used to add a second amplification adaptor to a second position in the target polynucleotide, while promoting the activity of the first and second transposases 732, 742. For example, the activity of the transposases 732, 742 can be promoted using a second condition of the fluid, in a manner as described with reference to Figures 4A-4D. Thus, a composition 705 as shown in Figure 7E can be provided, in which the transposases 732, 742 act on the target polynucleotide P3 to couple the first and second amplification adaptors thereto. The polynucleotide P3 can be released from the first and second fusion proteins 730, 740 to provide a composition 706 as shown in Figure 7F, comprising a fragment 760 of the target polynucleotide P3 having a first amplification adaptor at one end and a second amplification adaptor at the other end. Such release may be performed using proteinase K, sodium dodecyl sulfate (SDS), or both proteinase K and SDS; by denaturing the Cas-gRNA RNP 731, 741, decoupling the tag 733 from the tag partner 751, cleaving the linker between the tag partner 751 and the substrate 750, etc. Alternatively, the fragments 760 may remain coupled to the substrate 750 for subsequent processing. In either example, the resulting enriched fragments 760 shown in FIG. 7F (with any coupling to the substrate 750 not specifically shown) may be further analyzed in a manner as described with reference to FIGS. 5G-5H or 5I-5J.

[0393] The amplification adaptor-coupled fragment 760 may be amplified and sequenced. In a manner as described with reference to FIGS. 4A-4E, the length of the fragment 760 may be closely related to the first sequence 710 and the second sequence 720, e.g., the approximate distance between them. It will be understood that the fragment 760 shown in FIG. 7G may have any suitable length, e.g., approximately defined by the distance between the sequences 710, 720. For example, the fragment 760 may have a length of about 100 base pairs to about 1000 base pairs, e.g., about 500 base pairs to about 700 base pairs (e.g., about 600 base pairs), or about 200 base pairs to about 700 base pairs (e.g., about 300 base pairs), or about 100 base pairs to about 200 base pairs (e.g., about 150 base pairs), or about 1000 base pairs to about 3000 base pairs (e.g., about 2000 base pairs).

[0394] It will be appreciated that any suitable tag 733 and tag partner 751 may be used to pull down the target polynucleotide P3 to the substrate 750. For example, the tag partner 751 may comprise a SNAP protein and the tag 733 may comprise O-benzylguanine; the tag partner may comprise a CLIP protein and the tag may comprise O-benzylcytosine; the tag partner may comprise SpyTag and the tag may comprise SpyCatcher; the tag partner may comprise SpyCatcher and the tag may comprise SpyTag; the tag partner may comprise biotin and the tag may comprise streptavidin; the tag partner may comprise streptavidin and the tag may comprise biotin; the tag partner may comprise NTA, The tag may comprise a His tag; the tag partner may comprise a His tag, and the tag may comprise an NTA; the tag partner may comprise an antibody (e.g., an anti-FLAG antibody), and the tag may comprise an antigen (e.g., a FLAG tag) for which the antibody is selective; the tag partner may comprise an antigen (e.g., a FLAG tag), and the tag may comprise an antibody (e.g., an anti-FLAG antibody) selective for the antigen; or the tag partner may comprise a first oligonucleotide, and the tag may comprise a second oligonucleotide that is complementary to the first oligonucleotide and hybridizes to the first oligonucleotide. The tag partner 751 may be coupled to the substrate 750 via any suitable bond, for example, via a covalent bond or a non-covalent bond. Similarly, the tag 733 may be coupled to the Cas-gRNA RNPs 731, 732, respectively, via any suitable coupling, for example, via a covalent bond or a non-covalent bond, for example, in a manner similar to that described with reference to Figures 4F-4I. In some examples, the gRNA 734, 744 in the first and second fusion proteins 730, 740, respectively, can be coupled to a tag 733 in a manner as shown in Figure 7G. For example, RNA oligonucleotides coupled to tags can be purchased commercially and their preparation is known in the art.

[0395] It will be understood that any suitable Cas and any suitable transposase may be used in the fusion proteins 730, 740. Illustratively, the Cas may include dCas9 (e.g., to prevent the Cas from cleaving the target polynucleotide P3 before the transposase is activated) and the transposase may include Tn5 (e.g., so that the activity of the transposase can be well controlled through fluid conditions, such as adding a sufficient amount of magnesium ions). In other examples, the Cas may include Cas12k and the transposase may include Tn7 or a Tn7-like transposase (e.g., so that the activity of the transposase can be well controlled through fluid conditions, such as adding a sufficient amount of magnesium ions). The Cas and the transposase may be coupled to each other via any suitable bond, such as via a covalent bond or via a non-covalent bond, for example, as described in Figures 4F-I or in Strecker et al.

[0396] For example, Figures 6A and 6B show schematic compositions and operations in an exemplary process for ShCAST (Scytonema hofmanni CRISPR-associated transposase) targeted library preparation and enrichment. ShCAST 6000 includes Cas12k 6001 and Tn7-like transposase 6002 that can insert DNA 6003 into specific sites in the E. coli genome using RNA guides 6004. Some examples provided herein utilize ShCAST or a modified version of ShCAST incorporating Tn5 transposase (ShCAST-Tn5) for targeted amplification of specific genes. In this way, library preparation and enrichment steps are combined, thus simplifying, improving and facilitating automation of workflow efficiency of targeted library sequencing.

[0397] Illustratively, gRNA 6004 may be designed to target a specific gene (sequence) and gRNA spacing may control insert size. In some examples, gRNA 6004 and / or ShCAST / ShCAST-Tn5 6002 may be coupled to a tag 6005, e.g., biotinylated. In a manner as shown in FIG. 6A, a transposable element having gRNA 6004 and an adaptor 6003 (e.g., an Illumina adaptor) can be included on ShCAST transposase 6002 to obtain complex 6000. In a manner as shown in process flow 6010 of FIG. 6B, the resulting ShCAST / ShCAST-Tn5 complexes 6000 can be mixed with genomic DNA (target polynucleotide) 6011 under fluidic conditions (e.g., low or no magnesium, Mg2+) that allow the complexes to bind to their respective sequences in the target DNA while inhibiting tagmentation, in a manner similar to that described with reference to FIGS. 4A-4J and 7A-7G. The complexes can then be isolated using a substrate coupled to a tag partner, such as a tagged (e.g., biotinylated) gRNA and / or streptavidin beads 6012 to which ShCAST / ShCAST-Tn5 is coupled. Any unbound DNA can be washed away, for example to reduce or minimize off-target tagmentation. Fluidic conditions can then be altered (e.g., increase magnesium sufficiently) to promote tagmentation, in a manner similar to that described with reference to FIGS. 4A-4J. A gap-filling ligation step followed by thermal dissociation can be used to release the library from the beads in preparation for sequencing.

[0398] It should be noted that in compositions and operations as shown in Figures 6A-6B, the transposase portion 6002 of complex 6000 may be able to randomly insert into DNA. Such insertions may be inhibited or minimized by mixing the ShCAST / ShCAST-Tn5 complex with genomic DNA under fluid conditions (e.g., low or no magnesium) that inhibit tagmentation and thereby allow the target to be bound.

[0399] For further details regarding ShCAST, which contains Cas12k and Tn7, see Strecker et al., "RNA-Guided DNA insertion with CRISPR-associated transposases," Science 365(6448):48-53 (2019), the entire contents of which are incorporated herein by reference.

[0400] It should be understood that the tag 733 or tag 6005 may be coupled to the tag partner (and thus to the substrate) at any suitable time, and such coupling does not necessarily have to occur after the fusion protein or complex has been coupled to the target polynucleotide, and may in fact occur before the fusion protein or complex binds to the target polynucleotide. Illustratively, a gRNA 734, 744 coupled to the tag 733 in the manner illustrated in FIG. 7G may be coupled to the substrate 750 using the interaction between the tag 733 and the tag partner 751. The Cas (which also includes the transposase) of the fusion protein or complex may then be coupled to the substrate-bound gRNA. The target polynucleotide may then be coupled to the Cas, thus coupling the target polynucleotide to the substrate.

[0401] It should also be appreciated that the process flow described with reference to Figure 4J may be modified to include the use of tags in the manner described with reference to Figures 6A-6B and 7A-7G. For example, at any suitable time point relative to operations 4001 and 4002, the target polynucleotide may be pulled down onto a substrate using tags coupled to the Cas-gRNA RNPs, respectively. In the manner described with reference to Figures 7A-7F, the tag may be coupled to the Cas-gRNA RNPs prior to contacting the polynucleotide with the Cas-gRNA RNPs. Alternatively, the tag may be coupled to a tag partner coupled to the gRNA and the substrate, and a Cas-transposase fusion protein or complex is contacted with the gRNA. Operation 4003 may then be performed to promote activity of the transposase and add an amplification adaptor to the target polynucleotide.

[0402] Thus, it will be appreciated that a polynucleotide can be cleaved at any suitable pair of positions to form fragments, and any suitable amplification primers can be attached to the ends of the resulting fragments using the Cas-gRNA RNP / transposase fusion protein. The fragments can then be amplified and sequenced.

[0403] Compositions and methods for targeted epigenetic assays Some examples herein provide enrichment of polynucleotides (such as DNA) to generate epigenetic fragments of interest using Cas-gRNA RNPs, and assaying for proteins at loci along those fragments. Some non-limiting examples of assays are given with specific workflow operations and sequencing, but other examples can be easily envisioned. In this example, proteins along the fragments can be labeled using oligonucleotides, which can then be sequenced and the proteins can be characterized using the oligonucleotides. For example, the sequence of the oligonucleotides can provide information about the presence of a protein at a given fragment locus, can provide information about the location of a protein at a given fragment locus, can provide information about the amount of a protein at a given fragment locus, or can provide any suitable combination of such information. Fragments can be enriched, e.g., specifically selected from a given polynucleotide, while other portions of that polynucleotide and portions of other polynucleotides can be discarded. Such locus-associated proteomic analyses can be used, illustratively, to provide a genome-wide proteome atlas that complements whole genome sequencing to improve characterization of genotype-phenotype relationships, or to better characterize epigenetic signatures associated with specific loci and understand epigenetic mechanisms important for research or clinical applications and treatments.

[0404] For example, Figures 5A-5K show schematics of exemplary compositions and operations in a process flow for targeted epigenetic assays. As shown in Figure 5A, composition 501 may include a target polynucleotide P2 (such as double-stranded DNA) that includes a first subsequence 511 that may be targeted using a first Cas-gRNA RNP (i.e., includes a sequence to which a gRNA of the Cas-gRNA RNP may hybridize), and a second subsequence 512 that may be targeted using a second Cas-gRNA RNP. Target polynucleotide P2 may include a fragment generated in a manner as described in more detail elsewhere herein, for example, with reference to Figures 2A-2K, 3A-3E, or 4A-4J, or may include an entire chromosome or a portion thereof. Proteins 521, 522, and chromatin 523 may be coupled to the respective loci of target polynucleotide P2 between the first subsequence 511 and the second subsequence 512. Optionally, proteins 521, 522 may be crosslinked to increase their stability during subsequent processing operations, e.g., to leave them in place along the target polynucleotide P2 while preserving their ability to be selectively targeted by corresponding antibodies in the manner described below.

[0405] In the exemplary composition 502 shown in FIG. 5B, the target polynucleotide P2 may be contacted in a fluid with a first Cas-gRNA RNP 531 and a second Cas-gRNA RNP 532. The first Cas-gRNA RNP 531 and the second Cas-gRNA RNP 532 may each include a respective tag 533 that may be used to selectively pull down a portion of the target polynucleotide P2 between the first subsequence 511 and the second subsequence 512, thus concentrating that portion of the polynucleotide in a manner as described in more detail below with reference to FIGS. 5D-5F. In a non-limiting example, the Cas includes Cas9 or other suitable Cas capable of cleaving the target polynucleotide P2. The first and second Cas-gRNA RNPs 531, 532 hybridize to the first and second subsequences 511, 512 within the target polynucleotide P2 and cleave the target polynucleotide at the first and second subsequences, respectively, to form fragments. Illustratively, the resulting composition 503 shown in FIG. 5C includes a fragment 540 having one first protein 521, two second proteins 522, and chromatin 523 coupled to its respective locus, and first and second Cas-gRNA RNPs 531, 532 each coupled to a tag 533 and hybridized to subsequences 511, 512, respectively, thus coupling the tag 533 to the fragment 540. The fragment 540 can have any suitable length, for example, from about 100 base pairs to about 1000 base pairs, for example, from about 500 base pairs to about 700 base pairs, or from about 200 base pairs to about 400 base pairs, or from about 100 base pairs to about 200 base pairs, or from about 1000 base pairs to about 3000 base pairs (illustratively, about 2000 base pairs). The remaining portions 541, 542 of polynucleotide P2 may be of any length and in some instances may form the remainder of the chromosome after removal of fragment 540.

[0406] The fragments 540 can be enriched using the tag 533. For example, as shown in FIG. 5D, the hybridized fragments 540 of the first and second Cas-gRNA RNPs 531, 532 (each coupled to a tag 533), and the remaining portions 541, 542 of the polynucleotide P2 can be contacted with a substrate 550 coupled to a tag partner 551 via a respective linker. The tag partner 551 can be selected to be covalently or non-covalently coupled to the tag 533, forming a composition as shown in FIG. 5E, where the fragments 540 are coupled to the substrate 550 via the tag 533 and the tag partner 551, while the remaining portions 541, 542 are not coupled to the substrate 550 and can be washed away. The fragments 540 can then be released from the substrate 550, for example, by denaturing the Cas-gRNA RNPs 531, 532 (in which case the proteins 521, 522 may be pre-crosslinked to inhibit their denaturation), by decoupling the tag 533 from the tag partner 551, by cleaving a linker between the tag partner 551 and the substrate, etc. Alternatively, the fragments 540 may remain coupled to the substrate 550 for subsequent processing. In either example, the resulting enriched fragments 540 shown in FIG. 5F (with any coupling to the substrate 550 not specifically shown) can be further analyzed in a manner as described with reference to FIGS. 5G-5H or 5I-5J.

[0407] It will be appreciated that any suitable tag 533 and tag partner 551 may be used to pull down fragments 540. For example, the tag partner 551 may comprise a SNAP protein and the tag 533 may comprise O-benzylguanine; the tag partner may comprise a CLIP protein and the tag may comprise O-benzylcytosine; the tag partner may comprise SpyTag and the tag may comprise SpyCatcher; the tag partner may comprise SpyCatcher and the tag may comprise SpyTag; the tag partner may comprise biotin and the tag may comprise streptavidin; the tag partner may comprise streptavidin and the tag may comprise biotin; the tag partner may comprise NTA, The tag may comprise a His tag; the tag partner may comprise a His tag, and the tag may comprise an NTA; the tag partner may comprise an antibody (e.g., an anti-FLAG antibody), and the tag may comprise an antigen (e.g., a FLAG tag) for which the antibody is selective; the tag partner may comprise an antigen (e.g., a FLAG tag), and the tag may comprise an antibody (e.g., an anti-FLAG antibody) selective for the antigen; or the tag partner may comprise a first oligonucleotide, and the tag may comprise a second oligonucleotide that is complementary to the first oligonucleotide and hybridizes to the first oligonucleotide. The tags 533 may each be coupled to the Cas-gRNA RNPs 531, 532 via any suitable coupling, e.g., via a covalent bond or via a non-covalent bond, in a manner similar to that described with reference to Figures 4F-4I or 7G. Similarly, the tag partner 551 may be coupled to the substrate 550 via any suitable bond, e.g., via a covalent bond or via a non-covalent bond.

[0408] As provided herein, corresponding oligonucleotides may be used to label each of the proteins 521, 522 coupled to each locus of the fragments (which may be prepared and enriched in the manner described with reference to Figures 5A-5F), and such oligonucleotides may then be sequenced. The corresponding oligonucleotides may be used to identify the proteins, to identify the loci, and / or to quantify the proteins.

[0409] In some examples described herein with reference to Figures 5G-5H, using corresponding oligonucleotides to label each of the proteins may include contacting the enriched fragments 540 with a mixture of antibodies specific for different proteins, each of the antibodies coupled to a corresponding oligonucleotide that may be used to label the protein in a manner that characterizes the protein. For example, the composition 504 shown in Figure 5G includes the enriched fragments 540 in contact with a plurality of first, second, third, and fourth antibodies 551, 552, 553, 554, each of which is coupled to a corresponding first, second, third, and fourth oligonucleotide, respectively. Each of the antibodies 551, 552, 553, 554 is specific for a different protein. It will be understood that the enriched fragments 540 may be contacted with any suitable number and type of different antibodies specific for different proteins or other chromatin that may potentially be coupled to loci along the fragment and may be epigenetic targets. For any antibodies in the mixture specific for proteins coupled to the respective loci of the enriched fragment 540, those antibodies and corresponding oligonucleotides can become non-covalently coupled to those proteins via antibody / target binding. In the non-limiting exemplary composition 505 shown in FIG. 5E, a first antibody 551 is specific for and coupled to a first protein 521, while a second antibody 552 is specific for and coupled to a second protein 522. A plurality of second proteins 522 are coupled to respective ones of the loci, and a plurality of second antibodies 552 in the mixture are coupled to proteins at that locus. In this example, the enriched fragment 540 does not include the proteins for which the third and fourth antibodies 553, 554 are specific, and therefore these antibodies (and their respective oligonucleotides) are not coupled to the fragment.

[0410] Custom oligonucleotide-conjugated antibodies are commercially available or can be prepared using known techniques, for example, as described in the following references, the entire contents of each of which are incorporated herein by reference: Gong et al., "Simple method to prepare oligonucleotide-conjugated antibodies and its application to multiplex protein detection in single cells", Bioconjugate Chem. 27:217-225 (2016); and Stoeckius et al., "Simultaneous epitope and transcriptome measurement in single cells", Nature Methods 14:865-868 (2017).

[0411] The first and second oligonucleotides coupled to the antibodies 551, 552, respectively, may be sequenced and used to identify the presence, and optionally the amount, of the proteins 521, 522, respectively, in the enriched fragment 540. In some examples, the first and second oligonucleotides may be released from the fragment 540, for example, by applying a protease that digests the proteins 521, 522 and the antibodies 551, 552, and then amplified and sequenced. Such sequencing may be performed in any suitable manner. For example, sequencing the corresponding oligonucleotides may include hybridizing the corresponding oligonucleotides to a bead array, for example, using Illumina BeadArray™ technology (San Diego, Calif.), or performing sequencing by synthesis (SBS) on the corresponding oligonucleotides. The oligonucleotides may optionally include amplification adapters (e.g., P5 and P7 adapters, or Y-shaped adapters) and / or UMIs, or such amplification adapters and / or UMIs may be added to the oligonucleotides using known techniques, such as PCR, prior to amplification and sequencing.

[0412] Regardless of the particular sequencing method used, the presence of each of the corresponding oligonucleotides can be used to identify and optionally quantify the proteins coupled to the enriched fragment 540. For example, the presence of the first and second oligonucleotides can be detected using bead arrays or SBS, and based on such presence it can be deduced that the first and second proteins 521, 522 were present in the fragment 540. The amount of each of the corresponding oligonucleotides can also be used to quantify the proteins. For example, since the enriched fragment 540 contained two second proteins 522, two copies of the second antibody 552 will bind to it along with two copies of the second oligonucleotide, compared to one first protein 521 that will bind to one copy of the first antibody 551 and one copy of the first oligonucleotide. The relative amounts of the first oligonucleotide (1 copy) and the second oligonucleotide (2 copies) indicate the relative amounts of the first protein 521 (1 copy) and the second protein 522 (2 copies) in the enriched fragment 540. The absence of the third and fourth oligonucleotides indicates that the proteins for which the third and fourth antibodies 553, 554, respectively, are selective were not present in the enriched fragment 540. Thus, the method of the present invention provides an assay of the epigenetic signature of the enriched fragment 540, and more specifically, the proteins coupled to loci along the enriched fragment 540.

[0413] In other examples described herein with reference to Figures 5I-5J, using a corresponding oligonucleotide to label each of the proteins may include contacting the fragment with a plurality of transposases, each of which is coupled to a corresponding oligonucleotide that may be used to label the protein in a manner that characterizes the protein. For example, composition 506 shown in Figure 5I includes enriched fragment 540 (which may be prepared in a manner described with reference to Figures 5A-5F) that is contacted with a plurality of transposases 561, each of which comprises an oligonucleotide. In a non-limiting example, the transposase may include Tn5.

[0414] Proteins coupled to the respective loci of the enriched fragments may inhibit activity of the transposase at that locus. Thus, transposase 561 may become coupled to fragment 540 at a location other than the locus. At the location where transposase 561 is coupled to fragment 540, the transposase may bind a corresponding oligonucleotide to the fragment. Such a process may split fragment 540 into subfragments. In a non-limiting example of composition 507 shown in FIG. 5J, subfragment 571 includes first protein 521 and an oligonucleotide, subfragment 572 includes chromatin 523 and an oligonucleotide, and subfragment 573 includes protein 522 and an oligonucleotide. In this regard, it should be noted that transposase 561 (shown in FIG. 5I) can bind to fragment 540 at any location that is not inhibited by the presence of proteins 521, 522, or chromatin 523 (i.e., is not specific to a given protein or portion of the fragment), and therefore such transposases can add their respective oligonucleotides to any such location.

[0415] The oligonucleotides coupled to the second, first and third fragments 571, 572, 573, respectively, can be sequenced, for example in a manner as described with reference to Figures 5G-5H, and used to identify the presence and, optionally, the amount of proteins 521, 522 and chromatin 523, respectively. The respective positions in the oligonucleotide fragments 571, 572, 573 can be used to identify the respective loci of the proteins and / or chromatin. For example, in the purely exemplary diagram shown in Figures 5I and 5J, protein 521 inhibits any transposases from acting at that protein's locus, protein 522 inhibits any transposases from acting at those proteins' loci, and chromatin 523 inhibits any transposases from acting where that chromatin is located. Thus, the respective positions of protein 522, 521 and / or chromatin 523 in the second, first and third oligonucleotides in fragments 572, 571, 573 may be understood to be positions other than the positions to which the oligonucleotides are attached.

[0416] FIG. 5K shows an exemplary flow of operations in a method 5000 for characterizing proteins coupled to respective loci of a target polynucleotide. Method 5000 may include contacting a target polynucleotide with a first and a second Cas-gRNA RNP (operation 5001), for example, in a manner as described with reference to FIGS. 5A-5C. Optionally, method 5000 may include enriching the fragments before labeling each of the proteins coupled to the respective loci of the fragments, respectively, using corresponding oligonucleotides. For example, each of the first and second Cas-gRNA RNPs may be coupled to a tag, such that the fragments are coupled to the tag via the first and second Cas-gRNA RNPs, for example, in a manner as described with reference to FIGS. 5B-5C. Enriching may include contacting the fragments coupled to the tag via the first and second Cas-gRNA RNPs with a substrate coupled to a tag partner, for example, in a manner as described with reference to FIG. 5D. The enriching may further include coupling the tag to a tag partner and coupling the fragments to a substrate, for example, in a manner as described with reference to Figure 5E. The enriching may further include removing any portion of the target polynucleotide that is not coupled to the substrate, for example, in a manner as described with reference to Figure 5F.

[0417] Method 5000 may include hybridizing a first and a second Cas-gRNA RNP to a first and a second subsequence in a target polynucleotide, respectively, and a protein is coupled to each locus of the target polynucleotide between the first and the second subsequence (operation 5002), for example, in a manner as described with reference to Figures 5A-5C. Method 5000 may include cleaving the target polynucleotide at the first subsequence using the first Cas-gRNA RNP and at the second subsequence using the second Cas-gRNA RNP to form fragments, and a protein is coupled to each locus of the fragments (operation 5003), for example, in a manner as described with reference to Figures 5A-5C. Method 5000 may include using corresponding oligonucleotides to label each of the proteins coupled to each locus of the fragment (operation 5004), for example, in a manner as described with reference to Figures 5G-5H and / or in a manner as described with reference to Figures 5I-5J, and sequencing the corresponding oligonucleotides (operation 5005).

[0418] It will be appreciated that process flows as described with reference to Figures 5G-5H and 5I-5J, respectively, may be performed using polynucleotides of any suitable length, and need not necessarily be performed using fragments generated using process flows as described with reference to Figures 5A-5C, and therefore operations 5001-5003 of method 5000 described with reference to Figure 5K should be understood to be optional.

[0419] Thus, from Figures 5A-5K, it can be seen that in some examples herein, Cas-gRNA RNPs can be used to generate and enrich polynucleotide fragments coupled to proteins, and the location, amount, and / or identity of those proteins can be characterized using epigenetic assays as described herein.

[0420] Enrichment of selected fragments of polynucleotides using Cas-gRNA RNP nickase Some methods provided herein solve the problem of long and tedious workflows for targeted sequencing of intact dsDNA fragments. As is evident from the present disclosure, Cas-gRNA RNPs can provide rapid and specific cleavage of target regions in polynucleotides, e.g., dsDNA. As described herein with reference to Figures 8A-8H, Cas-gRNA RNP nickase and polymerase extension can be used to selectively enrich dsDNA fragments by elution from the substrate. Such methods and compositions can be used to recover intact source fragments. This can be particularly useful in applications where complete dsDNA cleavage by Cas-gRNA RNPs may not be desirable, e.g., cell-free DNA (cfDNA) sequencing. This can also, or alternatively, be useful because the underlying size of the sequencing library is not altered by CRISPR cleavage, which means that the generation of very short products is reduced or avoided.

[0421] More specifically, Figures 8A and 8H show schematics of exemplary compositions and operations in a process flow for enriching selected polynucleotide fragments using Cas-gRNA RNP nickase. Figure 8A shows an overview of an exemplary process flow of CRISPR nickase extension for selective elution of target regions. In operation A of the process flow, dsDNA fragment P4 (which may optionally be generated by methods as described elsewhere herein) may be 3' functionalized ("B") to facilitate binding of the fragment to beads. For example, the fragment may be 3' biotinylated using methods as described below with reference to Figure 8C. Some of the fragments P4 may contain the respective target sequences desired to be enriched and detected, while other fragments may not necessarily contain such sequences. For example, fragment P4 shown in Figure 8A contains target sequence 810, while other fragments may contain other target sequences or may not contain such target sequences.

[0422] In operation B of the process flow shown in FIG. 8A, the 3′-functionalized fragment P4 may be coupled to one or more substrates, such as beads that have been functionalized to couple to the 3′-functionalized fragment P4. In one non-limiting example, the beads 820 may include streptavidin to which the 3′-biotinylated fragment P4 is coupled. In the illustrated example, each of the functionalized 3′ ends of the dsDNA fragment P4 is coupled to a different bead 820, although it will be understood that in other examples, the 3′-functionalized ends of a given fragment P4 may be coupled to the same bead as each other. The beads 820 may be pulled out of solution (e.g., the beads may be ferromagnetic or paramagnetic and may be pulled out of solution using an external magnet), and the beads may then be washed to provide purified dsDNA fragment P4 coupled to the beads, while any other dsDNA may be substantially washed away.

[0423] As shown in FIG. 8A, in operation C, the bead-bound fragment P4 may be contacted with multiple Cas-gRNA RNP nickases (also referred to herein as CRISPR nickases). Each gRNA of the Cas-gRNA RNP nickases may target a specific region (subsequence) within each single strand of the dsDNA, which may be staggered so that the nickases cleave each strand at positions that are offset from each other and on either side of the double-stranded target region 810 that is desired to be enriched. For example, in a manner as shown in operation C of FIG. 8A, the gRNA of the first Cas-gRNA RNP nickase 851 may target a region that is forward ("fwd") of the target sequence 810, and the gRNA of the second Cas-gRNA RNP nickase 852 may target a region that is reverse ("rev") of the target sequence 810. Thus, the guide sequences of the first and second nickases 851, 852 may be considered to be "adjacent" to the target sequence 810 in the forward and reverse directions. The first Cas-gRNA RNP nickase 851 creates a nick (cut) in one strand of the bead-bound dsDNA fragment P4, and the second Cas-gRNA RNP nickase 852 creates a nick in the other strand of the bead-bound dsDNA fragment P4 at a position offset from that created by the nickase 851. It will be appreciated that any suitable number of gRNAs may be designed such that the corresponding Cas-gRNA RNP nickase cuts each strand at a position adjacent to a particular sequence within the dsDNA fragment. For example, multiple different gRNAs (e.g., 1000-100,000 gRNAs, or more than 100,000 gRNAs) may be used to simultaneously enrich for many different sequences of interest within a sample. Note that gRNAs do not necessarily have to be "adjacent" to a given target sequence 810, rather at least two guides per target sequence may bind to opposing strands within a given fragment P4 and form a nick.

[0424] As shown in step D of Figure 8A, the Cas-gRNA RNP nickases 851, 852 are removed to expose the 3' ends of the nicks, e.g., using mild heat and / or a reagent to destroy the Cas-gRNA RNP nickases, e.g., proteinase K, protease, or SDS detergent. Because the strands of each given dsDNA fragment P4 remain hybridized to each other, the fragments remain substantially coupled to the corresponding beads 820.

[0425] Target sequences 810 adjacent to opposing nicks in the strands of dsDNA can then be selectively eluted into solution, while the remainder of fragment P4 remains coupled to beads 820. For example, nicked fragment P4 can be contacted with a polymerase and nucleotides (not specifically shown). In a manner as shown in operation E of FIG. 8A, the polymerase can extend each strand of the fragment from the 3' end exposed by the nick, and such extension can displace the bound strand, resulting in elution of target sequence 810. The non-targeted regions remain coupled to beads 820 and are separated from the eluted target sequence 810, for example, using magnetic or other separation techniques. The polymerase extension results in elution of intact sequence 810, regardless of where in fragment P4 the nicks occurred.

[0426] Example workflow for enriching targets in lambda DNA and eluting them from a substrate using Cas 9 nickase and polymerase extension. Figure 8A shows an exemplary workflow that can be used to enrich targets in lambda DNA using Cas9 nickase. Specific guide RNA sequences are used that target four regions of the lambda genome. Figures 12-16 provide schematics of the library structure after various steps of the workflow, as described in more detail below. Table 1 provides the guide RNA sequences as well as the regions that they target. [Table 1]

[0427] Load the guide RNA sequence onto the Cas9 enzyme. Load the guide sequence onto the Casp separately in a final volume of 50ul containing 1uM guide, 1uM Cas9 nickase (Integrated DNA Technologies, Alt-R® SpCas9 D10A nickase V3, 1081062) and 1x phosphate buffered saline. Leave the components at room temperature for 10 minutes and then pool equal amounts to create the Cas9 nicking mix. Store the solution on ice until use.

[0428] Library preparation by tagged fragmentation using bead-linked transposomes

[0429] Libraries attached by their 3' ends to small surfaces were prepared using bead-linked transposomes.

[0430] Step 1: 500 ng of lambda DNA was incubated with 10 uL of TB1 and 10 uL of eBLT from the Illumina DNA Prep with Enrichment kit in a total volume of 50 uL. The mixture was heated to 41° C. for 5 minutes.

[0431] Step 2: Tn5 was removed by adding 10 uL of ST2 and heating at 37° C. for 5 minutes.

[0432] Figure 12 shows the library structure after step 2. Element 1200 indicates the PAM site in the DNA insert.

[0433] Step 3: The reaction plate was placed on a magnetic stand and the beads were pelleted. The supernatant was removed and the beads were washed by adding 150 uL of TWB. The magnet was then removed and the solution mixed by pipetting. The beads were pelleted again on the magnet and then the magnet was removed. The supernatant was discarded.

[0434] Step 4: 50 uL of ELM (from the Illumina DNA Prep PCR Free kit) was added to the solution. The solution was incubated at 37° C. for 15 minutes to allow gap filling and ligation between the 3′ end of the insert and the non-transferred strand of the transposon.

[0435] Figure 13 shows the library structure after step 4. Element 1200 indicates the PAM site in the DNA insert.

[0436] Step 5: The beads were pelleted on a magnet. The supernatant was removed and washed with TWB.

[0437] Step 6: Any incompletely gap-filled and ligated fragments that may contribute to background were removed by adding 0.5 μL of Exonuclease III (New England Biolabs, M0206) in 1× NEBuffer 1 (New England Biolabs) in a volume of 50 μL. Beads were resuspended by pipetting to mix and heating to 37° C. for 10 min.

[0438] Cas9 nicking reaction

[0439] Step 1: 2 uL of pooled loaded Cas9 nickase was added with 1× NEBuffer2.1 (New England Biolabs) in a total volume of 20 uL, the supernatant was removed, the beads were resuspended by pipetting and heating to 37° C. for 30 min.

[0440] Figure 14 shows how Cas9 nicks the target fragment on each strand.

[0441] Step 2: Cas9 was removed by adding 10 uL ST2 and heating at 37° C. for 5 min. The beads were pelleted and washed twice with TWB. The supernatant was discarded.

[0442] Figure 15 shows the library structure at this point, with one nick on each strand. Element 1200 indicates the PAM site in the DNA insert.

[0443] Polymerase extension to elute target fragments from beads.

[0444] 0.5 uL of DNA polymerase I (New England Biolabs, M0210) or Bsu DNA polymerase (New England Biolabs, M0330) was added to the solution. 1× NEBuffer 2 (New England Biolabs) was used and 200 uM of each dNTP was added in a total volume of 50 uL. The solution was stirred at 37° C. for 10 hours.

[0445] Figure 16 shows after polymerase extension. As shown, the fragment no longer has a 3' biotin and is therefore released into solution. Element 1200 indicates the PAM site in the DNA insert.

[0446] Purification and PCR

[0447] Step 1: The beads were pelleted and 40 uL of the supernatant containing the selected target fragment was transferred to a new tube. Illumina Purification Beads (IPB) were used to purify the beads by adding 100 uL of ITB, mixing thoroughly and incubating at room temperature for 5 minutes. The beads were pelleted on a magnet and washed twice with 180 μL of 80% ethanol. The supernatant was removed, allowed to dry for 2 minutes and then resuspended in 27 μL of water. The solution was mixed well, the beads were pelleted and 25 μL of the supernatant was transferred to a new tube.

[0448] Step 2: The library was amplified by adding 20 μL of EPM and 5 μL of index primer mix using the following PCR program. - 98°C for 1 min. - 12 cycles of 98°C for 20 seconds - 60℃ for 30 seconds - 72°C for 30 seconds Cool to -10℃

[0449] Sequencing

[0450] The libraries were quantified using a Qubit kit (dsDNA BR Assay Kit, Thermo Scientific) and a fluorometer, then sequenced on a MiSeq at a loading concentration of 12 pM.

[0451] FIG. 17 shows the sequencing depth across the lambda genome after enrichment of the four targets.

[0452] FIG. 8B shows further details regarding the use of at least two CRISPR events on fragment P4 to cause selective elution as described with reference to FIG. 8A. In operation A shown in FIG. 8B, no nicking event has occurred, and therefore fragment P4 remains coupled to bead 820. In operation B shown in FIG. 8B, it can be seen that two nicking events have occurred, in the manner described with reference to operation C described with reference to FIG. 8A, and subsequent extension of the nicks using a polymerase displaces both ends coupled to the respective beads, thus eluting the target sequence 810. In operation C shown in FIG. 8C, for example, only a single nicking event has occurred, since the Cas-gRNA RNP nickase nicked the off-target sequence 811 of fragment P4, and therefore the corresponding nickase did not nick the opposing portion of fragment P4 adjacent to sequence 811. It can be seen that the subsequent 3' extension of the nicks using polymerase displaces one of the ends coupled to the respective beads, while the other end remains coupled to the bead and is therefore not eluted. Thus, it can be seen from FIG. 8B that fragments nicked on opposing strands on either side of the target sequence 810 may be preferentially eluted compared to unnicked fragments or fragments nicked only on one strand. Note that while gRNAs may be designed to bind to regions where the corresponding nickase can generate nicks at respective positions that are 3' to the target sequence 810, and thus may be successfully eluted using polymerase extension, any nicks generated at positions that are 5' to the target sequence may not be able to extend beyond the nick on the template strand, for example, in a manner described in more detail below with reference to FIG. 8G. Note that the Cas-gRNA RNP nickases may optionally target different strands. Although the figure may show a single nickase that targets the strand hybridized to the gRNA, another nickase that nicks the other strand may be used. This can provide improved selection of sequences for nicking, since both strands in the genome can be used.

[0453] FIG. 8C shows an exemplary process flow for enriching dsDNA fragments from a sequencing library that has undergone PCR amplification prior to the nicking and extension operations as described with reference to FIGS. 8A-8B. Such PCR amplification may be useful, for example, when the Cas-gRNA RNP nickase binding and nicking steps are not 100% efficient and / or when relatively few dsDNA fragments are present, for example, when the dsDNA is obtained from a cell-free DNA (cfDNA) sequencing library, to enhance sensitivity and / or to perform quality control and amplify enough material to sequence from a small panel. In operation A shown in FIG. 8C, amplification adaptors are added via any suitable method (e.g., as described with reference to FIGS. 1J, 3D, 4A-4J, 6A-6B, or 7A-7G). The amplification adaptors may optionally be Y-shaped in the manner described with reference to FIGS. 1J and 3D to provide read 1 and read 2 sequencing primers, respectively. In one non-limiting example, the amplification adaptor may include A14 and B15 amplification adaptors (complements that are A14' and B15') in addition to the double stranded ME, ME' region. For example, as shown in operation A of FIG. 8C, the 3' end of the first strand of dsDNA fragment P4 may be coupled to the B15' amplification adaptor via the ME' sequence, and the 5' end of that strand may be coupled to the A14 amplification adaptor via the ME sequence. The 3' end of the second strand of fragment P4 may be coupled to the B15' amplification adaptor via the ME' sequence, and the 5' end of that strand may be coupled to the A14 amplification adaptor via the ME sequence. However, it will be understood that any other sequences and / or amplification adaptors (e.g., UMI, sample index, cluster amplification primer, etc.) may be added to the strands.

[0454] Following preparation of the library with the amplification adaptors, a PCR amplification is performed to amplify both strands of the initial fragment P4 separately, as shown in operation B of FIG. 8C. During or at the end of this operation, the fragments can be functionalized (e.g., biotinylated) at the 3' end in a manner similar to that described with reference to operation A of FIG. 8A. Exemplarily, non-template addition (e.g., using Taq polymerase) or terminal transferase can be used to add biotinylated nucleotides to the 3' end of the amplified strand, as shown in operation B of FIG. 8C. Subsequent operations can be performed similarly to those described with reference to FIG. 8A. For example, as shown in operation C of FIG. 8C, the entire library can be coupled to one or more substrates (such as beads 820) via the 3' functional group of fragment P4 in a manner as described with reference to operation B of FIG. 8A, and a Cas-gRNA RNP nickase is used to generate nicks 3' adjacent to each target sequence 810 in a manner as described with reference to operation C of FIG. 8A. As shown in operation D of FIG. 8C, the Cas-gRNA RNP nickase can then be removed in a manner as described with reference to operation D of FIG. 8A, and a polymerase can be added to extend from the nick and cause elution of the target sequence 810 in a manner as described with reference to operation E of FIG. 8A. The eluted target sequence can then be further amplified using, for example, PCR or cluster amplification, during which a UMI, a sample index, and / or a clustered adaptor can optionally be added, for example, if such sequences were not added during operation A of FIG. 8C. Non-limiting examples of sample indexes include Illumina i5 and i7 indexes. Non-limiting examples of clustered adaptors include P5 and P7 primers. The eluted fragments coupled with any suitable sequence can be sequenced on any suitable platform (e.g., Illumina's sequencing-by-synthesis platform) as part of a targeted sequencing assay.

[0455] PCR can be used to couple an appropriate adaptor to fragment P4 and amplify the fragment prior to Cas-gRNA-mediated elution, although it will be understood that PCR need not necessarily be so used. For example, FIG. 8D shows a process flow for enriching fragments from a PCR-free fragmented and ligated sequencing library. Here, in operation A of FIG. 8D, fragment P4 is generated and an amplification adaptor (e.g., ME / ME' region and 5' amplification adaptor) is added via any suitable method, such as described with reference to, for example, FIG. 1J, 3D, 4A-4J, 6A-6B, or 7A-7G. In a non-limiting example illustrated in FIG. 8D, a 3' functional group (such as biotin) can be added by adaptor ligation, for example, using a simplified adaptor including ME / ME' and a single A14 adaptor. The adaptor can be modified to include uracil (U), which can stall polymerase extension in a manner further described below with reference to operations C and D of FIG. 8D. In operation B of FIG. 8D, the 3' functional group is coupled to a substrate such as a bead 820 in a manner as described with reference to operation B of FIG. 8A, and a Cas-gRNA RNP nickase is used to generate a nick 3' adjacent to each target sequence 810 in a manner as described with reference to operation C of FIG. 8A. As shown in operation C of FIG. 8D, the Cas-gRNA RNP nickase can then be removed in a manner as described with reference to operation D of FIG. 8A, and a polymerase can be added to extend from the nick and cause elution of the target sequence 810 in a manner as described with reference to operation E of FIG. 8A. However, the uracil in the modified adaptor (e.g., A14-U) causes the polymerase to stall at the uracil position. As shown in operation C of FIG. 8D, a template switch oligonucleotide containing a second sequencing primer (e.g., B15) allows the stalled extension product to prime off and add a 3' amplification adaptor to the eluted target fragment.The eluted target fragments 810 may then be PCR amplified, optionally in a manner as described elsewhere herein, including the addition of cluster amplification adaptors (e.g., P5 and P7), UMIs, and / or sample indexes. However, it will be understood that a process flow that does not include PCR may be performed as appropriate, for example, by adding full sequencing / cluster amplification adaptors and a sample index at operations A and D of the process flow shown in FIG. 8D. For further details regarding selected operations described with reference to FIG. 8D, see International Publication No. WO 2021 / 252617, entitled "Methods for Increasing Yield of Sequencing Libraries," the entire contents of which are incorporated herein by reference.

[0456] It will be understood that the process flow as described with reference to Figures 8A-8D may be suitably adapted for use with any type of library, instrument, or workflow. Figure 8E shows a non-limiting example of a process flow for use with an Illumina Nextera workflow, where a sample library may be prepared using a Nextera system via simultaneous fragmentation and 5' adapter addition. The Nextera system may be coupled to a substrate (e.g., beads 820) in a manner as described with reference to operation B of Figure 8A, such that an initial fragmentation event may be used to bind fragment P4 to the substrate. As shown in operation A of Figure 8E, a Nextera library may be generated that is coupled to beads 820 via a 3' functional group, such as biotin. In some examples, the library may be generated using a mixture of transposomes that contain respective amplification adapters, such as A14 and B15 adapters, where a portion (e.g., about half) of fragment P4 may include A14 and B15 adapters at either end (as shown herein). The other fragment may not necessarily contain both an A14 and a B15 adaptor, for example it may lack a B15 adaptor but contain two A14 adaptors, or it may lack an A14 adaptor but contain two B15 adaptors.

[0457] As a result of the Nextera fragmentation process, each fragment P4 may contain a gap between the 3' end and the ME region that is approximately 9 base pairs long. The gap may be sealed by, for example, extension ligation using a polymerase and a ligase, as shown in operation B of FIG. 8E. Note that sealing of the nick may inhibit any non-specific extension and elution by the polymerase. Alternatively, a terminal base may be added to inhibit undesired extension and subsequent elution by TdT or polymerase and dideoxy bases. Cas-gRNA RNP nickase may then be applied to the fragments on the substrate, as described with reference to operation C of FIG. 8A, to create a targeted nick adjacent to the target sequence 810, as shown in operation C of FIG. 8E. Polymerase may then be added, as described with reference to operation E of FIG. 8A, to elute the target sequence, as shown in operation D of FIG. 8E. After elution, further amplification adapters and / or sample indexes may be coupled to the fragments, as described elsewhere herein, for example, using PCR or cluster amplification prior to sequencing. In this regard, any fragment P4 having two B15 adaptors or two A14 adaptors will not be amplified during such PCR amplification and therefore will not be sequenced. It will be appreciated that by exchanging adaptors to provide both A14 and B15 adaptors using a template switching mechanism as described with reference to operation D in Figure 8D, the loss of such B15-B15 and A14-A14 fragments can be reduced, such that such fragments can be amplified using PCR or cluster amplification and subsequently sequenced.

[0458] Figure 8F shows polymerase options for a nick extension elution operation as described with reference to operation E of Figure 8A, operation B of Figure 8B, operation D of Figure 8C, operation C of Figure 8D, and operation D of Figure 8E. In example A of Figure 8F, the use of a strand displacement polymerase results in displacement of the 3' functionalized (e.g., 3' biotinylated) strand from the target sequence 810, resulting in targeted elution. In example B of Figure 8F, a nick translation approach including the use of a polymerase with 5' exonuclease activity causes 5' to 3' degradation of the 3' functionalized (e.g., 3' biotinylated) strand, resulting in targeted elution of the target sequence 810.

[0459] 8G compares the use of a nick that is 3' to the target sequence (operation A) with the use of a nick that is 5' to the target sequence (operation B). As can be seen from operation A, two nicking events that are 3' to the target sequence 810 result in elution of the target sequence from the substrate, e.g., bead 820. As can be seen from operation B, two nicking events that are 5' to the target sequence 810 can stall the polymerase at the nick, leaving the target sequence bound to the substrate, e.g., bead 820.

[0460] It should be noted that numerous separation techniques are compatible with the process flows as described with reference to Figures 8A-8G and are not limited to the use of magnetic separation of beads as described. For example, the substrate may be provided within a flow system such as a packed column or flow cell. The target fragments may be eluted using a flow in such a system.

[0461] It will be further understood that fragment P4 may be functionalized to include any suitable tag and the substrate may be functionalized to include any suitable tag partner for pulling down fragment P4 to the substrate. For example, the tag partner may include a SNAP protein and the tag may include O-benzylguanine; the tag partner may include a CLIP protein and the tag may include O-benzylcytosine; the tag partner may include SpyTag and the tag may include SpyCatcher; the tag partner may include SpyCatcher and the tag may include SpyTag; the tag partner may include biotin and the tag may include streptavidin; the tag partner may include streptavidin and the tag may include biotin; the tag partner may include NTA and the tag may include The tag partner may comprise a His tag; the tag partner may comprise a His tag, and the tag may comprise an NTA; the tag partner may comprise an antibody (e.g., an anti-FLAG antibody), and the tag may comprise an antigen (e.g., a FLAG tag) for which the antibody is selective; the tag partner may comprise an antigen (e.g., a FLAG tag), and the tag may comprise an antibody (e.g., an anti-FLAG antibody) selective for the antigen; or the tag partner may comprise a first oligonucleotide, and the tag may comprise a second oligonucleotide that is complementary to the first oligonucleotide and hybridizes to the first oligonucleotide. The tag partner may be coupled to the substrate via any suitable bond, for example, via a covalent bond or via a non-covalent bond. Similarly, each tag may be 3'-coupled to fragment P4 via any suitable bond, for example, via a covalent bond or via a non-covalent bond.

[0462] The compositions and operations as described with reference to Figures 8A-8G can be used in any suitable manner or context. For example, Figure 8H shows a flow of operations in an exemplary method 8000 for generating fragments of a double-stranded polynucleotide. Although method 8000 may describe operations performed on a particular polynucleotide, it will be understood that the method may be applied to a mixture including several different polynucleotides that may be simultaneously manipulated in the manner described. In some examples, the double-stranded polynucleotide may include dsDNA, and optionally cfDNA.

[0463] Method 8000 may include coupling the double-stranded polynucleotide to a substrate (operation 8001). For example, the 3' end of the double-stranded polynucleotide may be functionalized (e.g., coupled to a tag or tag partner) in a manner as described with reference to operation A of FIG. 8A, operation B of FIG. 8C, operation A of FIG. 8D, or operation A of FIG. 8E. Further, the 3' functionalized end of the double-stranded polynucleotide may be coupled to a substrate, e.g., a substrate that couples to a tag partner or tag that becomes coupled to the tag or tag partner of the double-stranded polynucleotide, in a manner as described with reference to operation B of FIG. 8A, operation C of FIG. 8C, operation B of FIG. 8D, or operation A of FIG. 8E. Some examples described with reference to FIGS. 8A-8G may include streptavidin beads as the substrate and biotin as the 3' functional group, although many other examples of substrates and tag / tag partner pairs may be readily envisioned.

[0464] The method 8000 shown in Figure 8H may also include hybridizing a first and a second CRISPR-associated protein guide RNA ribonucleoprotein (Cas-gRNA RNP) nickase to a first and a second subsequence, respectively, in the double-stranded polynucleotide (operation 8002). The first subsequence may be 3' to a target sequence along a first strand of the double-stranded polynucleotide, and the second subsequence may be 3' to a target sequence along a second strand of the double-stranded polynucleotide. For example, the gRNA of the first Cas-gRNA RNP nickase 851 may be selectively coupled to a first strand of the double-stranded polynucleotide P4 at the "fwd" 3' position, and the gRNA of the second Cas-gRNA RNP nickase 852 may be selectively coupled to a second strand of the double-stranded polynucleotide P4 at the "rev" 3' position, in a manner as described with reference to operation C of FIG. 8A, operation B of FIG. 8B, operation C of FIG. 8C, operation B of FIG. 8D, operation C of FIG. 8E, example A of FIG. 8F, example B of FIG. 8F, and example A of FIG. 8G. The nickase may be considered to be 3' to the target sequence. As described above, the nickase may target either the strand to which the gRNA is hybridized or the opposite strand.

[0465] The method 8000 shown in FIG. 8H may also include cleaving the first strand at the first subsequence using a first Cas-gRNA RNP nickase and cleaving the second strand at the second subsequence using a second Cas-gRNA RNP nickase (operation 8003). For example, the nickase of the first Cas-gRNA RNP nickase 851 can selectively nick the first strand of the double-stranded polynucleotide P4 at a position defined by the subsequence to which its gRNA is coupled, and the nickase of the second Cas-gRNA RNP nickase 852 can selectively nick the second strand of the double-stranded polynucleotide P4 at a position defined by the subsequence to which its gRNA is coupled, in a manner as described with reference to operation C of FIG. 8A, operation B of FIG. 8B, operation C of FIG. 8C, operation B of FIG. 8D, operation C of FIG. 8E, example A of FIG. 8F, example B of FIG. 8F, and example A of FIG. 8G. The resulting nickase can be considered to be 3' to the target sequence. Such two instances of cleavage can be performed simultaneously or at different times from each other. For example, a large pool of first and second strand CRISPR nickase complexes can be incubated with the sample at one time. It will be appreciated that operations 8002 and 8003 may be performed using any suitable Cas-gRNA RNP nickase, illustratively, S. pyogenes Cas9 having a first mutation D10A and a second mutation H840A.

[0466] Method 8000 may also include using a polymerase to extend the first and second strands from their respective breaks and eluting the target sequence from the substrate (operation 8004). For example, the Cas-gRNA RNP nickase may be removed to expose the 3' end of the nick generated in operation 8003, and a suitable polymerase may be added to extend the target sequence, which is double stranded, from the 3' end, in a manner as described with reference to operations D and E of FIG. 8A, operation B of FIG. 8B, operation D of FIG. 8C, operation C of FIG. 8D, operation D of FIG. 8E, example A of FIG. 8F, example B of FIG. 8F, and example A of FIG. 8G. Such extension displaces the portion of the double stranded polynucleotide coupled to the substrate that remains coupled to the substrate, and elutes the target sequence. The target sequence is thus released from the substrate. It will be understood that operation 8004 may be performed using any suitable polymerase. For example, the polymerase can include a strand-displacing polymerase, illustratively Vent or Bsu, as described with reference to Example A of Figure 8F, or, for example, the polymerase can have 5' exonuclease activity, illustratively Taq, Bst, or DNA polymerase I.

[0467] Method 8000 may also include sequencing the eluted target sequences (operation 8005). Such sequencing may be performed in any suitable manner and using any suitable equipment, such as equipment commercially available from Illumina, Inc. At any suitable time prior to sequencing, the target sequences may be suitably attached to amplification adaptors, for example as described with reference to operations A-D of FIG. 8C, operations A-D of FIG. 8D, or operations A-D of FIG. 8E. Such amplification adaptors may be added before or after any suitable operation of operations 8001, 8002, 8003, and 8004. Furthermore, at any suitable time prior to sequencing, the target sequences may be amplified, for example using PCR or cluster amplification. Such amplification may be performed before or after any suitable one of operations 8001, 8002, 8003, and 8004.

[0468] Ligation of amplification adapters to selected polynucleotide fragments using Cas-gRNA RNPs Some methods provided herein solve the problem of long and tedious workflows for targeted sequencing of intact dsDNA fragments. As evident from the present disclosure, Cas-gRNA RNPs can provide rapid and specific hybridization of target regions in polynucleotides, e.g., dsDNA. As described herein with reference to Figures 9A-9F, a complex comprising Cas-gRNA RNPs and amplification adaptors can be used to ligate the amplification adaptors to selected fragments, such that these fragments can be subsequently amplified and sequenced, while other fragments will not be ligated to such adaptors and thus will not be amplified an...

Claims

1. 1. A method for treating a mixture of a first double-stranded polynucleotide derived from a first species and a second double-stranded polynucleotide derived from a second species, the method comprising: protecting an end of the first double-stranded polynucleotide and any end of the second double-stranded polynucleotide; selectively generating a free end within the first double-stranded polynucleotide after protecting the ends of the first and second double-stranded polynucleotides; degrading the first double-stranded polynucleotide from the free end toward the protected end; A method comprising:

2. 2. The method of claim 1, wherein selectively generating the free ends within the first double-stranded polynucleotide comprises hybridizing a CRISPR-associated protein guide RNA ribonucleoprotein (Cas-gRNA RNP) to a sequence present in the first double-stranded polynucleotide and absent in the second double-stranded polynucleotide, and cleaving the sequence with the Cas-gRNA RNP.

3. The method of claim 2 , wherein the sequence comprises a mammalian-specific repetitive element.

4. The method of claim 3, wherein the mammalian-specific repeat elements comprise human-specific repeat elements.

5. 2. The method of claim 1, wherein the first double-stranded nucleotide sequence comprises a plurality of chromosomes derived from the first species.

6. The method of claim 1 , wherein the second species is a bacterium, a fungus, or a virus.

7. 7. The method of claim 1, wherein protecting the ends of the first and second double-stranded polynucleotides comprises (a) ligating hairpin adaptors to the ends, (b) 5' dephosphorylating the ends, and / or (c) adding modified bases to the ends.

8. The method of claim 7, wherein the modified base comprises a phosphorothioate linkage.

9. The method of claim 7, wherein the modified base is added using a terminal transferase.

10. 10. The method of claim 1, wherein degrading the first double-stranded polynucleotide is performed using an exonuclease.

11. The method of claim 1 , wherein the free end comprises a 3′ end or the free end comprises a 5′ end.

12. 12. The method of claim 11, wherein degrading the first double-stranded polynucleotide is performed using Exonuclease III.

13. 12. The method of claim 11, wherein degrading the first double-stranded polynucleotide is performed using lambda exonuclease.

14. 14. The method of any one of claims 1 to 13, further comprising subsequently ligating amplification adaptors to the ends of any remaining double-stranded polynucleotides in the mixture.

15. 15. The method of claim 14, wherein the amplification adapter comprises a unique molecular identifier (UMI).

16. 16. The method of claim 14 or claim 15, further comprising subsequently amplifying and sequencing the double-stranded polynucleotide.

17. 17. The method of any one of claims 1 to 16, wherein the first double-stranded polynucleotide comprises double-stranded DNA and / or the second double-stranded polynucleotide comprises double-stranded DNA.

18. 18. The method of any one of claims 1 to 17, wherein the second double-stranded polynucleotide comprises circular DNA.

19. 19. The method of any one of claims 1 to 18, wherein the Cas comprises Cas9.

20. 1. A composition comprising: a first double-stranded polynucleotide derived from a first species, wherein an end of the first double-stranded polynucleotide is protected; a second double-stranded polynucleotide derived from a second species, wherein any terminus of said second double-stranded polynucleotide is protected; and a CRISPR-associated protein guide RNA ribonucleoprotein (Cas-gRNA RNP) hybridized to a sequence present in the first double-stranded polynucleotide and absent in the second double-stranded polynucleotide, the Cas-gRNA RNP for cleaving the sequence to selectively generate a free end within the first double-stranded polynucleotide; A composition comprising:

21. The composition of claim 20 , wherein the sequence comprises a mammalian-specific repetitive element.

22. The composition of claim 21 , wherein the mammalian-specific repetitive elements comprise human repetitive elements.

23. 23. The composition of any one of claims 20 to 22, wherein the second species is a bacterium, a fungus, or a virus.

24. 24. The composition of any one of claims 20 to 23, wherein the ends of the first and second double-stranded polynucleotides are protected by (a) using hairpin adaptors, (b) using 5' dephosphorylation, and / or (c) using modified bases.

25. The composition of claim 24 , wherein the modified base comprises a phosphorothioate linkage.

26. 26. The composition of any one of claims 20 to 25, wherein the free end comprises a 3' end, or the free end comprises a 5' end.

27. 27. The composition of any one of claims 20 to 26, wherein the first double-stranded polynucleotide comprises double-stranded DNA and / or the second double-stranded polynucleotide comprises double-stranded DNA.

28. 28. The composition of any one of claims 20 to 27, wherein the second double-stranded polynucleotide comprises circular DNA.

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