WO2015075056A1 - Enzymes programmables pour l'isolement de fragments d'adn spécifiques - Google Patents

Enzymes programmables pour l'isolement de fragments d'adn spécifiques Download PDF

Info

Publication number
WO2015075056A1
WO2015075056A1 PCT/EP2014/074986 EP2014074986W WO2015075056A1 WO 2015075056 A1 WO2015075056 A1 WO 2015075056A1 EP 2014074986 W EP2014074986 W EP 2014074986W WO 2015075056 A1 WO2015075056 A1 WO 2015075056A1
Authority
WO
WIPO (PCT)
Prior art keywords
cas9
crrna
dsdna
complex
nuclease
Prior art date
Application number
PCT/EP2014/074986
Other languages
English (en)
Inventor
Arvydas Lubys
Original Assignee
Thermo Fisher Scientific Baltics Uab
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Thermo Fisher Scientific Baltics Uab filed Critical Thermo Fisher Scientific Baltics Uab
Publication of WO2015075056A1 publication Critical patent/WO2015075056A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Definitions

  • dsDNA double-stranded DNA
  • the invention uses an in vitro assembled, catalytically inactive, programmable nuclease to recognize and bind those DNA fragments that contain sequences recognized by that nuclease.
  • an in vitro assembled, catalytically inactive, programmable nuclease to recognize and bind those DNA fragments that contain sequences recognized by that nuclease.
  • programmable nuclease is ternary ribonucleoprotein complex Cas9/crRNA tracRNA.
  • An artificially engineered nuclease with customized target dsDNA sequence specificity may also be used. Examples of such programmable enzymes include engineered homing
  • DNA fragments bound by such catalytically inactive programmable site specific nucleases are easily isolated using the affinity tag that may be located either on the nuclease protein molecule, or in the case of Cas9 complexes on crRNA, or tracrRNA, or in the case of TFO complexes on the guiding oligonucleotide.
  • affinity tag may be located either on the nuclease protein molecule, or in the case of Cas9 complexes on crRNA, or tracrRNA, or in the case of TFO complexes on the guiding oligonucleotide.
  • Such specific DNA fragments isolated according to the inventive method may be used in any downstream application including, but not limited to, targeted sequencing by any of existing next-generation sequencing techniques. The method may be used for removal of undesired DNA sequences, such as those encoding ribosomal RNA in RNA sequencing experiments.
  • PCR polymerase chain reaction
  • a methods that resolves issues specific for oligonucleotide-directed hybridization- based techniques is desirable.
  • Type II CRISPR-Cas systems or other site specific nucleases, that may be engineered to recognize different DNA sequences, could serve the same function and likely overcome obstacles specific for hybridization-based techniques.
  • CRISPR Clustered, regularly interspaced, short palindromic repeats
  • Cas CRISPR Associated
  • Systems evolved as bacterial and archaeal adaptive defense systems directed towards invading phages and plasmids (Terns and Terns, CRISPR-based adaptive immune systems. Curr Opin Microbiol. 14 (201 1 ) 321 ; Bhaya et al., CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annu Rev Genet. 45 (201 1 ) 273).
  • These defense systems include two major components: enzymes encoded by genes of Cas operon, and small RNAs which are transcribed from the CRISPR region into a long transcript and then processed into a set of individual short CRISPR RNAs
  • crRNAs Each crRNA has bipartite structure: one is a repeat that is the same in all crRNAs of a particular CRISPR/Cas system, the other, protospacer, is a variable spacer sequence complementary to the invading nucleic acid.
  • Bacteria harboring functionally active CRISPR- Cas systems can incorporate short sequences of invading elements within the CRISPR loci.
  • crRNAs produced from CRISPR loci form complexes with Cas protein(s) and guide them to recognize and inactivate those invading elements that have sequences complementary to crRNAs, thus providing a "memory" for bacteria or archaea to recognize and inactivate phages and plasmids already encountered.
  • CRISPR-Cas systems Based on structural and functional peculiarities of CRISPR-Cas systems, they are divided into three major types (Makarova et al., Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol. 9 (201 1 ) 467).
  • crRNAs and Cas proteins are the only components required for expression and maturation of crRNAs, while processing of crRNAs in Type II CRISPR-Cas systems requires the presence of one additional short RNA of fixed size, termed trans-activating crRNA (tracrRNA), which is encoded in the vicinity of the cas genes and CRISPR region (Deltcheva et al., CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature.
  • Cas9-crRNA ribonucleoprotein complex may be isolated from bacterial cells by using the protein purification tag added to the Cas9, and the isolated complex retained its ability to recognize and introduce double-stranded breaks into those DNA fragments which contained sequences complementary to the crRNA (Gasiunas et al., Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci U S A 109 (2012) E2579-86).
  • Electrophoretic Mobility Shift Assays demonstrated that the isolated wild-type Cas9-crRNA complex in the absence of magnesium ions binds dsDNA fragment bearing a nucleotide sequence complementary to the protospacer. Magnesium ions are required for DNA cleavage, thus their absence in the binding reaction mixture prevents bound DNA from cleavage. Binding was efficient only if the target dsDNA included not only a sequence complementary to the protospacer of crRNA, but also had the so-called conserved
  • Catalytically active complexes include crRNA, tracrRNA and Cas9, and it was concluded that the PAM sequence is obligatory for binding of dsDNA targets.
  • EMSA demonstrated that adding tracrRNA enhances target DNA binding by catalytically inactive Cas9-crRNA substantially, providing evidence that tracrRNA is required for target DNA recognition.
  • the role of tracrRNA was speculated to orient crRNA properly for interaction with the
  • the workflow was described as follows: (i) introduction of DNA or RNA coding for both the catalytically inactive Cas9 and the guide RNA into cells of interest, (ii) co-expression of both Cas9 and guide RNA to form complexes in vivo between expressed Cas9/guide RNA and genomic DNA region of interest, (iii) cross-linking macromolecules by treatment with formaldehyde, (iv) isolation of all Cas9 molecules, including those complexed with the target DNA, using immunoprecipitation using antibody against the tag added to the Cas9 mutant protein, and (v) analysis of all proteins which were co-purified with Cas9 by mass-spectrometry, attempting to identify those cellular proteins which were in close vicinity to the Cas9 presumably bound to the genomic DNA at a specific genome location.
  • the invention describes new technique of selective isolation of double-stranded DNA fragments of interest.
  • the technique has three major steps.
  • In the first step there is in vitro formation of complexes between Cas9, tracrRNA and one, few, or as many as necessary different crRNA molecules which predetermine the specificity of the formed
  • Cas9/tracrRNA crRNA complexes towards sequence targets located within the double stranded DNA In the second step double-stranded DNA under investigation is mixed with preformed complexes for the time required for formation of higher-order complexes between
  • DNA fragments bound by Cas9/crRNA tracRNA are isolated by using the afinity tag-solid particle interaction.
  • Affinity tags that may be used are known in the art, such as a biotin moiety, which may be located predominantly in synthetic tracrRNA, but potentially could be located in crRNA, in hybrid tracrRNA crRNA molecule, or on the surface of Cas9 protein, including polyhistidine tag, MBP maltose binding protein (MBP) tag, chitin binding domain (CBD) tag, or strepavidin tag fused to Cas9, or an antibody raised against Cas9 protein.
  • MBP MBP maltose binding protein
  • CBD chitin binding domain
  • Complexes bound by streptavidin-bearing particles are then purified from the mixture of DNA fragments and released from particles. Double-stranded DNA isolated thereby from released complexes may be used in any downstream application including, but not limited to, targeted sequencing by any of existing next-generation sequencing techniques.
  • the inventive method may be used for removal of undesired DNA sequences, such as those which code for ribosomal RNA in RNA sequencing experiments.
  • the dsDNA Cas9/crRNA tracRNA complexes are isolated from the reaction mixture using the affinity tag incorporated into the structure of any of Cas9, crRNA, and/or tracRNA of the ternary ribonucleoprotein complex before its in vitro assembly.
  • desired DNA sequences were enriched from mixtures of dsDNA fragments.
  • the method depletes undesired DNA sequences from mixtures of dsDNA fragments.
  • inventive method can be applied to other members of Type II CRISPR-Cas systems, or other catalytically inactive programmable nucleases.
  • catalytic mutants of Cas9, or other programmable nucleases may be used.
  • Catalytically inactive mutants of Cas9 useful in practicing this invention may be generated by introducing D31A mutation in the RuvC active center, or N891A mutation in the HNH domain in S. thermophilus Cas9, (Gasiunas et al., Cas9- crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl.Acad. Sci.
  • the present invention relates to a method for selective in vitro isolation of double-stranded DNA, the method comprising (a) contacting a sample, preferably a biological sample, containing double-stranded DNA (dsDNA) with a catalytically inactive nuclease, where the catalytically inactive nuclease comprises a target dsDNA sequence binding specificity, preferably a customizable target dsDNA sequence binding specificity, and optionally an affinity tag, (b) incubating the catalytically inactive nuclease with the sample under conditions sufficient for forming a complex comprising the catalytically inactive nuclease and a target (or selected) dsDNA, and (c) isolating the complex from the sample.
  • dsDNA double-stranded DNA
  • the sample containing dsDNA is contacted with a catalytically inactive nuclease in a solution.
  • a solution can be, but is not limited to, a solution as used in the Examples described herein.
  • the term -biological sample refers to a sample obtained from a biological subject, including sample of biological tissue or fluid origin obtained in vivo or in vitro. Such samples can be, but are not limited to, body fluid (e.g., blood, blood plasma, serum, or urine), organs, tissues, fractions, cells isolated from mammals including, humans and cell organelles.
  • Biological samples can further be, but are not limited to, fragmented dsDNA, for example fragmented dsDNA plasmids or vectors, for instance by cleavage with one or more restriction enzymes.
  • Biological samples also may include sections of the biological sample including tissues (e.g., sectional portions of an organ or tissue).
  • Biological samples may also include extracts from a biological sample.
  • Biological samples may further comprise proteins, carbohydrates or nucleic acids.
  • a biological sample may be of prokaryotic origin, archaeal origin, or eukaryotic origin (e.g., insects, protozoa, birds, fish, and reptiles).
  • the biological sample is mammalian (e.g., rat.
  • the biological sample is of primate origin (e.g., example, chimpanzee, or human).
  • double-stranded DNA or "dsDNA” as used herein refers to a deoxyribonucleotide polymer (DNA strand) hybridized to its complement through Watson- Crick bonding.
  • the dsDNA can be of any length and can be associated with additional components (e.g., histone proteins or proteins involved in replication or transcription).
  • additional components e.g., histone proteins or proteins involved in replication or transcription.
  • the two strands of DNA may not be 100% complementary, so long as the percentage is high enough in the given conditions for the two strands to remain associated.
  • nucleic acid in a polynucleotide refers to the ability of a nucleic acid in a polynucleotide to form a base pair with another nucleic acid in a second polynucleotide.
  • sequence A-G-T is complementary to the sequence T-C-A.
  • catalytically inactive nuclease comprising customizable target dsDNA sequence binding specificity refers to a catalytically inactive nuclease for which the target dsDNA sequence binding specificity can be customized, such as for example demonstrated in the examples, but not limited thereto.
  • condition sufficient for forming a complex refers to the conditions, such as for example, but not limited to, temperature, incubation time, buffers, that are sufficient to allow formation of a complex comprising the catalytically inactive nuclease and the target (or selected) dsDNA.
  • the catalytically inactive nuclease is selected from the group consisting of a Type II CRISPR-Cas system, a homing nuclease, a triple helix forming oligonucleotide (TFO)-linked nuclease, a zinc-finger nuclease, a transcription-activator like effector nuclease (TALEN), and combinations thereof.
  • a Type II CRISPR-Cas system a homing nuclease, a triple helix forming oligonucleotide (TFO)-linked nuclease, a zinc-finger nuclease, a transcription-activator like effector nuclease (TALEN), and combinations thereof.
  • the Type II CRISPR-Cas system is ribonucleoprotein complex Cas9/crRNA tracRNA and prior to step (a), forming a complex in vitro between Cas9, a synthetic tracrRNA and at least one synthetic crRNA molecule that predetermines the specificity of the formed Cas9/tracrRNA crRNA complex towards a sequence target located within the double stranded DNA.
  • the Type II CRISPR-Cas system more preferably the Cas9/crRNA/tracrRNA, is obtained from or modified from Streptococcus thermophilius and/or Streptococcus pyogenes.
  • the homing nuclease is either l-Crel or l-Scel.
  • the complex is isolated from the sample using the affinity tag.
  • the affinity tag is selected from the group consisting of biotin, desthiobiotin, streptavidin, polyhistidine, maltose binding protein (MBP), chitin binding domain (CBD), and combinations thereof.
  • the complex is isolated using an antibody having affinity for the catalytically inactive nuclease, or a portion thereof.
  • the affinity tag is attached to the Cas9 protein, the synthetic tracrRNA, the synthetic crRNA, and/or a tracrRNA crRNA hybrid duplex molecule.
  • step (c) of the method of the invention the complex is isolated from the sample by an interaction between the affinity tag and a binding molecule, the binding molecule capable of binding with the affinity tag, and the binding molecule attached to a solid support.
  • the method further comprises releasing the bound complex from the solid support.
  • the isolated, selected dsDNA is used in targeted sequencing.
  • the sample substantially depleted of the selected dsDNA is used in downstream applications.
  • the nuclease is rendered catalytically inactive by at least one of: (a) at least one mutation in the nuclease or, when the nuclease is a complex, at least one component of the complex, where the mutation at least substantially abolishes catalytic activity; or (b) the solution is at least substantially devoid of an agent required for catalytic activity of the nuclease, or (c) a catalytic inhibitor is present.
  • the solution lacks or substantially lacks Mg 2+ .
  • the catalytically inactive nuclease is Cas9/crRNA tracRNA and the Cas9 protein is mutated in a RuvC active center or a HNH domain to render the nuclease catalytically inactive.
  • the dsDNA is a collection of fragmented dsDNA.
  • the catalytically inactive nuclease or catalytically inactive ribonucleoprotein retains binding affinity for the selected dsDNA.
  • the present invention relates to a method for in vitro targeted sequence- specific double-stranded DNA enrichment or depletion, the method comprising: (a) contacting a sample, preferably a biological sample, containing double-stranded DNA (dsDNA) with a catalytically inactive ribonucleoprotein Cas9/crRNA tracRNA in a solution, where the catalytically inactive ribonucleoprotein Cas9/crRNA tracRNA comprises a target dsDNA sequence binding specificity, preferably a customizable target dsDNA sequence binding specificity, and optionally an affinity tag, (b) incubating the catalytically inactive
  • ribonucleoprotein Cas9/crRNA tracRNA with the sample under conditions sufficient for forming a complex comprising the catalytically inactive ribonucleoprotein Cas9/crRNA tracRNA and a selected dsDNA, and (c) isolating the complex from the sample.
  • the ribonucleoprotein Cas9/crRNA tracRNA is rendered catalytically inactive by at least (a) at least one mutation in the Cas9 protein, where the mutation at least substantially abolishes catalytic activity, or (b) the solution is at least substantially devoid of an agent required for catalytic activity of the ribonucleoprotein Cas9/crRNA tracRNA, or (c) a catalytic inhibitor is present.
  • the solution lacks or substantially lacks Mg 2+ .
  • the Cas9 protein is mutated in a RuvC active center or a HNH domain to render the nuclease catalytically inactive.
  • the complex is isolated from the sample using the affinity tag, the affinity tag selected from the group consisting of biotin, desthiobiotin, streptavidin, polyhistidine, maltose binding protein (MBP), chitin binding domain (CBD), and combinations thereof.
  • the affinity tag selected from the group consisting of biotin, desthiobiotin, streptavidin, polyhistidine, maltose binding protein (MBP), chitin binding domain (CBD), and combinations thereof.
  • step (c) of the method of the invention the complex is isolated from the sample by an interaction between the affinity tag and a binding molecule, the binding molecule capable of binding with the affinity tag, and the binding molecule attached to a solid support.
  • the method further comprises releasing the bound complex from the solid support.
  • the affinity tag is attached to the Cas9 protein, the synthetic tracrRNA, the synthetic crRNA, and/or a tracrRNA crRNA hybrid duplex molecule.
  • the complex is isolated using an antibody having affinity for at least a portion of the catalytically inactive ribonucleoprotein Cas9/crRNA tracRNA.
  • the isolated selected dsDNA is used in targeted sequencing.
  • the substantially depleted of dsDNA sample is used in downstream applications.
  • the method of the invention results in up to 70% enrichment of the selected dsDNA from the sample.
  • the method of the invention results in up to 70% depletion of the selected dsDNA from the sample.
  • the dsDNA is a collection of fragmented dsDNA.
  • the catalytically inactive nuclease or catalytically inactive ribonucleoprotein retains binding affinity for the selected dsDNA.
  • FIG. 1 illustrates experimental workflow
  • FIG. 2 schematically represents crRNA tracrRNA molecules used for formation of tertiary Cas9/crRNA tracrRNA complex.
  • FIG. 3 demonstrates catalytic activity of Cas9/crRNA tracrRNA complexes having biotinylated tracrRNA.
  • FIG. 4 demonstrates specific binding of biotinylated Cas9/crRNA tracrRNA to target DNA and formation of higher complexity complexes.
  • FIGS. 5A, 5B show gel results demonstrating capture of biotinylated
  • FIGS. 6A, 6B show the potential of biotinylated Cas9/crRNA tracrRNA complexes in both target enrichment and target depletion.
  • FIG. 7 illustrates the ability of biotinylated Cas9/crRNA tracrRNA to bind specifically to the target DNA and to form higher complexity complexes in more complex DNA mixtures than shown in FIG. 4.
  • FIG. 8 shows biotinylated Cas9/crRNA/tracrRNA complexes used for both target enrichment and target depletion as in FIG. 6, but with more complex mixture of DNA fragments.
  • FIG. 9 shows Cas9-RNA binding with target DNA.
  • FIG. 10 shows isolation of specific DNA fragments.
  • FIG. 1 1 is an experimental workflow of chloroplast DNA enrichment or depletion.
  • FIG. 12 shows relative enrichment normalized to control sample of specific DNA fragments from Arabidopsis thaliana.
  • Cas9/crRNA tracrRNA complex is shown in FIG. 2.
  • Cas9 protein is complexed with 42-nt-long crRNA and 74-nt-long tracrRNA which carries the biotin moiety at its very 3' end.
  • Twenty S'- terminal nucleotides of crRNA are complementary to a 20 nucleotide length region in the target DNA, and crRNA guides Cas9 protein to its target DNA.
  • PAM sequence required for Cas9 interaction with the double-stranded DNA, is located.
  • PAM sequence is NGGNG for Streptococcus thermophilus (underlined) or NGG for Streptococcus pyogenes (bold) Cas9 proteins.
  • tracrRNA 5'-terminal part of tracrRNA makes complementary interactions with the 3' end of crRNA, while the rest of tracrRNA potentially form secondary hairpin structures.
  • the biotin label on tracrRNA is used for capturing of complexes with bound target DNA on streptavidin-coated magnetic beads.
  • FIG. 3 illustrates the catalytic activity of Cas9/crRNA tracrRNA complexes having biotinylated tracrRNA.
  • Cas9/crRNA tracrRNA complex shown in Figure 2 while two others, 231 1 bp and 1310 bp in length, don't have such targets; lane 2 - reaction products resulting after the incubation of fragments shown in Lane 1 with biotin-free Streptococcus thermophilus Cas9/crRNA tracrRNA complex.
  • the DNA fragment of 847 bp is cleaved into two fragments, 712 bp and 135 bp in length, of which the smaller one is masked by tracrRNA and crRNA which both are present in the reaction mixture; lane 3 - reaction products resulting after the incubation of fragments shown in Lane 1 with biotin-free Streptococcus pyogenes Cas9/crRNA tracrRNA; lane 4 - the same as in Lane 2 except biotin-labeled tracrRNA was used for complex formation; lane 5 - the same as in Lane 3 except biotin-labeled tracrRNA was used for complex formation.
  • M - O'GeneRuler 1 kb Plus DNA Ladder The results show that biotin doesn't interfere with the catalytic activity of Cas9/crRNA tracrRNA complexes.
  • FIG. 4 illustrates the ability of biotinylated Cas9/crRNA tracrRNA to bind specifically to the target DNA and to form higher complexity complexes.
  • FIGS. 5A, 5B are results from SDS-PAGE gels illustrate the ability to capture biotinylated Cas9/crRNA tracrRNA from Streptococcus thermophilius (FIG. 5A) and
  • Streptococcus pyogenes FIG. 5B using streptavidin coated magnetic beads.
  • Streptococcus thermophilus Cas9/crRNA tracrRNA biotin-free complex after incubation with streptavid in-coated magnetic beads MB (lane 2) stands for the fraction of Cas9 bound and later on eluted from magnetic beads, while Sup (lane 3) shows Cas9 which remained in supernatant after removal of streptavidin-coated magnetic beads; lanes 4 and 5 - Streptococcus thermophilus Cas9/crRNA tracrRNA biotin-bearing complexes after their incubation with streptavidin-coated magnetic beads.
  • M - PageRuler Plus Prestained Protein Ladder M - PageRuler Plus Prestained Protein Ladder.
  • biotinylated tracrRNA acted as an affinity tag to capture Cas9 molecules which are included into tertiary complexes, while non-specific binding of biotin- free Cas9/crRNA tracrRNA complexes to streptavidin-coated magnetic beads (lanes 2 and 7) was much less efficient.
  • FIGS. 6A, 6B The potential of biotinylated Cas9/crRNA tracrRNA complexes in both target enrichment and target depletion is shown in FIGS. 6A, 6B from Streptococcus thermophilius (FIG. 6A) and Streptococcus pyogenes (FIG. 6B).
  • the fragment of interest in this case the 847 bp in length fragment, may be either enriched (lanes 2 and 4) or depleted (lanes 3 and 5), depending on which DNA fraction, the magnetic bead-bound DNA or DNA that remained in supernatant after removal of the magnetic bead-bound DNA, was used for further studies.
  • FIG. 7 illustrates the ability of biotinylated Cas9/crRNA tracrRNA to bind specifically to the target DNA and to form higher complexity complexes as in FIG. 4, but in more complex DNA mixture.
  • FIG. 8 demonstrates the use of biotinylated Cas9/crRNA tracrRNA complexes in both target enrichment and target depletion, as in FIG. 6, but using more complex mixture of DNA fragments.
  • Lane 4 - electrophoretic analysis of DNA bound to Streptococcus thermophilus Cas9/crRNA tracrRNA biotinylated complexes which were then captured by magnetic beads and later on released from magnetic beads; lane 5 - DNA fragments left in the
  • plasmid pMTC- eGFP-N (4468 bp) was digested into three fragments (231 1 , 1310, and 847 bp) using Fast Digest restriction endonucleases BamHI, Mfel (Muni), and Rsrll (Cpol). Only one fragment (847 bp length) had a target for Cas9/tracrRNA crRNA.
  • first crRNA tracrRNA duplex (10 ⁇ ) was made by annealing equimolar amounts of tracrRNA and crRNA in 10 mM Tris-HCI (pH 7.5 at 37°C), 100 mM NaCI, 1 mM EDTA, then mixing with 1.55 ⁇ Cas9 protein and 10X complex formation buffer (100 mM Tris-HCI (pH 7.5 at 37°C), 1000 mM NaCI, 10 mM EDTA, 0.5 mg/ml BSA, 10 mM DTT) and incubating for one hour at 37°C for the complex to form.
  • reaction products were mixed with 6X DNA loading dye and used for DNA electrophoresis in the 1 % agarose gel containing 5 ⁇ g ml ethidium bromide in 1X TAE buffer.
  • Cas9/tracrRNA crRNA complex (using Cas9 from both Streptococcus thermophilus and Streptococcus pyogenes) for one hour at 37°C in the same reaction buffer as in Example I except lacking Mg 2+ .
  • the reaction mixture was then mixed with 6X DNA loading dye and loaded into 1 % agarose gel with 5 ⁇ g ml ethidium bromide in 1X TAE buffer.
  • Cas9/tracrRNA crRNA target presumably due to Cas9/tracrRNA crRNA binding to it and forming higher complex of lowered electrophoretic mobility. In contrast, mobility of target-free fragments remained unchanged.
  • biotinylated Cas9/crRNA tracrRNA complexes and non- biotinylated complexes as controls were mixed with 5 ⁇ TBST buffer (25 mM Tris-HCI, 0, 15 M NaCI, 0,05 % Tween-20, pH 7,47) prewashed streptavidin-coated magnetic beads (Pierce, Thermo Scientific), suspended in 20 ⁇ PBS buffer (137 mM NaCI, 2.3 mM KCI, 4.3 mM Na 2 HP0 4 , 1.76 mM KH 2 P0 4 , pH 7.4). This mixture was incubated for 15 min at 4°C with constant mixing.
  • Example II To demonstrate that Cas9/tracrRNA crRNA forms higher complexes with DNA fragment in a sequence specific manner in the absence of magnesium ions, the experiment described in Example II was repeated using additional DNA of higher complexity. 100 ng of the same digested plasmid as in Example I was mixed with 200 ng of FastDigest Styl (Eco130l) digested ⁇ DNA (10 fragments). The mixture of DNA fragments was incubated with 500 ng Cas9/tracrRNA crRNA complex (using Cas9 from both Streptococcus thermophilus and Streptococcus pyogenes) for one hour at 37°C in the same reaction buffer as in Example I except without Mg 2+ . Subsequently, the reaction mixture was mixed with 6X DNA loading dye and loaded into 1 % agarose gel with 5 ⁇ g ml ethidium bromide in 1 X TAE buffer.
  • Results shown in FIG. 7, indicated shift of the fragment possessing the target for Cas9/tracrRNA crRNA, likely due to Cas9/tracrRNA crRNA binding to it, and forming a higher- complexity complex of lowered mobility. Other non-specific fragments did not exhibit a shift.
  • Cas9/tracrRNA crRNA complexes in buffer without Mg 2+ described in Example IV Five ⁇ streptavidin-coated magnetic particles (pre-washed with 1 ml of TBST buffer and suspended in 20 ⁇ PBS buffer) was then added to Cas9/crRNA/tracrRNA and DNA mixture. The reaction mixtures were incubated for 15 min at 4°C with constant mixing. The tubes were placed into a magnetic rack and the supernatant was transferred to fresh vials. The magnetic beads presumably containing bound Cas9/crRNA tracrRNA were suspended in nuclease free water and heated for five min at 70°C to disrupt streptavidin-biotin interaction and to release bound DNA fragments from Cas9/tracRNA crRNA complexes.
  • Magnetic particles were then placed into a magnetic rack, and the water which contained the eluted DNA was transferred to a new vial. Supernatant and the collected elution mixture were mixed with 6X DNA loading dye and loaded into 1 % agarose gel with 5 ⁇ g ml ethidium bromide in 1X TAE buffer.
  • Results shown in FIG. 8, demonstrated that only the specific desired fragment was isolated, while all other DNA fragments remained in the supernatant.
  • Homing endonuclease l-Scel is chemically labeled with biotin and incubated with the mixture of double-stranded DNA fragments, of which only one contains the target for l-Scel, under magnesium-free conditions. Formation of the enzyme:DNA complex is visualized by observing the shift in electrophoretic mobility. The enzyme together with the bound DNA fragment is captured by streptavidin-coated magnetic beads, and subsequently the bound DNA is eluted from the magnetic particles. Gel electrophoresis of eluted fragments reveals that only the fragment possessing the target for l-Scel is extracted from the mixture of DNA fragments, indicating that homing endonucleases and their mutants of altered specificity may be explored in DNA enrichment or depletion experiments.
  • TALEN meganuclease of predesigned specificity is chemically labeled with biotin and is incubated with a mixture of double-stranded DNA fragments, of which only one contains the target for TALEN meganuclease, under magnesium-free conditions. Formation of the enzyme:DNA complex is visualized by observing the shift in electrophoretic mobility. The enzyme together with the bound DNA fragment is captured by streptavidin-coated magnetic beads, and subsequently the bound DNA is eluted from magnetic particles.
  • Catalytically inactive TFO - nuclease of predesigned specificity is chemically labeled with biotin and is incubated with a mixture of double-stranded DNA fragments, of which only one contains the target for TFO - nuclease. Formation of the enzyme: target DNA complex is visualized by observing the shift in electrophoretic mobility. The enzyme together with the bound DNA fragment is captured by streptavidin-coated magnetic beads, and subsequently the bound DNA is eluted from magnetic particles.
  • Plasmid pMTC-eGFP-N (4468 bp) was digested into three fragments (231 1 , 1310, and 847 bp) using Fast Digest restriction endonucleases BamHI, Mfel (Muni) and RsrII (Cpol). Only the 847 bp fragment contained the target for Cas9-RNA.
  • ⁇ DNA was digested into ten fragments using FastDigest Styl (Eco130l). Fragments from both plasmid and ⁇ DNA were mixed and incubated with Cas9-RNA complexes assembled with S.thermophilus or
  • FIG. 9 shows electrophoretic mobility of the 847 bp fragment was altered (arrows), while the mobility of other fragments remained unchanged.
  • Example IX The same digested plasmid and ⁇ DNA were used as described in Example IX. They were mixed and incubated with Cas9-RNA complexes assembled with both Cas9 proteins in a buffer without Mg +2 . The Cas9 proteins with bound target DNA were captured using streptavidin-coated magnetic beads.
  • FIG. 10 shows that only the 847 bp fragment was captured and eluted from magnetic beads (lane MB), while other non-specific fragments remained in supernatant (lane sup). Specific fragment isolation was observed with both Cas9 proteins from S. thermophilus and S. pyogenes. Biotinylated dsDNA Cas9-RNA complexes were captured by streptavidin-coated magnetic beads.
  • A. thaliana DNA was purified using Thermo Scientificrt, GeneJETIF Plant Genomic
  • DNA Purification Mini Kit (Thermo Fisher Scientific). Fifty ng DNA was fragmented using MuSeek Library Preparation Kit, lllumina TM compatible. Seven crRNAs targeting chloroplast DNA with a minimum number of off-targets in genomic DNA were chosen according to Gibbs free energy calculations (Di et al. Cell. 152 (2013)1 173-83). Isolated DNA fragments were PCR amplified and size selected before sequencing on MiSeq (lllumina). Bioinformatical analysis was done using scripts in Python with some functions from HTSeq (EMBL
  • FIG. 1 Experimental workflow of chloroplast DNA enrichment or depletion from total A. thaliana DNA is shown in FIG. 1 1.
  • Chl_1-7 Total DNA from A.thallana was extracted and fragmented. Seven targets in chloroplast DNA were chosen and complementary crRNAS (Chl_1-7) were synthesized. Chl_1 and Chl_5 had two targets in DNA because of inverted repeat sequences in the chloroplast genome. Cas9-RNA complex with multiple crRNAs and S. thermophilus Cas9 was made and used to capture specific DNA fragments. dsDNA Cas9-RNA complexes were then isolated using magnetic beads. Eluate and supernatant were prepared for next generation sequencing with MiSeq (Illumine) platform.
  • MiSeq Illumine
  • A. thaliana DNA was purified using Thermo ScientificTM GeneJETTM Plant Genomic DNA Purification Mini Kit. Purified DNA was fragmented using MuSeek Library Preparation Kit, llluminaTM compatible (Thermo Fisher Scientific) and purified with Thermo ScientificTM
  • Streptococcus thermophilus Cas9 and complex formation buffer (10 mM Tris-HCI (pH 7.5 at 37°C), 100 mM NaCI, 1 mM EDTA, 0.05 mg/ml BSA, 1 mM DTT) and incubated for one hour at 37°C.
  • One hundred ng DNA fragments were incubated with 490 ng Cas9/tracrRNA/crRNA complexes in a 20 ⁇ reaction volume for one hour at 37°C in a reaction buffer containing 10 mM Tris-HCI (pH 7.5 at 37°C), 100 mM NaCI, 0.05 mg/ml BSA, 1 mM DTT).
  • Magnetic beads were suspended in nuclease free water and heated for five min at 70°C to disrupt streptavidin-biotin interaction and release bound DNA fragments from Cas9/tracRNA/crRNA complexes. Magnetic particles were then placed into a magnetic rack, and the water with eluted DNA was transferred to a new vial. Eluted DNA was purified with Thermo ScientificTM MagJETTM NGS Cleanup and Size Selection Kit, protocol A. Isolated DNA fragments were PCR amplified with MuSeek Indexes and MuSeek Library Preparation Kit, llluminaTM compatible (Thermo Fisher Scientific). After amplification, DNA fragments were size selected using Thermo ScientificTM MagJETTM NGS Cleanup and Size Selection Kit, protocol C.
  • Binding Mix Three hundred ⁇ of Binding Mix was used for size selection procedure. The same procedure except for treatment with Cas9/crRNA tracrRNA complex was performed with control sample. DNA libraries were quantified with KAPA Library Quantification Kit (Kapa Biosystems) and sequenced with lllumina MiSeq. Relative enrichment was determined by comparing number of reads of specific sequence in sample treated with Cas9/crRNA tracrRNA complex and control sample.
  • FIG. 12 shows relative enrichment, normalized to control sample and calculated from bioinformatics analysis data, of specific DNA fragments from Arabidopsis thaliana total DNA fragment mixture was achieved using the inventive method and could be used in more complex applications.
  • Chl_1 - Chl_7 represents seven different crRNAs and their targets in A. thaliana chloroplast DNA. The results demonstrated feasability of enrichment of specific DNA fragments from a chloroplast DNA fragment mixture.
  • Chloroplast DNA from A.thaliana total DNA mixture was successfully isolated using multiple crRNAs, with enrichment varying from 0 to 42 times compared to control.

Abstract

Cette invention concerne l'isolement sélectif simultané in vitro de multiples fragments d'ADN double brin (dsDNA) d'intérêt à partir de mélanges complexes de fragments dsDNA faisant appel à des nucléases spécifiques de séquences programmables catalytiquement inactives. Les complexes ribonucléoprotéiques ternaires Cas9/crRNA/tracRNA assemblés in vitro reconnaissent et se lient aux fragments dsDNA qui contiennent des séquences nucléotidiques complémentaires des séquences crRNA de guidage formant les complexes dsDNA/Cas9/crRNA/tracRNA.
PCT/EP2014/074986 2013-11-19 2014-11-19 Enzymes programmables pour l'isolement de fragments d'adn spécifiques WO2015075056A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201361906132P 2013-11-19 2013-11-19
US61/906,132 2013-11-19

Publications (1)

Publication Number Publication Date
WO2015075056A1 true WO2015075056A1 (fr) 2015-05-28

Family

ID=51932345

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2014/074986 WO2015075056A1 (fr) 2013-11-19 2014-11-19 Enzymes programmables pour l'isolement de fragments d'adn spécifiques

Country Status (1)

Country Link
WO (1) WO2015075056A1 (fr)

Cited By (46)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016014409A1 (fr) * 2014-07-21 2016-01-28 Illumina, Inc. Enrichissement de polynucléotides à l'aide de systèmes crispr-cas
US9340800B2 (en) 2013-09-06 2016-05-17 President And Fellows Of Harvard College Extended DNA-sensing GRNAS
US9359599B2 (en) 2013-08-22 2016-06-07 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
US9388430B2 (en) 2013-09-06 2016-07-12 President And Fellows Of Harvard College Cas9-recombinase fusion proteins and uses thereof
US9526784B2 (en) 2013-09-06 2016-12-27 President And Fellows Of Harvard College Delivery system for functional nucleases
US9834791B2 (en) 2013-11-07 2017-12-05 Editas Medicine, Inc. CRISPR-related methods and compositions with governing gRNAS
US9840699B2 (en) 2013-12-12 2017-12-12 President And Fellows Of Harvard College Methods for nucleic acid editing
WO2017218512A1 (fr) * 2016-06-13 2017-12-21 Grail, Inc. Enrichissement d'acides nucléiques acellulaires mutés pour la détection du cancer
US10077453B2 (en) 2014-07-30 2018-09-18 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US10113163B2 (en) 2016-08-03 2018-10-30 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
WO2018231952A1 (fr) * 2017-06-13 2018-12-20 Genetics Research, Llc, D/B/A Zs Genetics, Inc. Enrichissement d'une cible dans du plasma/sérum
US10167457B2 (en) 2015-10-23 2019-01-01 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US10323236B2 (en) 2011-07-22 2019-06-18 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US10337001B2 (en) 2014-12-03 2019-07-02 Agilent Technologies, Inc. Guide RNA with chemical modifications
US10370700B2 (en) 2017-06-13 2019-08-06 Genetics Research, Llc Detection of targeted sequence regions
US10508298B2 (en) 2013-08-09 2019-12-17 President And Fellows Of Harvard College Methods for identifying a target site of a CAS9 nuclease
WO2020005846A1 (fr) * 2018-06-25 2020-01-02 Bionano Genomics, Inc. Marquage de l'adn
US10527608B2 (en) 2017-06-13 2020-01-07 Genetics Research, Llc Methods for rare event detection
US10538758B2 (en) 2015-08-19 2020-01-21 Arc Bio, Llc Capture of nucleic acids using a nucleic acid-guided nuclease-based system
US10669571B2 (en) 2014-12-20 2020-06-02 Arc Bio, Llc Compositions and methods for targeted depletion, enrichment, and partitioning of nucleic acids using CRISPR/Cas system proteins
US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
US10767175B2 (en) 2016-06-08 2020-09-08 Agilent Technologies, Inc. High specificity genome editing using chemically modified guide RNAs
CN111868255A (zh) * 2018-03-15 2020-10-30 特温斯特兰德生物科学有限公司 用于富集用于测序应用和其他核酸材料询问的核酸材料的方法和试剂
US10947599B2 (en) 2017-06-13 2021-03-16 Genetics Research, Llc Tumor mutation burden
US11085078B2 (en) 2016-09-29 2021-08-10 Oxford Nanopore Technologies Limited Method for nucleic acid detection by guiding through a nanopore
US11142788B2 (en) 2017-06-13 2021-10-12 Genetics Research, Llc Isolation of target nucleic acids
US11268082B2 (en) 2017-03-23 2022-03-08 President And Fellows Of Harvard College Nucleobase editors comprising nucleic acid programmable DNA binding proteins
US11306309B2 (en) 2015-04-06 2022-04-19 The Board Of Trustees Of The Leland Stanford Junior University Chemically modified guide RNAs for CRISPR/CAS-mediated gene regulation
US11306324B2 (en) 2016-10-14 2022-04-19 President And Fellows Of Harvard College AAV delivery of nucleobase editors
US11319532B2 (en) 2017-08-30 2022-05-03 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11447770B1 (en) 2019-03-19 2022-09-20 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
US11542496B2 (en) 2017-03-10 2023-01-03 President And Fellows Of Harvard College Cytosine to guanine base editor
US11560566B2 (en) 2017-05-12 2023-01-24 President And Fellows Of Harvard College Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation
US11661590B2 (en) 2016-08-09 2023-05-30 President And Fellows Of Harvard College Programmable CAS9-recombinase fusion proteins and uses thereof
US11680261B2 (en) 2018-11-15 2023-06-20 Grail, Inc. Needle-based devices and methods for in vivo diagnostics of disease conditions
WO2023137292A1 (fr) * 2022-01-12 2023-07-20 Jumpcode Genomics, Inc. Procédés et compositions pour l'analyse du transcriptome
US11732274B2 (en) 2017-07-28 2023-08-22 President And Fellows Of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE)
US11739367B2 (en) 2017-11-08 2023-08-29 Twinstrand Biosciences, Inc. Reagents and adapters for nucleic acid sequencing and methods for making such reagents and adapters
WO2023164252A1 (fr) * 2022-02-28 2023-08-31 Trustees Of Boston University Dosages de détection isotherme d'acides nucléiques et leurs utilisations
US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
EP4276195A1 (fr) * 2022-05-10 2023-11-15 Genartis S.r.l. Procédés pour déterminer la présence ou le risque de développer la dystrophie myotonique de type 1 (md1) ou la dystrophie myotonique de type 2 (md2)
US11845985B2 (en) 2018-07-12 2023-12-19 Twinstrand Biosciences, Inc. Methods and reagents for characterizing genomic editing, clonal expansion, and associated applications
US11884915B2 (en) 2021-09-10 2024-01-30 Agilent Technologies, Inc. Guide RNAs with chemical modification for prime editing
US11898179B2 (en) 2017-03-09 2024-02-13 President And Fellows Of Harvard College Suppression of pain by gene editing
US11912985B2 (en) 2020-05-08 2024-02-27 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006027099A2 (fr) 2004-09-03 2006-03-16 Justus-Liebig-Universität Giessen Conjugues enzymatiques a specificite programmable interagissant de maniere hautement specifique avec l'adn
WO2011053987A1 (fr) * 2009-11-02 2011-05-05 Nugen Technologies, Inc. Compositions et procédés de sélection et d'amplification de séquences d'acide nucléique ciblées
WO2013142578A1 (fr) * 2012-03-20 2013-09-26 Vilnius University Clivage d'adn dirigé par arn par le complexe cas9-arncr
US20140038241A1 (en) * 2012-08-05 2014-02-06 Zhaohui Zhou Genomic enrichment method, composition, and reagent kit
US20140356867A1 (en) * 2013-05-29 2014-12-04 Agilent Technologies, Inc. Nucleic acid enrichment using cas9

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006027099A2 (fr) 2004-09-03 2006-03-16 Justus-Liebig-Universität Giessen Conjugues enzymatiques a specificite programmable interagissant de maniere hautement specifique avec l'adn
WO2011053987A1 (fr) * 2009-11-02 2011-05-05 Nugen Technologies, Inc. Compositions et procédés de sélection et d'amplification de séquences d'acide nucléique ciblées
WO2013142578A1 (fr) * 2012-03-20 2013-09-26 Vilnius University Clivage d'adn dirigé par arn par le complexe cas9-arncr
US20140038241A1 (en) * 2012-08-05 2014-02-06 Zhaohui Zhou Genomic enrichment method, composition, and reagent kit
US20140356867A1 (en) * 2013-05-29 2014-12-04 Agilent Technologies, Inc. Nucleic acid enrichment using cas9

Non-Patent Citations (22)

* Cited by examiner, † Cited by third party
Title
ALBERT ET AL.: "Direct selection of human genomic loci by microarray hybridization", NAT METHODS., vol. 4, 2007, pages 903
BAINBRIDGE ET AL.: "Whole exome capture in solution with 3 Gbp of data", GENOME BIOL., 2010, pages 11
BHAYA ET AL.: "CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation", ANNU REV GENET., vol. 45, 2011, pages 273
DELTCHEVA ET AL.: "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase", NATURE, vol. 471, 2011, pages 602
DI ET AL., CELL, vol. 152, 2013, pages 1173 - 83
DURAI ET AL.: "Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells", NUCLEIC ACIDS RESEARCH, vol. 33, 2005, pages 5978
EISENSCHMIDT ET AL.: "Developing a programmed restriction endonuclease for highly specific DNA cleavage", NUCLEIC ACIDS RESEARCH, vol. 33, 2005, pages 7039
FUJITA; FUJII: "Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR", BIOCHEM BIOPHYS RES COMMUN, vol. 11, 11 August 2013 (2013-08-11), XP028715147 *
FUJITA; FUJII: "Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR", BIOCHEM BIOPHYS RES COMMUN, vol. 11, 2013
GASIUNAS ET AL.: "Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria", PROC NATL ACAD SCI U S A, vol. 109, 2012, pages E2579 - 86
GASIUNAS ET AL.: "Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria", PROC. NATL.ACAD. SCI. USA, vol. 109, 2012, pages E2579 - E2586
HODGES ET AL.: "Genome-wide in situ exon capture for selective re-sequencing", NAT GENET., vol. 39, 2007, pages 1522
JINEK ET AL.: "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity", SCIENCE, vol. 337, 2012, pages 816
KARVELIS ET AL., BIOCHEM. SOC. TRANS., vol. 41, 2013, pages 1401 - 1406
KARVELIS ET AL.: "crRNA and tracrRNA guide Cas9-mediated DNA interference in Streptococcus thermophilus", RNA BIOL., vol. 10, 2013, pages 841
KARVELIS ET AL.: "Programmable DNA cleavage in vitro by Cas9 + Supplementary Online Data", BIOCHEM. SOC. TRANS., vol. 41, 2013, pages 1401 - 1406+4pp, XP002735715 *
LI ET AL.: "TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and Fokl DNA-cleavage domain", NUCLEIC ACIDS RESEARCH, vol. 39, 2011, pages 359
MAKAROVA ET AL.: "Evolution and classification of the CRISPR-Cas systems", NAT REV MICROBIOL., vol. 9, 2011, pages 467
QI LEI S ET AL: "Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression", CELL, vol. 152, no. 5, 28 February 2013 (2013-02-28), CELL PRESS, US, pages 1173 - 1183, XP028987304, ISSN: 0092-8674, DOI: 10.1016/J.CELL.2013.02.022 *
RICHTER ET AL.: "Exploiting CRISPR/Cas: interference mechanisms and applications", INT J MOL SCI., vol. 14, 2013, pages 14518
SAPRANAUSKAS ET AL.: "The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli", NUCLEIC ACIDS RES., vol. 39, 2011, pages 9275
TERNS; TERNS: "CRISPR-based adaptive immune systems", CURR OPIN MICROBIOL., vol. 14, 2011, pages 321

Cited By (92)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10323236B2 (en) 2011-07-22 2019-06-18 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US11920181B2 (en) 2013-08-09 2024-03-05 President And Fellows Of Harvard College Nuclease profiling system
US10954548B2 (en) 2013-08-09 2021-03-23 President And Fellows Of Harvard College Nuclease profiling system
US10508298B2 (en) 2013-08-09 2019-12-17 President And Fellows Of Harvard College Methods for identifying a target site of a CAS9 nuclease
US10227581B2 (en) 2013-08-22 2019-03-12 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
US9359599B2 (en) 2013-08-22 2016-06-07 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
US11046948B2 (en) 2013-08-22 2021-06-29 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
US9999671B2 (en) 2013-09-06 2018-06-19 President And Fellows Of Harvard College Delivery of negatively charged proteins using cationic lipids
US10912833B2 (en) 2013-09-06 2021-02-09 President And Fellows Of Harvard College Delivery of negatively charged proteins using cationic lipids
US10597679B2 (en) 2013-09-06 2020-03-24 President And Fellows Of Harvard College Switchable Cas9 nucleases and uses thereof
US9737604B2 (en) 2013-09-06 2017-08-22 President And Fellows Of Harvard College Use of cationic lipids to deliver CAS9
US11299755B2 (en) 2013-09-06 2022-04-12 President And Fellows Of Harvard College Switchable CAS9 nucleases and uses thereof
US9526784B2 (en) 2013-09-06 2016-12-27 President And Fellows Of Harvard College Delivery system for functional nucleases
US9388430B2 (en) 2013-09-06 2016-07-12 President And Fellows Of Harvard College Cas9-recombinase fusion proteins and uses thereof
US10682410B2 (en) 2013-09-06 2020-06-16 President And Fellows Of Harvard College Delivery system for functional nucleases
US9340800B2 (en) 2013-09-06 2016-05-17 President And Fellows Of Harvard College Extended DNA-sensing GRNAS
US10858639B2 (en) 2013-09-06 2020-12-08 President And Fellows Of Harvard College CAS9 variants and uses thereof
US9340799B2 (en) 2013-09-06 2016-05-17 President And Fellows Of Harvard College MRNA-sensing switchable gRNAs
US10190137B2 (en) 2013-11-07 2019-01-29 Editas Medicine, Inc. CRISPR-related methods and compositions with governing gRNAS
US9834791B2 (en) 2013-11-07 2017-12-05 Editas Medicine, Inc. CRISPR-related methods and compositions with governing gRNAS
US10640788B2 (en) 2013-11-07 2020-05-05 Editas Medicine, Inc. CRISPR-related methods and compositions with governing gRNAs
US11390887B2 (en) 2013-11-07 2022-07-19 Editas Medicine, Inc. CRISPR-related methods and compositions with governing gRNAS
US11053481B2 (en) 2013-12-12 2021-07-06 President And Fellows Of Harvard College Fusions of Cas9 domains and nucleic acid-editing domains
US11124782B2 (en) 2013-12-12 2021-09-21 President And Fellows Of Harvard College Cas variants for gene editing
US10465176B2 (en) 2013-12-12 2019-11-05 President And Fellows Of Harvard College Cas variants for gene editing
US9840699B2 (en) 2013-12-12 2017-12-12 President And Fellows Of Harvard College Methods for nucleic acid editing
AU2015294354B2 (en) * 2014-07-21 2021-10-28 Illumina, Inc. Polynucleotide enrichment using CRISPR-Cas systems
WO2016014409A1 (fr) * 2014-07-21 2016-01-28 Illumina, Inc. Enrichissement de polynucléotides à l'aide de systèmes crispr-cas
EP3172321B1 (fr) 2014-07-21 2019-08-21 Illumina, Inc. Enrichissement de polynucléotides à l'aide de systèmes crispr-cas
US10457969B2 (en) 2014-07-21 2019-10-29 Illumina, Inc. Polynucleotide enrichment using CRISPR-Cas systems
EP3172321B2 (fr) † 2014-07-21 2023-01-04 Illumina, Inc. Enrichissement de polynucléotides à l'aide de systèmes crispr-cas
US11578343B2 (en) 2014-07-30 2023-02-14 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US10077453B2 (en) 2014-07-30 2018-09-18 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US10704062B2 (en) 2014-07-30 2020-07-07 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US10337001B2 (en) 2014-12-03 2019-07-02 Agilent Technologies, Inc. Guide RNA with chemical modifications
US10900034B2 (en) 2014-12-03 2021-01-26 Agilent Technologies, Inc. Guide RNA with chemical modifications
US11692213B2 (en) 2014-12-20 2023-07-04 Arc Bio, Llc Compositions and methods for targeted depletion, enrichment, and partitioning of nucleic acids using CRISPR/Cas system proteins
US10774365B2 (en) 2014-12-20 2020-09-15 Arc Bio, Llc Compositions and methods for targeted depletion, enrichment, and partitioning of nucleic acids using CRISPR/Cas system proteins
US10669571B2 (en) 2014-12-20 2020-06-02 Arc Bio, Llc Compositions and methods for targeted depletion, enrichment, and partitioning of nucleic acids using CRISPR/Cas system proteins
US11535846B2 (en) 2015-04-06 2022-12-27 The Board Of Trustees Of The Leland Stanford Junior University Chemically modified guide RNAS for CRISPR/Cas-mediated gene regulation
US11306309B2 (en) 2015-04-06 2022-04-19 The Board Of Trustees Of The Leland Stanford Junior University Chemically modified guide RNAs for CRISPR/CAS-mediated gene regulation
US11851652B2 (en) 2015-04-06 2023-12-26 The Board Of Trustees Of The Leland Stanford Junior Compositions comprising chemically modified guide RNAs for CRISPR/Cas-mediated editing of HBB
US10538758B2 (en) 2015-08-19 2020-01-21 Arc Bio, Llc Capture of nucleic acids using a nucleic acid-guided nuclease-based system
US10167457B2 (en) 2015-10-23 2019-01-01 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US11214780B2 (en) 2015-10-23 2022-01-04 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US10767175B2 (en) 2016-06-08 2020-09-08 Agilent Technologies, Inc. High specificity genome editing using chemically modified guide RNAs
CN109415727A (zh) * 2016-06-13 2019-03-01 格里尔公司 富集突变的无细胞核酸以供癌症检测的方法
US11624064B2 (en) 2016-06-13 2023-04-11 Grail, Llc Enrichment of mutated cell free nucleic acids for cancer detection
WO2017218512A1 (fr) * 2016-06-13 2017-12-21 Grail, Inc. Enrichissement d'acides nucléiques acellulaires mutés pour la détection du cancer
US10947530B2 (en) 2016-08-03 2021-03-16 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US10113163B2 (en) 2016-08-03 2018-10-30 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US11702651B2 (en) 2016-08-03 2023-07-18 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US11661590B2 (en) 2016-08-09 2023-05-30 President And Fellows Of Harvard College Programmable CAS9-recombinase fusion proteins and uses thereof
US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
US11739379B2 (en) 2016-09-29 2023-08-29 Oxford Nanopore Technologies Plc Method for nucleic acid detection by guiding through a nanopore
US11085078B2 (en) 2016-09-29 2021-08-10 Oxford Nanopore Technologies Limited Method for nucleic acid detection by guiding through a nanopore
US11306324B2 (en) 2016-10-14 2022-04-19 President And Fellows Of Harvard College AAV delivery of nucleobase editors
US11820969B2 (en) 2016-12-23 2023-11-21 President And Fellows Of Harvard College Editing of CCR2 receptor gene to protect against HIV infection
US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
US11898179B2 (en) 2017-03-09 2024-02-13 President And Fellows Of Harvard College Suppression of pain by gene editing
US11542496B2 (en) 2017-03-10 2023-01-03 President And Fellows Of Harvard College Cytosine to guanine base editor
US11268082B2 (en) 2017-03-23 2022-03-08 President And Fellows Of Harvard College Nucleobase editors comprising nucleic acid programmable DNA binding proteins
US11560566B2 (en) 2017-05-12 2023-01-24 President And Fellows Of Harvard College Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation
EP3638781A4 (fr) * 2017-06-13 2021-03-17 Genetics Research, LLC, D/B/A ZS Genetics, Inc. Enrichissement d'une cible dans du plasma/sérum
US10527608B2 (en) 2017-06-13 2020-01-07 Genetics Research, Llc Methods for rare event detection
US10947599B2 (en) 2017-06-13 2021-03-16 Genetics Research, Llc Tumor mutation burden
US10370700B2 (en) 2017-06-13 2019-08-06 Genetics Research, Llc Detection of targeted sequence regions
US11421263B2 (en) 2017-06-13 2022-08-23 Genetics Research, Llc Detection of targeted sequence regions
US11142788B2 (en) 2017-06-13 2021-10-12 Genetics Research, Llc Isolation of target nucleic acids
WO2018231952A1 (fr) * 2017-06-13 2018-12-20 Genetics Research, Llc, D/B/A Zs Genetics, Inc. Enrichissement d'une cible dans du plasma/sérum
US11732274B2 (en) 2017-07-28 2023-08-22 President And Fellows Of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE)
US11319532B2 (en) 2017-08-30 2022-05-03 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11932884B2 (en) 2017-08-30 2024-03-19 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
US11739367B2 (en) 2017-11-08 2023-08-29 Twinstrand Biosciences, Inc. Reagents and adapters for nucleic acid sequencing and methods for making such reagents and adapters
CN111868255A (zh) * 2018-03-15 2020-10-30 特温斯特兰德生物科学有限公司 用于富集用于测序应用和其他核酸材料询问的核酸材料的方法和试剂
JP2021515579A (ja) * 2018-03-15 2021-06-24 ツインストランド・バイオサイエンシズ・インコーポレイテッドＴｗｉｎｓｔｒａｎｄ Ｂｉｏｓｃｉｅｎｃｅｓ， Ｉｎｃ． 配列決定用途および他の核酸物質インテロゲーションのための核酸物質を濃縮するための方法および試薬
EP3765063A4 (fr) * 2018-03-15 2021-12-15 Twinstrand Biosciences, Inc. Procédés et réactifs pour l'enrichissement de matériau d'acide nucléique pour des applications de séquençage et d'autres interrogations de matériau d'acide nucléique
WO2020005846A1 (fr) * 2018-06-25 2020-01-02 Bionano Genomics, Inc. Marquage de l'adn
EP4159872A1 (fr) * 2018-06-25 2023-04-05 Bionano Genomics, Inc. Marquage de l'adn
CN112469834A (zh) * 2018-06-25 2021-03-09 生物纳米基因公司 Dna的标记
US20210230664A1 (en) * 2018-06-25 2021-07-29 Bionano Genomics, Inc. Labeling of dna
US11845985B2 (en) 2018-07-12 2023-12-19 Twinstrand Biosciences, Inc. Methods and reagents for characterizing genomic editing, clonal expansion, and associated applications
US11680261B2 (en) 2018-11-15 2023-06-20 Grail, Inc. Needle-based devices and methods for in vivo diagnostics of disease conditions
US11795452B2 (en) 2019-03-19 2023-10-24 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11643652B2 (en) 2019-03-19 2023-05-09 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11447770B1 (en) 2019-03-19 2022-09-20 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11912985B2 (en) 2020-05-08 2024-02-27 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence
US11884915B2 (en) 2021-09-10 2024-01-30 Agilent Technologies, Inc. Guide RNAs with chemical modification for prime editing
WO2023137292A1 (fr) * 2022-01-12 2023-07-20 Jumpcode Genomics, Inc. Procédés et compositions pour l'analyse du transcriptome
WO2023164252A1 (fr) * 2022-02-28 2023-08-31 Trustees Of Boston University Dosages de détection isotherme d'acides nucléiques et leurs utilisations
EP4276195A1 (fr) * 2022-05-10 2023-11-15 Genartis S.r.l. Procédés pour déterminer la présence ou le risque de développer la dystrophie myotonique de type 1 (md1) ou la dystrophie myotonique de type 2 (md2)

Similar Documents

Publication Publication Date Title
WO2015075056A1 (fr) Enzymes programmables pour l'isolement de fragments d'adn spécifiques
US20200172959A1 (en) Method for Fragmenting Genomic DNA Using CAS9
AU2016319110B2 (en) Full interrogation of nuclease DSBs and sequencing (FIND-seq)
KR102425438B1 (ko) 서열결정에 의해 평가된 DSB의 게놈 전체에 걸친 비편향된 확인 (GUIDE-Seq)
Hale et al. Essential features and rational design of CRISPR RNAs that function with the Cas RAMP module complex to cleave RNAs
JP2018532419A (ja) ＣＲＩＳＰＲ−Ｃａｓ ｓｇＲＮＡライブラリー
JP2018529353A (ja) 配列決定法による切断事象の包括的生体外報告（ＣＩＲＣＬＥ−ｓｅｑ）
JP2018535689A (ja) ガイド核酸を作製および使用するための方法および組成物
WO2013078470A2 (fr) Isolation multiplex d'acides nucléiques associés à des protéines
JP2020524491A (ja) ガイド核酸の作製および使用
WO2018067447A1 (fr) Méthodes améliorées d'identification de sites de rupture de double-brin
US20200255823A1 (en) Guide strand library construction and methods of use thereof
US20230257799A1 (en) Methods of identifying and characterizing gene editing variations in nucleic acids
WO2021119550A1 (fr) Procédé de détermination d'une architecture de génome 3d avec une résolution de paire de base et utilisations supplémentaires associées
KR20220084322A (ko) 세포에서 하나 이상의 표적-특이적인 프로그램 가능한 뉴클레아제의 오프-타겟 활성을 프로파일링하는 진정한 편향되지 않은 시험관내 분석(abnoba-seq)
EP4001429A1 (fr) Analyse de sites de liaison crispr-cas et de clivage suivie d'un séquençage à haut débit (abc-seq)
JP2023538537A (ja) 核酸の標的化除去のための方法
WO2024081738A2 (fr) Compositions, méthodes et systèmes de modification d'adn
AU2022234741A1 (en) Genomic library preparation and targeted epigenetic assays using cas-grna ribonucleoproteins
EP2783001A2 (fr) Isolation multiplex d'acides nucléiques associés à des protéines
CN117015602A (zh) 分析细胞中蛋白质编码变体的表达

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14800051

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 14800051

Country of ref document: EP

Kind code of ref document: A1