METHOD, COMPOSITION, AND REAGENT KIT FOR TARGETED GENOMIC ENRICHMENT

• US 20140127752A1
• Filed: 11/07/2013
• Published: 05/08/2014
• Est. Priority Date: 11/07/2012
• Status: Abandoned Application

• Current Assignee: Recombitech, Inc.
• Original Assignee: Recombitech, Inc.
• Inventors: Zhou, Zhaohui, Shan, Qun, Xu, Yue
• Application Number:
  US14/074,679
• Publication Number:
  US 20140127752A1
• Time in Patent Office: Days
• US Class Current: 435/91.53
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Y 301/21001 Deoxyribonuclease I (3.1.21.1)

Abstract

A composition and method of cleaving a target DNA and isolating a DNA sequence of interest, directed by a targeting oligonucleotide (“ON”) including a DNA binding agent (stable or unstable), is disclosed. The targeting ON binds to the target DNA before or during DNA cleavage. After cleavage, the isolation of the DNA fragment of interest is facilitated by the affinity tag on the targeting ON or an affinity tag attached using either ligation or polymerase extension method.

Claims

1. A composition comprising an engineered stable-binding sequence specific DNA nuclease, comprising

one or more targeting oligonucleotides, wherein:

said engineered sequence specific DNA nuclease is capable of cutting a target double stranded DNA with sequence specificity greater than eight base pairs long;

said targeting oligonucleotide includes one or more affinity tags ; and

purification of a piece of DNA fragment of interest cut by said sequence specific DNA nuclease is facilitated by said affinity tag.

2. The composition of claim 1, wherein said engineered sequence specific DNA nuclease comprises:

a Cas9 protein or a variant thereof;

a targeting oligonucleotide;

wherein one of the above components includes an affinity tag or is bound to a solid support.

3. The composition of claim 2, comprising a Cas 9 protein.

4. The composition of claim 2, comprising a targeting DNA that is a single-stranded DNA or RNA 30-1,000 nucleotides in length, wherein a 30-150 nucleotides long sequence is complementary to a sequence on one strand of the target double stranded DNA.

5. The composition of claim 2, wherein said affinity tag is selected from the group consisting of biotin, azido group, acetylene group, HIS-tag, Calmodulin-tag, CBP, CYD, Strep II, FLAG-tag, HA-tag, Myc-tag, S-tag, SBP-tag, Softag-1, Softag-3, V5-tag, Xpress-tag, Isopeptag, SpyTag, B, HPC peptide tags, GST, MBP, biotin carboxyl carrier protein, glutathione-S-transferase-tag, green fluorescent protein-tag, maltose binding protein-tag, Nus-tag, Strep-tag, and thioredoxin-tag.

6. The composition of claim 2, wherein said solid support is a plate, membrane, gel, magnetic bead, or microbead.

7. A method for cutting out a DNA fragment of interest from a target double stranded DNA, comprising:

contacting said target double stranded DNA with a composition comprising an engineered sequence specific DNA nuclease that includes one or more targeting oligonucleotides, wherein said engineered sequence specific DNA nuclease is capable of cutting a double stranded DNA with sequence specificity greater than eight base pairs long; and

isolating the DNA fragment of interest.

8. The method of claim 7, wherein:

said composition includes an engineered stable-binding sequence specific DNA nuclease, and one or more components of the engineered stable-binding sequence specific DNA nuclease include one or more affinity tags or are bound to a solid support; and

purification of said DNA fragment of interest is facilitated by said affinity tag or solid support.

9. The method of claim 7, further comprising:

attaching one or more affinity tags to said DNA fragment of interest; and

isolating said DNA fragment of interest facilitated by said affinity tag.

10. The method of claim 7, wherein said composition includes one or more targeting oligonucleotides, and the composition causes the target DNA to be cut by said engineered sequence specific DNA nuclease at only one end of the DNA fragment of interest in such a way that an affinity tag can be attached to the cut DNA fragment and this DNA fragment can be isolated using said affinity tag.

11. The method of claim 8, wherein said composition includes at least one pair of targeting oligonucleotides, and the composition causes the target DNA to be cut by said engineered stable-binding sequence specific DNA nuclease at both ends of the DNA fragment of interest in such a way that at least one pair of targeting oligonucleotides remain bound to the DNA fragment of interest after the target DNA is cut.

12. The method of claim 8, wherein said composition includes at least one pair of targeting oligonucleotides, and the composition causes the target DNA to be cut by said engineered stable-binding sequence specific DNA nuclease at both ends of the DNA fragment of interest in such a way that only one targeting oligonucleotide remains bound to the DNA fragment of interest after the target DNA is cut, and wherein said targeting oligonucleotide that remains bound includes said affinity tag or is bound to said solid support.

13. A reagent kit comprising an engineered stable-binding sequence specific DNA nuclease, wherein:

said engineered stable-binding sequence specific DNA nuclease is capable of cutting a target double stranded DNA with sequence specificity greater than eight base pairs long;

said engineered stable-binding sequence specific DNA nuclease includes one or more targeting oligonucleotides that includes one or more stable-binding agent;

at least one component of the engineered stable-binding sequence specific DNA nuclease includes an affinity tag or is bound to a solid support; and

purification of a piece of DNA fragment of interest cut by said engineered stable-binding sequence specific DNA nuclease is facilitated by said affinity tag or solid support.

14. The reagent kit of claim 13, wherein said engineered stable-binding sequence specific DNA nuclease comprises a Cas9 protein or a variant thereof.

15. The reagent kit of claim 13, comprising a Cas 9 protein.

16. The reagent kit of claim 13, comprising a targeting DNA that is a single-stranded DNA or RNA 30-1,000 nucleotides in length, wherein a 30-150 nucleotides long sequence is substantially complementary to a sequence on one strand of the target double stranded DNA.

17. The reagent kit of claim 13, comprising a Cas 9 mutant protein.

18. The reagent kit of claim 13, wherein said affinity tag is selected from the group consisting of biotin, azido group, acetylene group, HIS-tag, Calmodulin-tag, CBP, CYD, Strep II, FLAG-tag, HA-tag, Myc-tag, S-tag, SBP-tag, Softag-1, Softag-3, V5-tag, Xpress-tag, Isopeptag, SpyTag, B, HPC peptide tags, GST, MBP, biotin carboxyl carrier protein, glutathione-S-transferase-tag, green fluorescent protein-tag, maltose binding protein-tag, Nus-tag, Strep-tag, and thioredoxin-tag.

19. The reagent kit of claim 13, wherein said solid support is a plate, membrane, gel, magnetic bead, or microbead.