Chimeric genome engineering molecules and methods

Patent metadata

Field	Value
Publication number	US-11293019-B2
Application number	US-201816955639-A
Country	US
Kind code	B2
Filing date	Dec 21, 2018
Priority date	Dec 22, 2017
Publication date	Apr 5, 2022
Grant date	Apr 5, 2022

Abstract

The present disclosure provides compositions and methods for increasing mutation efficiency and homologous recombination rates of site-specific endonucleases. The compositions and methods comprise a chimeric polypeptide comprising a site-specific endonuclease or a domain thereof and a functional moiety. The current inventions relate to functional enhancement of the CRISPR-Cas enzymes. Disclosed herein include possible variants and their intended improvements.

First claim

What is claimed is:

1. A method of targeting a single locus for mutagenesis, said method comprising:
selecting a locus for mutagenesis; and
contacting a genomic sample comprising the locus to a chimeric polypeptide comprising an exonuclease selected from the group consisting of: a RecE, a RecJ, a RecBCD, a Mungbean nuclease, an ExoIII, and an ExoVII, and a programmable endonuclease that binds to the locus and is selected from the group consisting of: a Cas9 and a Cpf 1, thereby generating a modified locus, thereby modifying an unique segment of the genomic sample, wherein the modifying comprises mutagenizing, inserting, or deleting at most 40 bases.

2. The method of claim 1 , wherein said selecting comprises identifying a unique segment of at least 10 bases in the genomic sample.

3. The method of claim 1 , wherein said contacting occurs in vivo.

4. The method of claim 3 , wherein said contacting comprises transfecting a cell using a vector encoding the enzyme.

5. The method of claim 3 , wherein said contacting comprises bombarding a cell using a nucleic acid encoding the enzyme.

6. The method of claim 5 , wherein said bombarding comprises contacting to at least one gold particle.

7. The method of claim 5 , wherein said bombarding comprises contacting to at least one tungsten particle.

8. The method of claim 3 , wherein said contacting comprises vacuum infiltration.

9. The method of claim 3 , wherein said contacting comprises Agrobacterium -mediated transformation.

10. The method of claim 3 , wherein said contacting comprises stable transformation.

11. The method of claim 3 , wherein said contacting comprises transient expression.

12. The method of claim 1 , wherein said exonuclease comprises an Exo1 exonuclease activity.

13. The method of claim 1 , wherein said exonuclease comprises 5′- 3 ′ overhang exonuclease activity.

14. The method of claim 1 , wherein said exonuclease comprises double-stranded nucleic acid exonuclease activity.

15. The method of claim 1 , wherein said exonuclease does not exhibit single stranded nucleic acid exonuclease activity.

16. The method of claim 1 , further comprises sequencing across the locus for identifying a mutation or a deletion relative to the locus prior to contacting.

17. The method of claim 1 , further comprises sequencing a substantial portion of the genomic sample aside from the locus comprising sequencing at least 1%, at least 5%, at least 10%, or at least 50% of a genome copy of the genomic sample.

18. The method of claim 1 , wherein said contacting occurs in vivo, and wherein said method comprises sequencing a substantial portion of the genomic sample aside from the locus is performed subsequent to at least one cell division subsequent to said contacting.

19. The method of claim 1 , comprising contacting the genomic sample to a composition comprising a buffer comprising Zinc ion.

20. The method of claim 1 , comprising contacting the genomic sample to a composition comprising a buffer comprising Zinc sulfate.

21. The method of claim 1 , wherein the exonuclease and the programmable endonuclease are fused in frame, wherein the chimeric polypeptide exhibits enhanced on target mutagenesis compared to a sequence specific endonuclease and wherein the chimeric polypeptide exhibits the same or lower off target mutagenesis compared to the sequence specific endonuclease when unfused.

22. A polypeptide comprising a sequence-specific endonuclease fused in frame to a DNA binding protein (DBP), wherein the DBP binds single-stranded DNA or double-stranded DNA, wherein the sequence-specific endonuclease is selected from the group consisting of: a Cas9 and a Cpf 1.

23. A polypeptide comprising a sequence-specific endonuclease fused in frame to a terminal deoxyribonucleotidyl transferase (TdT), wherein the sequence-specific endonuclease is selected from the group consisting of: a Cas9 and a Cpf 1.

24. The method of claim 1 , wherein the programmable endonuclease is the Cas9.

25. The method of claim 24 , wherein the Cas9 is SpCas9.

26. The method of claim 1 , wherein the programmable endonuclease is the Cpf1.

Assignees

• Gflas Life Sciences Inc
• Seoul Nat Univ R&Db Foundation

Inventors

• Park Jongjin
• Yoon Ji Young
• Choi Sunmee
• Park Mijin
• Park Slki
• Park Aiden Y
• Lee Jung Hyuk
• Lim Junghak
• Kim Dong Wook
• Choe Sunghwa

Classifications

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title

• C07K2319/80

  containing a DNA binding domain, e.g. Lacl or Tet-repressor · CPC title

• C12N9/22 Primary

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C07K2319/21

  containing a His-tag · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title