Chimeric genome engineering molecules and methods
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.
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.