Engineering and optimization of systems, methods and compositions for sequence manipulation with functional domains
The invention provides for engineering and optimization of systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. Provided are vectors and vector systems, some of which encode one or more components of a CRISPR complex, as well as methods for the design and use of such vectors with additional functional domains. Also provided are methods of directing CRISPR complex formation in prokaryotic and eukaryotic cells to ensure enhanced specificity for target recognition and avoidance of toxicity.
1. A non-naturally occurring or engineered composition for altering expression of at least one gene product comprising introducing into a eukaryotic cell containing a DNA molecule having a target sequence adjacent to a Protospacer Adjacent Motif (PAM) and encoding the gene product; said composition comprising one or more vectors encoding an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) system,
said one or more vectors comprising:
a) a first regulatory element operable in a eukaryotic cell operably linked to at least one nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with the target sequence, and
b) a second regulatory element operable in a eukaryotic cell operably linked to a nucleotide sequence encoding a fusion of a Type-II Cas9 protein and one or more protein domains,
wherein:
components (a) and (b) are located on same or different vectors of the system, the Cas9 protein comprises one or more mutations in a catalytic domain, the guide RNA comprises a tracr sequence which is 30 or more nucleotides in length, the Cas9 protein and the guide RNA do not naturally occur together,
whereby expression of the at least one gene product is altered through the CRISP R-Cas system acting as to the DNA molecule comprising the guide RNA directing sequence-specific binding of the CRISPR-Cas system and PAM recognition, and wherein
the one or more protein domains comprises an epitope tag, a reporter, or a domain having transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, nucleic acid or cellular molecule binding activity, activity as a light-responsive cytochrome heterodimer, transposase activity, integrase activity, recombinase activity, resolvase activity, invertase activity, protease activity, nuclease activity, transcription-protein recruiting activity, cellular uptake activity or antibody presentation activity.
2. A multiplexed non-naturally occurring or engineered composition for altering expression of at least one gene product comprising introducing into a eukaryotic cell containing a DNA molecule having a target sequence adjacent to a Protospacer Adjacent Motif (PAM) and encoding the gene product; said composition comprising one or more vectors encoding an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) system,
said one or more vectors comprising:
a) a first regulatory element operable in a eukaryotic cell operably linked to at least one nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with the target sequence, and
b) a second regulatory element operable in a eukaryotic cell operably linked to a nucleotide sequence encoding a fusion of a Type-II Cas9 protein and one or more protein domains,
wherein:
components (a) and (b) are located on same or different vectors of the system, the Cas9 protein comprises one or more mutations in a catalytic domain, the guide RNA comprises a tracr sequence which is 30 or more nucleotides in length, the Cas9 protein and the guide RNA do not naturally occur together,
whereby expression of the at least one gene product is altered through the CRISPR-Cas system acting as to the DNA molecule comprising the guide RNA directing sequence-specific binding of the CRISP R-Cas system and PAM recognition, and wherein
the one or more protein domains comprises an epitope tag, a reporter, or a domain having transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, nucleic acid or cellular molecule binding activity, activity as a light-responsive cytochrome heterodimer, transposase activity, integrase activity, recombinase activity, resolvase activity, invertase activity, protease activity, nuclease activity, transcription-protein recruiting activity, cellular uptake activity or antibody presentation activity, and the system includes at least two guide RNAs, each of which hybridizes with a different target sequence whereby the system is multiplexed.
3. A non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors for altering expression of at least one gene product comprising introducing into a eukaryotic cell containing a DNA molecule having a target sequence adjacent to a Protospacer Adjacent Motif (PAM) and encoding the gene product; said composition comprising one or more vectors encoding an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) system,
said one or more vectors comprising:
a) a first regulatory element operable in a eukaryotic cell operably linked to a guide sequence capable of hybridizing to a target sequence in the eukaryotic cell, and at least one or more tracr mate sequences, and
b) a second regulatory element operable in a eukaryotic cell operably linked to a nucleotide sequence encoding a fusion of a Type-II Cas9 protein and one or more protein domains, and
c) a third regulatory element operably linked to a tracr sequence,
wherein:
components (a), (b) and (c) are located on same or different vectors of the system, the Cas9 protein comprises one or more mutations in a catalytic domain, the guide RNA comprises a tracr sequence which is 30 or more nucleotides in length, the Cas9 protein and the guide RNA do not naturally occur together,
whereby expression of the at least one gene product is altered through the CRISPR-Cas system acting as to the DNA molecule comprising the guide RNA directing sequence-specific binding of the CRISP R-Cas system and PAM recognition, and wherein
the one or more protein domain comprises an epitope tag, a reporter, or a domain having transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, nucleic acid or cellular molecule binding activity, activity as a light-responsive cytochrome heterodimer, transposase activity, integrase activity, recombinase activity, resolvase activity, invertase activity, protease activity, nuclease activity, transcription-protein recruiting activity, cellular uptake activity or antibody presentation activity.
4. A multiplexed CRISPR enzyme system composition comprising a vector system comprising one or more vectors for altering expression of at least one gene product comprising introducing into a eukaryotic cell containing a DNA molecule having a target sequence adjacent to a Protospacer Adjacent Motif (PAM) and encoding the gene product; said composition comprising one or more vectors encoding an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) system,
said one or more vectors comprising:
a) a first regulatory element operable in a eukaryotic cell operably linked to a guide sequence capable of hybridizing to a target sequence in the eukaryotic cell, and at least one or more tract mate sequences,
b) a second regulatory element operable in a eukaryotic cell operably linked to a nucleotide sequence encoding a fusion of a Type-II Cas9 protein and one or more protein domains, and
c) a third regulatory element operably linked to a tracr sequence,
wherein:
components (a), (b) and (c) are located on same or different vectors of the system, the Cas9 protein comprises one or more mutations in a catalytic domain, the guide RNA comprises a tracr sequence which is 30 or more nucleotides in length, the Cas9 protein and the guide RNA do not naturally occur together,
whereby expression of the at least one gene product is altered through the CRISPR-Cas system acting as to the DNA molecule comprising the guide RNA directing sequence-specific binding of the CRISPR-Cas system and PAM recognition, and wherein
the one or more protein domain comprises an epitope tag, a reporter, or a domain having transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, nucleic acid or cellular molecule binding activity, activity as a light-responsive cytochrome heterodimer, transposase activity, integrase activity, recombinase activity, resolvase activity, invertase activity, protease activity, nuclease activity, transcription-protein recruiting activity, cellular uptake activity or antibody presentation activity, and the system comprises at least two different guide sequences, each capable of hybridizing to a different target sequence, whereby the system is multiplexed.
5. The composition of any one of claims 1 to 4 , wherein the one or more mutations comprise SpCas9 D10A or SpCas9 H840A.
6. The composition of any one of claims 1 to 4 , wherein the Cas9 protein comprises of SpCas9 D10, SpCas9 E762, SpCas9 H840, SpCas9 N854, SpCas9 N863, SpCas9 D986.
7. The composition of any one of claims 1 to 4 , comprising at least two or more nuclear localization sequences.
8. The composition of any one of claims 1 to 4 , wherein the one or more protein domain(s) has transcription activation activity.
9. The composition of claim 8 , wherein the one or more protein domain(s) comprises VP64.
10. The composition of any one of claims 1 to 4 , wherein there are one or more linker sequences between the Cas9 protein and the one or more protein domain(s).
11. The composition of any one of claims 1 to 4 , wherein the one or more protein domain(s) binds DNA.
12. The composition of any one of claims 1 to 4 , wherein the one or more protein domain(s) affects transcription of the target nucleic acid.
13. The composition of any one of claims 1 to 4 , wherein the eukaryotic cell is a mammalian cell.
14. The composition of claim 13 , wherein the mammalian cell is a human cell.
15. The composition of any one of claims 1 to 4 , wherein the Cas9 protein is codon optimized for expression in the eukaryotic cell.
16. The composition of any one of claims 1 to 4 , wherein the Cas9 protein is from an organism from a genus comprising Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium or Corynebacter.
17. The composition of any one of claims 1 to 4 , wherein the vectors of the system are viral vectors comprising a lentiviral vector, an adenoviral vector or an AAV vector.
18. A method of modulating gene expression at a genomic locus of interest in a cell comprising contacting the cell with the composition of any one of the claims 1 to 4 .
19. The composition of any one of claims 1 to 4 , wherein the epitope tag, comprises a histidine (His) tag, a V5 tag, a FLAG tag, an influenza hemagglutinin (HA) tag, a Myc tag, a VSV-G tag, or a thioredoxin (Trx) tag.
20. The composition of any one of claims 1 to 4 , wherein the reporter comprises glutathione-Stransferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), betagalactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyanfluorescent protein (CFP), yellow fluorescent protein (YFP), or an autofluorescent protein.
21. The composition of claim 20 , wherein the autofluorescent protein comprises blue fluorescent protein (BFP).
22. The composition of any one of claims 1 to 4 , wherein the one or more protein domain(s) has transcription repression activity.
23. The composition of any one of claims 1 to 4 , wherein the one or more protein domain(s) has transcription release factor activity.
24. The composition of any one of claims 1 to 4 , wherein the one or more protein domain(s) has histone modification activity.
25. The composition of claim 24 , wherein the histone modification activity comprises histone acetylase activity.
26. The composition of any one of claims 1 to 4 , wherein the one or more protein domain(s) has RNA cleavage activity.
27. The composition of any one of claims 1 to 4 , wherein, the one or more protein domain(s) has nucleic acid or cellular molecule binding activity.
28. The composition of claim 27 , wherein, the one or more protein domain(s) having nucleic acid or cellular molecule binding activity comprises maltose binding protein (MBP), S-tag, a Lex A DNA binding domain (DBD) fusion protein, a GAL4 DNA binding domain fusion protein, or a herpes simplex virus (HSV) BP16 fusion protein.
29. The composition of any one of claims 1 to 4 , wherein, the one or more protein domain(s) has activity as a light-responsive cytochrome heterodimer.
30. The composition of claim 29 , wherein the light-responsive cytochrome heterodimer is from Arabidopsis thaliana.
31. The composition of any one of claims 1 to 4 , wherein, the one or more protein domain(s) has transposase activity.
32. The composition of any one of claims 1 to 4 , wherein, the one or more protein domain(s) has integrase activity.
33. The composition of any one of claims 1 to 4 , wherein, the one or more protein domain(s) has recombinase activity.
34. The composition of any one of claims 1 to 4 , wherein, the one or more protein domain(s) has resolvase activity.
35. The composition of any one of claims 1 to 4 , wherein, the one or more protein domain(s) has invertase activity.
36. The composition of any one of claims 1 to 4 , wherein, the one or more protein domain(s) has protease activity.
37. The composition of any one of claims 1 to 4 , wherein, the one or more protein domain(s) has nuclease activity.
38. The composition of any one of claims 1 to 4 , wherein, the one or more protein domain(s) has transcription-protein recruiting activity.
39. The composition of any one of claims 1 to 4 , wherein, the one or more protein domain(s) has cellular uptake activity.
40. The composition of any one of claims 1 to 4 , wherein, the one or more protein domain(s) has antibody presentation activity.
41. The composition of any one of claims 1 to 4 , wherein, the guide directs CRISPR system formation at a promoter sequence.
42. The composition of any one of claims 1 to 4 , wherein, the one or more protein domain(s) comprises VP 16, VP64, p65, KRAB, EnR, or SID.
43. A CRISPR-Cas system obtained from expression of the vector system of any one of claims 1 to 4 .