ENGINEERING AND OPTIMIZATION OF IMPROVED SYSTEMS, METHODS AND ENZYME COMPOSITIONS FOR SEQUENCE MANIPULATION
The invention provides for engineering and optimization of systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. Provided are compositions and methods related to components of a CRISPR complex particularly comprising a Cas ortholog enzyme.
1 . (A) A non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising
I. a first regulatory element operably linked to a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the polynucleotide sequence comprises
(a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell,
(b) a tracr mate sequence, and
(c) a tracr sequence, and
II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences,
wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation,
wherein components I and II are located on the same or different vectors of the system,
wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, and
wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence,
or
(B) a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising
I. a first regulatory element operably linked to
(a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, and
(b) at least one or more tracr mate sequences,
II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, and
III. a third regulatory element operably linked to a tracr sequence,
wherein components I, II and III are located on the same or different vectors of the system,
wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence,
wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence, and
wherein the CRISPR enzyme is a Cas9 ortholog of a genus belonging to the group consisting of Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma and Campylobacter.
2 . The composition according to claim 1 wherein the Cas9 ortholog is a Sutterella wadsworthensis Cas9.
3 . The composition according to claim 1 wherein the Cas9 ortholog is a Filifactor alocis Cas9.
4 . The composition according to claim 1 wherein the Cas9 ortholog is a Lactobacillus johnsonii Cas9.
5 . The composition according to claim 1 wherein the Cas9 ortholog is a Campylobacter lari Cas9.
6 . The composition according to claim 1 wherein the Cas9 ortholog is a Corynebacter diptheriae Cas9.
7 . The composition according to claim 1 wherein the Cas9 ortholog is a Parvibaculum lavamentivorans Cas9.
8 . The composition according to claim 1 wherein the Cas9 ortholog is a Mycoplasma gallisepticum Cas9.
9 . The composition according to claim 1 wherein the Cas9 ortholog is a Staphylococcus aureus subsubspecies Aureus Cas9.
10 . The composition according to claim 1 wherein the Cas9 ortholog is a Legionella pneumophila Paris Cas9.
11 . The composition according to claim 1 wherein the Cas9 ortholog is a Treponema denticola Cas9.
12 . The composition according to claim 1 wherein the Cas9 ortholog is a Staphylococcus pseudintermedius Cas9.
14 . The composition according to claim 1 wherein the Cas9 ortholog is a Neisseria cinerea Cas9.
15 . The composition according to claim 1 , wherein the CRISPR enzyme is truncated in comparison to the corresponding wild type CRISPR enzyme or the CRISPR enzyme is comprised of 500-2000 amino acids.
16 . The composition according to claim 1 , wherein the CRISPR enzyme is a nuclease directing cleavage of both strands at the location of the target sequence, or the CRISPR enzyme is a nickase directing cleavage of one strand at the location of the target sequence.
17 . The composition according to claim 1 , wherein the guide sequence comprises at least fifteen nucleotides.
18 . The composition according to claim 1 , wherein the CRISPR enzyme is codon-optimized or codon-optimized for expression in a eukaryotic cell.
19 . The composition according to claim 1 , wherein the CRISPR enzyme has one or more mutations in any domain of the enzyme.
20 . The composition according to any of claim 19 , wherein the CRISPR enzyme has one or more mutations in a catalytic domain.
21 . The composition according to claim 1 , wherein the enzyme further comprises a functional domain.
22 . A modified CRISPR enzyme wherein the enzyme is a Cas9 ortholog of a genus belonging to the group consisting of Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma and Campylobacter and wherein differences from the corresponding wild type CRISPR enzyme comprise:
the modified CRISPR enzyme is truncated in comparison to a wild type CRISPR enzyme, or
the CRISPR enzyme has a length of at least 500 amino acids, at least 800-899 amino acids, at least 900-999 amino acids, at least 1000-1099 amino acids, at least 1100-1199 amino acids, at least 1200-1299 amino acids, at least 1300-1399 amino acids, at least 1400-1499 amino acids, at least 1500-1599 amino acids, at least 1600-1699 amino acids or at least 2000 amino acids, and/or
the CRISPR enzyme is a nuclease directing cleavage of both strands near the location of the target sequence, or the CRISPR enzyme is a nickase directing cleavage of one strand near the location of the target sequence, and/or
the CRISPR enzyme is codon-optimized or codon-optimized for expression in a eukaryotic cell, and/or
the CRISPR enzyme comprises one or more mutations, and/or
the CRISPR enzyme comprises a chimeric CRISPR enzyme.
23 . The modified CRISPR enzyme according to claim 22 , wherein the CRISPR enzyme has one or more mutations in any domain of the enzyme.
24 . The modified CRISPR enzyme according to claim 22 , wherein the CRISPR enzyme has one or more mutations in a catalytic domain.
25 . The modified CRISPR enzyme according to claim 22 , wherein the enzyme further comprises a functional domain.
26 . The modified CRISPR enzyme according to claim 22 , wherein the CRISPR enzyme is truncated in comparison to a wild type CRISPR enzyme or the CRISPR enzyme is comprised of 500-2000 amino acids.
27 . The modified CRISPR enzyme according to claim 22 , wherein the CRISPR enzyme is a nuclease directing cleavage of both strands at the location of the target sequence, or the CRISPR enzyme is a nickase directing cleavage of one strand at the location of the target sequence.
28 . The modified CRISPR enzyme according to claim 22 , wherein the guide sequence comprises at least fifteen nucleotides.
29 . The modified CRISPR enzyme according to claim 22 , wherein the CRISPR enzyme is codon-optimized or codon-optimized for expression in a eukaryotic cell.