ENGINEERING OF SYSTEMS, METHODS AND OPTIMIZED GUIDE COMPOSITIONS FOR SEQUENCE MANIPULATION
The invention provides for 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. Also provided are methods of directing CRISPR complex formation in eukaryotic cells and methods for selecting specific cells by introducing precise mutations utilizing the CRISPR-Cas system.
1 . A non-naturally occurring or engineered composition comprising:
A) 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
wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation,
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 a 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 CRISPR enzyme system, wherein the system is encoded by 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) one or more guide sequences capable of hybridizing to one or more target sequences in a eukaryotic
(b) a tracr mate sequence, and
(c) one or more tracr sequences, 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,
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
C) a multiplexed CRISPR enzyme system, wherein the system is encoded by 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) one or more guide sequences capable of hybridizing to one or more target sequences in a eukaryotic cell,
(b) tracr mate sequence, and
(c) one or more tracr sequences, 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,
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,
wherein in the multiplexed system multiple chiRNA polynucleotide sequences are used
or
D) a multiplexed CRISPR enzyme system, wherein the system is encoded by a vector system comprising one or more vectors comprising
I. a first regulatory element operably linked to
(a) one or more guide sequences capable of hybridizing to a target sequence in a 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 in the multiplexed system multiple guide sequences and a single tracr sequence is used;
and
wherein, in the polynucleotide sequence of A), or in the system of B), C) or D), one or more of the guide, tracr and tracr mate sequences are modified to improve stability.
2 . The CRISPR-Cas system chiRNA or CRISPR enzyme system of claim 1 wherein the modification comprises sequence optimization.
3 . The CRISPR-Cas system chiRNA or CRISPR enzyme system of claim 1 wherein the modification comprises reduction in polyT sequences in the tracr and/or tracr mate sequence.
4 . The CRISPR-Cas system chiRNA or CRISPR enzyme system of claim 3 wherein one or more Ts present in a poly-T sequence of the relevant wild type sequence have been substituted with a non-T nucleotide.
5 . The CRISPR-Cas system chiRNA or CRISPR enzyme system of claim 2 , 3 , or 4 wherein the modified sequence does not comprise any polyT sequence having more than 4 contiguous Ts.
6 . The CRISPR-Cas system chiRNA or CRISPR enzyme system of claim 1 wherein the modification comprises altering loops and/or hairpins.
7 . The CRISPR-Cas system chiRNA or CRISPR enzyme system of claim 6 wherein the modification comprises providing a minimum of two hairpins in the guide sequence.
8 . The CRISPR-Cas system chiRNA or CRISPR enzyme system of claim 6 wherein the modification comprises providing a hairpin formed by complementation between the tracr and tracr mate sequence.
9 . The CRISPR-Cas system chiRNA or CRISPR enzyme system of claim 6 wherein the modification comprises providing one or more further hairpin(s) at the 3′ end of the tracrRNA sequence.
10 . The CRISPR-Cas system chiRNA or CRISPR enzyme system of claim 6 wherein the modification comprises providing one or more additional hairpin(s) added to the 3′ of the guide sequence.
11 . The CRISPR-Cas system chiRNA or CRISPR enzyme system of claim 1 wherein the modification comprises extending the 5′ end of the guide sequence.
12 . The CRISPR-Cas system chiRNA or CRISPR enzyme system of claim 11 wherein the modification comprises providing one or more hairpins in the 5′ end of the guide sequence.
13 . The CRISPR-Cas system chiRNA or CRISPR enzyme system of claim 1 wherein the modification comprises two hairpins.
14 . The CRISPR-Cas system chiRNA or CRISPR enzyme system of claim 1 wherein the modification comprises three hairpins.
15 . The CRISPR-Cas system chiRNA or CRISPR enzyme system of claim 1 wherein the modification comprises at most five hairpins.
16 . The CRISPR-Cas system chiRNA or CRISPR enzyme system of claim 1 wherein the CRISPR enzyme is a type 11 CRISPR system enzyme.
17 . The CRISPR-Cas system chiRNA or CRISPR enzyme system of claim 1 wherein the CRISPR enzyme is a Cas9 enzyme.
18 . The CRISPR-Cas system chiRNA or CRISPR enzyme system of claim 1 wherein the CRISPR enzyme is comprised of less than one thousand amino acids.
19 . The CRISPR-Cas system chiRNA or CRISPR enzyme system of claim 1 wherein the CRISPR enzyme is comprised of less than four thousand amino acids.
20 . The CRISPR-Cas system chiRNA or CRISPR enzyme system of claim 1 wherein the Cas9 enzyme is StCas9 or St1Cas9.
21 . The CRISPR-Cas system chiRNA or CRISPR enzyme system of claim 1 wherein the Cas9 enzyme is a Cas9 enzyme from an organism selected from the group comprising of genus Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium or Corynebacter.
22 . The CRISPR-Cas system chiRNA or CRISPR enzyme system of claim 1 wherein the CRISPR enzyme is a nuclease directing cleavage of both strands at the location of the target sequence.
23 . The CRISPR-Cas system chiRNA or CRISPR enzyme system of claim 1 , wherein the first regulatory element and the third regulatory element is a polymerase III promoter.
24 . The CRISPR-Cas system chiRNA or CRISPR enzyme system of claim 1 , wherein the second regulatory element is a polymerase II promoter.
25 . The CRISPR-Cas system chiRNA or CRISPR enzyme system of claim 1 , wherein the guide sequence comprises at least fifteen nucleotides.
26 . The CRISPR-Cas system chiRNA or CRISPR enzyme system of claim 1 , wherein the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell.
27 . The composition of claim 1 wherein the composition comprises a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence.
28 . The composition of claim 27 , wherein the composition further comprises a polynucleotide sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences.
29 . The CRISPR enzyme system of claim 1 .
30 . The multiplexed CRISPR enzyme system of claim 1 .
31 . The transcription or translation product of the composition of claim 27 or 28 , the CRISPR enzyme system of claim 29 , or the multiplexed CRISPR enzyme system of claim 30 .