CRISPR-CAS COMPONENT SYSTEMS, METHODS AND 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 composition comprising (i) a population of eukaryotic cells, and (ii) a library comprising a plurality of 100 or more CRISPR-Cas9 system guide RNAs; wherein each of the eukaryotic cells contains one or more of the guide RNAs and thus the guide RNAs of the library are integrated into the population of eukaryotic cells; and each guide RNA comprises an engineered guide sequence that targets a unique genomic locus in a eukaryotic cell, whereby the library targets a plurality of target sequences of genomic loci in the population of eukaryotic cells.
2 . The composition of claim 1 , wherein the population of eukaryotic cells is a population of embryonic stem (ES) cells.
3 . The composition of claim 1 , wherein a target sequence of the plurality of target sequences of genomic loci is a non-coding sequence.
4 . The composition of claim 2 , wherein a target sequence of the plurality of target sequences of genomic loci has gene function, whereby when the guide RNA is assembled with a Cas9 protein, one or more gene products of the ES cells is altered by the targeting.
5 . The composition of claim 1 , wherein a target sequence of the plurality of target sequences of genomic loci has gene function, whereby when the guide RNA is assembled with a Cas9 protein the targeting results in a knockout of gene function.
6 . The composition of claim 1 , wherein a target sequence of the plurality of target sequences of genomic loci has function as to a pathway or, wherein through one or more of the plurality of 100 or more CRISPR-Cas9 system guide RNAs, the targeting is of sequences of a pathway.
7 . The composition of claim 6 , wherein the pathway is an immune pathway.
8 . The composition of claim 1 , wherein the plurality of 100 or more CRISPR-Cas9 system guide RNAs comprises 1000 or more guide RNAs.
9 . The composition of claim 1 , wherein the plurality of 100 or more CRISPR-Cas9 system guide RNAs comprises 20,000 or more guide RNAs.
10 . The composition of claim 6 , wherein the pathway is a cell division pathway.
11 . The composition of claim 1 , wherein cells of the population contain:
a vector system comprising one or more vectors comprising a CRISPR-Cas9 system comprising:
I. a nucleic acid molecule encoding a Cas9 protein, and
II. one or more a nucleic acid molecule(s) encoding guide RNA of the library,
wherein components I and II are on same or different vectors,
wherein the nucleic acid molecule encoding the Cas9 protein is operably linked to a regulatory element, and
wherein when transcribed, the guide RNA comprising the guide sequence directs sequence-specific binding of a CRISPR-Cas9 system to the target sequence
whereby cleavage is induced by the Cas9 protein.
12 . The composition of claim 11 , wherein the one or more vectors are plasmid vectors.
13 . The composition of claim 11 , wherein the regulatory element is an inducible promoter.
14 . The composition of claim 13 , wherein the inducible promoter is a doxycycline inducible promoter.
15 . The composition of claim 11 , wherein cells of the population of cells comprise different knockout mutations in a plurality of unique genes.
16 . The composition of claim 11 , wherein components I and II are on the same vector.
17 . The composition of claim 11 , wherein the plurality of 100 or more CRISPR-Cas9 system guide RNAs comprises 1000 or more guide RNAs; whereby knockout mutations are achieved in 1000 or more unique genes.
18 . The composition of claim 11 , wherein the plurality of 100 or more CRISPR-Cas9 system guide RNAs comprises 20,000 or more guide RNAs; whereby knockout mutations are achieved in 20,000 or more unique genes.
19 . The composition of claim 11 , wherein knockout of gene function is achieved in a plurality of unique genes which function in a particular physiological pathway or condition.
20 . The composition of claim 19 , wherein the pathway or condition is an immune pathway or condition.
21 . The composition of claim 19 , wherein the pathway or condition is a cell division pathway or condition.
22 . The composition of claim 1 , wherein cells of the population of eukaryotic cells contain or express a Cas9 protein.
23 . The composition of claim 22 , wherein the guide RNAs are on a vector.
24 . The composition of claim 23 , wherein the vector is a plasmid vector.
25 . A functional genomics method comprising:
delivering Cas9 or nucleic acid molecule(s) encoding and leading to expression of Cas9 to the eukaryotic cells of the population of claim 1 , so that the eukaryotic cells of the population contain Cas9,
whereby in eukaryotic cells of the population, guide RNA of the library is assembled with a Cas9 to thereby form CRISPR-Cas9 complex(es), and
generating data from the engineered guide sequence of the guide RNA in the CRISPR-Cas9 complex targeting a unique genomic locus and the library targeting a plurality of target sequences of genomic loci in the population of eukaryotic cells.
26 . The method of claim 25 , wherein:
the population of eukaryotic cells are ES, or
the target sequence comprises a non-coding sequence, or
the target sequence comprises a genomic locus that has gene function and the eukaryotic cells are ES whereby one or more gene products of the ES cells is altered by the targeting, or
the target sequence comprises a genomic locus that has gene function whereby the targeting results in a knockout of gene function, or
the target sequence comprises a genomic locus that has function as to a pathway, or
the target sequence comprises a genomic locus that has function as to an immune pathway, or
the target sequence comprises a genomic locus that has function as to an cell division pathway, or
the plurality of 100 or more CRISPR-Cas9 system guide RNAs comprises 1000 or more guide RNAs, or
the plurality of 100 or more CRISPR-Cas9 system guide RNAs comprises 20,000 or more guide RNAs, or
the plurality of 100 or more CRISPR-Cas9 system guide RNAs comprises guide RNAs that result in different knockout mutations in a plurality of unique genes, or
the method includes gene or genome editing, or modifying a target sequence, or cleavage of a target sequence, or inactivation of a target sequence, or altering a target sequence whereby an encoded protein has altered function, or lack of transcription of an encoded protein, or gene therapy, or drug discovery, or drug screening, or disease diagnosis, or prognosis, or the manufacture of a medicament, or inactivation of a control sequence, or target sequence modification such that disease development and/or progression is inhibited or reduced, or production of an altered protein, or contacting of a test compound and modulation of a cell signaling event associated with a disease gene and detecting a reduction or augmentation of the cell signaling event, or screening for a cellular function change.
27 . The method of claim 25 , further comprising operating an instrument that provides measurement or data.
28 . A kit comprising the composition of claim 1 and instructions for using the kit.
29 . A method of altering the expression of a genomic locus of interest in a eukaryotic cell comprising:
(a) contacting the genomic locus with the composition of claim 1 , and
(b) determining if the expression of the genomic locus has been altered.
30 . The method of claim 29 , wherein the guide sequence directs sequence-specific binding of the CRISPR complex to the target sequence based on the presence of a CRISPR motif sequence.
31 . The method of claim 30 , wherein the CRISPR motif sequence is NAG.
32 . A method of selecting one or more prokaryotic cell(s) by introducing one or more mutations in a gene in the one or more prokaryotic cell(s), the method comprising:
(a) introducing one or more vectors into the prokaryotic cell (s), wherein the one or more vectors drive expression of one or more of: a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, a tracr sequence, and an editing template;
wherein the editing template comprises the one or more mutations that abolish CRISPR enzyme cleavage;
(b) allowing homologous recombination of the editing template with the target polynucleotide in the cell(s) to be selected;
(b) allowing a CRISPR complex to bind to a target polynucleotide to effect cleavage of the target polynucleotide within the gene, wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence within the target polynucleotide, and (2) the tracr mate sequence that is hybridized to the tracr sequence,
wherein binding of the CRISPR complex to the target polynucleotide induces cell death, thereby allowing one or more prokaryotic cell(s) in which one or more mutations have been introduced to be selected.
33 . The method of claim 32 , wherein the CRISPR enzyme is a type II CRISPR system enzyme
34 . The method of claim 33 , wherein the CRISPR enzyme is Cas9.