CRISPR-Cas systems, crystal structure and uses thereof
The invention provides for systems, methods, and compositions for altering expression of target gene sequences and related gene products. Provided are structural information on the Cas protein of the CRISPR-Cas system, use of this information in generating modified components of the CRISPR complex, vectors and vector systems which encode one or more components or modified components of a CRISPR complex, as well as methods for the design and use of such vectors and components. Also provided are methods of directing CRISPR complex formation in eukaryotic cells and methods for utilizing the CRISPR-Cas system.
1. A method for synthesizing compounds to fit within or bind to a CRISPR-cas9 system or a functional portion thereof comprising:
(a) inputting into a programmed computer comprising a processor through an input device, data comprising a three-dimensional (3D) co-ordinates of a subset of amino acid residues from a CRISPR-cas9 crystal structure according to Table A, wherein the amino acid residues are located in a Cas9 domain of the CRISPR-cas9 system, thereby generating a 3D structure data set of the Cas9;
(b) comparing, using said processor, said data set to a computer database of structures of compounds that bind or putatively bind or that are desired to bind to one or more residues within the 3D structure of the Cas9 domain;
(c) selecting from said database, using computer methods, compounds with desired structures that bind to one or more residues within the Cas9 domain, based on the CRISPR-cas9 crystal structure according to Table A;
(d) constructing, using computer methods, a model of the selected compounds;
(e) outputting to an output device the selected compounds;
(f) synthesizing one or more of the selected compounds that bind to one or more residues within the Cas9 domain; and
(g) testing binding of said synthesized selected compounds to the CRISPR-cas9 system.
2. A method for synthesizing compounds to fit within or bind to a CRISPR-cas9 system or a Cas9 ortholog comprising:
providing co-ordinates of amino acid residues from a Cas9 domain of the CRISPR-cas9 crystal structure according to Table A on a computer, providing the structure of a candidate compound, fitting the structure of the candidate compound to the selected co-ordinates, to thereby obtain product data comprising desired compounds that bind to one or more residues within the Cas9 domain, with output thereof; synthesizing compound(s) from said product data that bind to one or more residues within the Cas9 domain; and testing binding of said synthesized compound(s) to a CRISPR-cas9 system.
3. The method of claim 1 or claim 2 , further comprising analyzing the binding or a desired function of a CRISPR-cas9 system resulting from said synthesized selected compound(s) or structure(s).
4. The method of claim 1 or claim 2 , wherein the Cas9 domain is a REC1 domain, a REC2 domain, a RuvC domain, a HNH domain, or a PI domain.
5. The method of claim 1 or claim 2 , wherein the amino acid residues located in the Cas9 domain are selected from R63, R65, R66, R69, R70, R74, R78, S104, F105, R115, H116, I135, H160, K163, R165, Y325, H328, R340, F351, D364, Q402, R403, T404, N407, L455, S460, T472, R487, N497, K510, W659, R661, Q695, H721, K742, R895, Q926, V1009, W1010, Y1013, V1100, E1108, I1110, R1122, Y1131, H1349, or a combination thereof.
6. The method of claim 1 or claim 2 , wherein the amino acid residues located in the Cas9 domain are selected from R63, R65, R66, R69, R70, R74, R78, K163, R165, N407, N497, K510, W659, R661, Q695, R895, Q926, V1009, W1010, Y1013, E1108, or a combination thereof.