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(12) Patent Application Publication (10) Pub. No.: US 2014/0186843 A1 ZHANG Et Al US 2014O186843A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0186843 A1 ZHANG et al. (43) Pub. Date: Jul. 3, 2014 (54) METHODS, SYSTEMS, AND APPARATUS FOR (22) Filed: Dec. 12, 2013 IDENTIFYING TARGET SEQUENCES FOR CAS ENZYMES OR CRISPR-CAS SYSTEMIS Related U.S. Application Data FORTARGET SEQUENCES AND (60) Provisional application No. 61/736,527, filed on Dec. CONVEYING RESULTS THEREOF 12, 2012, provisional application No. 61/748,427, filed on Jan. 2, 2013, provisional application No. (71) Applicants: Massachusetts Institute of Technology, 61/791,409, filed on Mar. 15, 2013, provisional appli Cambridge, MA (US): THE BROAD cation No. 61/835,931, filed on Jun. 17, 2013. INSTITUTE, INC., Cambridge, MA (US) Publication Classification (72) Inventors: Feng ZHANG, Cambridge, MA (US); (51) Int. C. Naomi HABIB, Cambridge, MA (US) CI2O I/68 (2006.01) (52) U.S. C. (73) Assignees: Massachusetts Institute of Technology, CPC ...................................... CI2O I/686 (2013.01) Cambridge, MA (US): THE BROAD USPC ....................................... 435/6.12: 435/287.2 INSTITUTE, INC., Cambridge, MA (US) (57) ABSTRACT Disclosed are locational or positional methods concerning (21) Appl. No.: 14/104,900 CRISPR-Cas systems, and apparatus therefor. 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Yor fixed length before the selected CRISPR motif which is to occur in the sequence before the selected motif Scan input sequence to identify all target sites which include the Selected motifs and defined fragments S106 Filter identified target sites based on number of times each appears in target genome or target DNA library Select unique target site S1 10 Patent Application Publication Jul. 3, 2014 Sheet 47 of 50 US 2014/O186843 A1 Patent Application Publication Jul. 3, 2014 Sheet 48 of 50 US 2014/O186843 A1 Define thermodynamic model having a set of weights linking effective free energy of hybridization Z to local free energies G -Define a training Yset - of guide-RNA target-DNA sequence pairs Input known values of G for each guide-RNA? target-DNA sequence pair in the training set S2O4. v Calculate value of Z for each guide-RNA/target DNA sequence pair in the training set S2O6 w (Optional) Normalize calculated values of Z S208 ------------------------------------------------------------------------------------------------------------------------------------------------------------------- (Optional) Fitter out values of Z having less than a minimum sequencing depth S210 Determine the position-weights using a machine learning algorithm Output weights (including determining of G-values for the testing set of guide-RNA target-DNA sequence-pairs and using these as well as the position-weights to output new values of Z for the testing-set) S214 FG. 33B Patent Application Publication Jul. 3, 2014 Sheet 49 of 50 US 2014/O186843 A1 input genome or DNA sequence S300 Locate a mismatch between a target sequence of the input sequence and the guide RNA of a particular CaS enzyme ----------------------------------------------------------------------------------------------Determine average cutting frequency at the particular position for the mismatch Determine average cutting frequency for the particular mismatch type of the mismatch S306 Create a product by multiplying together the determined average Cutting frequencies S308 O - is there s s - another mismatch?) S.
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