# US 2019/0169650 A1 · Using programmable dna binding proteins to enhance targeted genome modification > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2019/0169650 A1 - Document ID: US20190169650A1 - Status: Published application - Kind: A1 - Application number: 16/277,614 - Filed: 2019-02-15 - Published: 2019-06-06 - Earliest claimed: 2016-06-02 - Assignees: Sigma-Aldrich Co. - Applicants: Sigma-Aldrich Co. LLC - Inventors: Fuqiang Chen - Web page: https://patentgrep.com/patent/US20190169650A1 ## CPC - C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} - C12N 15/907: {in mammalian cells} - C12N 9/222: {Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes} - C12N 15/102: {Mutagenizing nucleic acids} - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract Compositions and methods for using programmable DNA binding proteins to increase the efficiency and/or specificity of targeted genome modification or to facilitate the detection of specific genomic loci in eukaryotic cells. ## Claims ### Claim 1 A composition comprising (a) a programmable DNA modification protein or nucleic acid encoding the programmable DNA modification protein, wherein the programmable DNA modification protein is a RNA-guided clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated (Cas) (CRISPR/Cas) nuclease system or the programmable DNA modification protein is a fusion protein comprising a catalytically inactive CRISPR/Cas system linked to a non-nuclease domain; and (b) at least one programmable DNA-binding protein or nucleic acid encoding the at least one programmable DNA-binding protein, wherein the at least one programmable DNA-binding protein is a CRISPR/Cas system lacking nuclease activity, wherein each CRISPR/Cas system comprises a CRISPR/Cas protein and a guide RNA. ### Claim 2 (depends on 1) The composition of claim 1 wherein the CRISPR/Cas system lacking nuclease activity is a catalytically inactive CRISPR/Cas protein or a CRISPR/Cas nickase. ### Claim 3 (depends on 1) The composition of claim 1, wherein the non-nuclease domain of the fusion protein has cytosine deaminase activity, histone acetyltransferase activity, transcriptional activation activity, or transcriptional repressor activity. ### Claim 4 (depends on 1) The composition of claim 1, wherein each CRISPR/Cas system is a type II CRISPR/Cas system or a type V CRISPR/Cas system. ### Claim 5 (depends on 1) The composition of claim 1, wherein nucleic acid encoding each CRISPR/Cas protein is mRNA or DNA. ### Claim 6 (depends on 1) The composition of claim 1, wherein nucleic acid encoding each CRISPR/Cas protein and/or encoding each guide RNA is part of a plasmid vector or a viral vector. ### Claim 7 (depends on 1) The composition of claim 1, wherein the guide RNA of each CRISPR/Cas system is at least partially chemically synthesized. ### Claim 8 (depends on 1) The composition of claim 1, wherein the guide RNA of each CRISPR/Cas system is enzymatically synthesized. ### Claim 9 (depends on 1) A kit comprising the composition of claim 1. ### Claim 10 A method for increasing targeted genome modification efficiency and/or specificity in a eukaryotic cell, the method comprising introducing into the eukaryotic cell: (a) a programmable DNA modification protein or nucleic acid encoding the programmable DNA modification protein, wherein the programmable DNA modification protein is a RNA-guided clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated (Cas) (CRISPR/Cas) nuclease system or the programmable DNA modification protein is a fusion protein comprising a catalytically inactive CRISPR/Cas system linked to a non-nuclease domain; and (b) at least one programmable DNA-binding protein or nucleic acid encoding the at least one programmable DNA-binding protein, wherein the at least one programmable DNA-binding protein is a CRISPR/Cas system lacking nuclease activity, wherein each CRISPR/Cas system comprises a CRISPR/Cas protein and a guide RNA, wherein the programmable DNA modification protein is targeted to a target chromosomal sequence and each of the at least one programmable DNA-binding protein is targeted to a site proximal to the target chromosomal sequence, and wherein binding of the at least one programmable DNA-binding protein to the site proximal to the target chromosomal sequence increases accessibility of the programmable DNA modification protein to the target chromosomal sequence, thereby increasing targeted genome modification efficiency and/or specificity. ### Claim 11 (depends on 10) The method of claim 10, wherein the site proximal to the target chromosomal sequence is located within about 250 base pairs on either side of the target chromosomal sequence. ### Claim 12 (depends on 11) The method of claim 11, wherein the wherein the site proximal to the target chromosomal sequence is located within about 100 base pairs on either side of the target chromosomal sequence. ### Claim 13 (depends on 12) The method of claim 12, wherein the wherein the site proximal to the target chromosomal sequence is located within about 75 base pairs on either side of the target chromosomal sequence. ### Claim 14 (depends on 13) The method of claim 13, wherein the wherein the site proximal to the target chromosomal sequence is located within about 50 base pairs on either side of the target chromosomal sequence. ### Claim 15 (depends on 14) The method of claim 14, wherein the wherein the site proximal to the target chromosomal sequence is located within about 25 base pairs on either side of the target chromosomal sequence. ### Claim 16 (depends on 10) The method of claim 10, wherein the CRISPR/Cas system lacking nuclease activity is a catalytically inactive CRISPR/Cas protein or a CRISPR/Cas nickase. ### Claim 17 (depends on 10) The method of claim 10, wherein the non-nuclease domain of the fusion protein has cytosine deaminase activity, histone acetyltransferase activity, transcriptional activation activity, or transcriptional repressor activity. ### Claim 18 (depends on 10) The method of claim 10, wherein the eukaryotic cell is in vitro. ### Claim 19 (depends on 10) The method of claim 10, wherein the eukaryotic cell is in vivo. ### Claim 20 (depends on 10) The method of claim 10, wherein the eukaryotic cell is a mammalian cell. ### Claim 21 (depends on 20) The method of claim 20, wherein the mammalian cell is a human cell.