# US 11,078,498 B2 · CRISPR enabled multiplexed genome engineering > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,078,498 B2 - Document ID: US11078498B2 - Status: Granted - Kind: B2 - Application number: 16/938,739 - Filed: 2020-07-24 - Published: 2020-12-17 - Granted: 2021-08-03 - Earliest claimed: 2014-02-11 - Assignees: The Regents of the University of Colorado, a body corporate - Applicants: The Regents of the University of Colorado, a Body Corporate - Inventors: Andrew Garst; Ryan T Gill - Web page: https://patentgrep.com/patent/US11078498B2 ## CPC - C12N 15/902: {using homologous recombination} - C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} - C12N 9/222: {Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes} - C12N 15/102: {Mutagenizing nucleic acids} - C12N 15/1024: {In vivo mutagenesis using high mutation rate "mutator" host strains by inserting genetic material, e.g. encoding an error prone polymerase, disrupting a gene for mismatch repair} - C12N 15/1082: {Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors} - C12N 15/1093: {General methods of preparing gene libraries, not provided for in other subgroups} - C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} - C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids} - C40B 40/08: Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries - C40B 50/06: Biochemical methods, e.g. using enzymes or whole viable microorganisms - C12N 2310/10: Type of nucleic acid - C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR] - C12N 2320/11: for the determination of target sites, i.e. of active nucleic acids - C12N 2320/12: in functional genomics, i.e. for the determination of gene function - C12N 2330/31: Libraries, arrays - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60) ## Abstract Described herein are method for generating a vector for editing a cell. The method comprises ligating into a vector that encodes a portion of a gRNA a cassette comprising at least one editing cassette, a promoter, and a gene encoding another portion of the gRNA. Upon ligation, the portion of the gRNA from the editing cassette and the other portion of the gRNA are ligated and form a functional gRNA. ## Claims ### Claim 1 A method for generating a vector for editing a cell comprising: obtaining a vector that comprises a nucleotide sequence encoding a first region of a gRNA; and ligating into the vector that encodes the first region of the gRNA a second nucleotide sequence encoding: a region homologous to a target region in a cell comprising a change in sequence relative to the target region; a site conferring immunity to nuclease-mediated editing; a promoter; and a gene encoding a second region of the gRNA, whereupon the ligation forms a functional gRNA; thus generating the vector for editing the cell. ### Claim 2 (depends on 1) The method of claim 1, wherein the first region of the gRNA comprises a nucleotide sequence encoding a tracrRNA. ### Claim 3 (depends on 2) The method of claim 2, wherein the second region of the gRNA comprises a nucleotide sequence encoding a crRNA. ### Claim 4 (depends on 1) The method of claim 1, wherein the first region is at least 15-30 nucleotides in length. ### Claim 5 (depends on 1) The method of claim 1, wherein the first region is at least 20 nucleotides in length. ### Claim 6 (depends on 1) The method of claim 1, wherein said functional gRNA is a chimeric guide RNA. ### Claim 7 (depends on 1) The method of claim 1, wherein the promoter and the nucleotide sequence encoding the functional gRNA are operably linked. ### Claim 8 (depends on 1) The method of claim 1, wherein the vector further comprises a nucleotide sequence encoding an endonuclease. ### Claim 9 (depends on 1) The method of claim 1, wherein said first region of the gRNA comprises a nucleotide sequence that interacts with, or recruits, an endonuclease. ### Claim 10 (depends on 1) The method of claim 1, wherein the site conferring immunity to nuclease-mediated editing comprises a protospacer adjacent motif (PAM) mutation. ### Claim 11 (depends on 1) The method of claim 1, wherein the cell is a bacterial cell. ### Claim 12 (depends on 1) The method of claim 1, wherein the cell is a yeast cell. ### Claim 13 (depends on 1) The method of claim 1, wherein the cell is a mammalian cell. ### Claim 14 (depends on 1) The method of claim 1, wherein the target region is within a non-coding region. ### Claim 15 (depends on 1) The method of claim 1, wherein the target region is within a coding region. ### Claim 16 (depends on 1) The method of claim 1, wherein the change in sequence relative to the target region comprises one or more substitutions relative to the target sequence. ### Claim 17 (depends on 1) The method of claim 1, wherein the change in sequence relative to the target region comprises one or more insertions relative to the target sequence. ### Claim 18 (depends on 1) The method of claim 1, wherein the change in sequence relative to the target region comprises one or more deletions relative to the target sequence. ### Claim 19 (depends on 1) A vector library comprising a plurality of vectors created with the method of claim 1. ### Claim 20 (depends on 19) The vector library of claim 19, wherein the plurality ofvectors encode a plurality of functional gRNAs that target at least two different target regions.