# US 11,111,506 B2 · Compositions and methods of engineered CRISPR-Cas9 systems using split-nexus Cas9-associated polynucleotides > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,111,506 B2 - Document ID: US11111506B2 - Status: Granted - Kind: B2 - Application number: 15/965,921 - Filed: 2018-04-28 - Published: 2018-08-30 - Granted: 2021-09-07 - Earliest claimed: 2015-08-07 - Assignees: Caribou Biosciences, Inc. - Applicants: Caribou Biosciences, Inc. - Inventors: Paul Daniel Donohoue; Andrew Paul May - Web page: https://patentgrep.com/patent/US11111506B2 ## CPC - C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} - C12N 15/87: Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation - C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein} - C12N 9/96: Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates - C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids} - C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} - C12N 2310/10: Type of nucleic acid - C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR] - C12N 2310/3513: Protein; Peptide - C12N 2310/531: Stem-loop; Hairpin - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract The present specification discloses engineered Type II CRISPR-Cas9 systems comprising split-nexus Cas9-associated polynucleotides (sn-casPNs), including systems comprising three split-nexus Cas9-associated polynucleotides (sn1-casPN/sn2-casPN/sn3-casPN) and systems comprising two split-nexus Cas9-associated polynucleotides (sn1-casPN/sn2-casPN). Together with a Cas9 protein, the sn-casPNs facilitate site-specific modifications, including cleavage and mutagenesis, of a target polynucleotide in vitro and in vivo. Furthermore, the engineered Type II CRISPR-Cas9 systems comprising sn-casPNs are useful in methods of regulating expression of a target nucleic acid. Methods are described herein for the creation of a variety of engineered Type II CRISPR-Cas9 systems comprising two or more sn-casPNs. Polynucleotide sequences, expression cassettes, vectors, compositions, and kits for carrying out a variety of methods are also described. Furthermore, the present specification provides genetically modified cells, compositions of modified cells, transgenic organisms, pharmaceutical compositions, as well as a variety of compositions and methods involving the engineered Type II CRISPR-Cas9 systems. ## Claims ### Claim 1 One or more expression cassettes comprising: one or more regulatory sequences operably linked to one or more polynucleotides encoding, a first Type II CRISPR-Cas9-associated split-nexus polynucleotide having a 5′ end and a 3′ end (sn1-casPN) comprising, in the 5′ to 3′ direction, a first stem element nucleotide sequence I and a nexus stem element nucleotide sequence I, a second Type II CRISPR-Cas9-associated split-nexus polynucleotide having a 5′ end and a 3′ end (sn2-casPN) comprising, in the 5′ to 3′ direction, a nexus stem element nucleotide sequence II and a second stem element nucleotide sequence I, wherein the nexus stem element nucleotide sequence I of the sn1-casPN and the nexus stem element nucleotide sequence II of the sn2-casPN are capable of forming a nexus stem element by base-pair hydrogen bonding between the nexus stem element nucleotide sequence I and the nexus stem element nucleotide sequence II, a third Type II CRISPR-Cas9-associated polynucleotide having a 5′ end and a 3′ end (sn3-casPN) comprising, in the 5′ to 3′ direction, a DNA target binding sequence and a first stem element nucleotide sequence II, wherein the first stem element nucleotide sequence I of the sn1-casPN and the first stem element nucleotide sequence II of the sn3-casPN are capable of forming a first stem element by base-pair hydrogen bonding between the first stem element nucleotide sequence I and the first stem element nucleotide sequence II, a first adjunct polynucleotide having a 5′ end and a 3′ end comprising a second stem element nucleotide sequence II, wherein the second stem element nucleotide sequence I of the sn2-casPN and the second stem element nucleotide sequence II of the first adjunct polynucleotide are capable of forming a second stem element by base-pair hydrogen bonding between the second stem element nucleotide sequence I and the second stem element nucleotide sequence II, wherein the one or more regulatory elements are capable of facilitating expression of a Type II CRISPR-Cas9-associated polynucleotide composition comprising the sn1-casPN, the sn2-casPN, the sn3-casPN, and the first adjunct polynucleotide. ### Claim 2 (depends on 1) The one or more expression cassettes of claim 1, wherein the nexus stem element nucleotide sequence I comprises a first auxiliary polynucleotide 3′ of the nexus stem element nucleotide sequence I. ### Claim 3 (depends on 1) The one or more expression cassettes of claim 1, wherein the nexus stem element nucleotide sequence II comprises a second auxiliary polynucleotide 5′ of the nexus stem element nucleotide sequence II. ### Claim 4 (depends on 1) The one or more expression cassettes of claim 1, wherein the nexus stem element nucleotide sequence I comprises a first auxiliary polynucleotide 3′ of the nexus stem element nucleotide sequence I and the nexus stem element nucleotide sequence II comprises a second auxiliary polynucleotide 5′ of the nexus stem element nucleotide sequence II. ### Claim 5 (depends on 4) The one or more expression cassettes of claim 4, wherein the first auxiliary polynucleotide further comprises an effector binding element nucleotide sequence I; the second auxiliary polynucleotide further comprises an effector binding element nucleotide sequence II; and the effector binding element nucleotide sequence I of the first auxiliary polynucleotide and the effector binding element nucleotide sequence II of the second auxiliary polynucleotide are capable of forming an effector binding element by base-pair hydrogen bonding between the effector binding element nucleotide sequence I and the effector binding element nucleotide sequence II. ### Claim 6 (depends on 4) The one or more expression cassettes of claim 4, wherein the first auxiliary polynucleotide further comprises, in the 5′ to 3′ direction, a linker element nucleotide sequence I and the effector binding element nucleotide sequence I; the second auxiliary polynucleotide further comprises, in the 5′ to 3′ direction, the effector binding element nucleotide sequence II and a linker element nucleotide sequence II; and the linker element nucleotide sequence I of the first auxiliary polynucleotide and the linker element nucleotide sequence II of the second auxiliary polynucleotide are capable of forming a linker element by base-pair hydrogen bonding between the linker element nucleotide sequence I and the linker element nucleotide sequence II. ### Claim 7 (depends on 4) The one or more expression cassettes of claim 4, wherein the first auxiliary polynucleotide further comprises a hairpin and the second auxiliary polynucleotide further comprises a hairpin. ### Claim 8 (depends on 1) The one or more expression cassettes of claim 1, wherein the first adjunct polynucleotide further comprises, in the 5′ to 3′ direction, a loop element nucleotide sequence and the second stem element nucleotide sequence II; and the 5′ end of the first adjunct polynucleotide is covalently bonded to the 3′ end of the sn2-casPN. ### Claim 9 (depends on 8) The one or more expression cassettes of claim 8, wherein the first stem element nucleotide sequence I further comprises, in the 5′ to 3′ direction, a loop element nucleotide sequence and the first stem element nucleotide sequence I; and the 5′ end of the first stem element nucleotide sequence I is covalently bonded to the 3′ end of the first stem element nucleotide sequence II. ### Claim 10 (depends on 9) The one or more expression cassettes of claim 9, wherein the one or more polynucleotides encoding further comprise, a second adjunct polynucleotide having a 5′ end and a 3′ end comprising third stem element nucleotide sequence II; wherein the first adjunct polynucleotide comprises, in the 5′ to 3′ direction, the loop element nucleotide sequence, the second stem element nucleotide sequence II, and a third stem element nucleotide sequence I; and wherein the third stem element nucleotide sequence I of the first adjunct polynucleotide and the third stem element nucleotide sequence II of the second adjunct polynucleotide are capable of forming a third stem element by base-pair hydrogen bonding between the third stem element nucleotide sequence I and third stem element nucleotide sequence II. ### Claim 11 (depends on 10) The one or more expression cassettes of claim 10, wherein the second adjunct polynucleotide further comprises, in the 5′ to 3′ direction, a loop element nucleotide sequence and the third stem element nucleotide sequence II; and 5′ end of the second adjunct polynucleotide is covalently bonded to the 3′ end of the first adjunct polynucleotide. ### Claim 12 (depends on 1) The one or more expression cassettes of claim 1, wherein the first stem element nucleotide sequence I of the sn1-casPN further comprises, in the 5′ to 3′ direction, an upper stem element nucleotide sequence I, a bulge element nucleotide sequence I, and a lower stem element nucleotide sequence I; the first stem element nucleotide sequence II of the sn3-casPN further comprises, in the 5′ to 3′ direction, a lower stem element nucleotide sequence II, a bulge element nucleotide sequence II, and an upper stem element nucleotide sequence II; and the upper stem element nucleotide sequence I of the sn1-casPN and the upper stem element nucleotide sequence II of the sn3-casPN are capable of forming an upper stem element by base-pair hydrogen bonding between the upper stem element nucleotide sequence I and the upper stem element nucleotide sequence II and the lower stem element nucleotide sequence I of the sn1-casPN and the lower stem element nucleotide sequence II of the sn3-casPN are capable of forming a lower stem element by base-pair hydrogen bonding between the lower stem element nucleotide sequence I and the lower stem element nucleotide sequence II. ### Claim 13 (depends on 1) The one or more expression cassettes of claim 1, further comprising an expression cassette comprising one or more regulatory sequences operably linked to a polynucleotide encoding a Cas9 protein. ### Claim 14 (depends on 13) The one or more expression cassettes of claim 13, wherein the Cas9 protein comprises an inactive RuvC domain and an inactive HNH domain. ### Claim 15 (depends on 13) The one or more expression cassettes of claim 13, wherein the Cas9 protein comprises an active RuvC domain, an active HNH domain, or an active RuvC domain and an active HNH domain. ### Claim 16 The one or more expression cassettes of 13, wherein the one or more regulatory sequences facilitate expression of the Type II CRISPR-Cas9-associated polynucleotide composition and the Cas9 protein in a eukaryotic cell. ### Claim 17 The one or more expression cassettes of 1, wherein the one or more regulatory sequences facilitate expression of the Type II CRISPR-Cas9-associated polynucleotide composition in a eukaryotic cell. ### Claim 18 (depends on 1) One or more vectors comprising: the one or more expression cassettes of claim 1. ### Claim 19 (depends on 18) The one or more vectors of claim 18, wherein the one or more vectors are derived from a mammalian virus. ### Claim 20 (depends on 19) The one or more vectors of claim 19, wherein the mammalian virus is at least one mammalian virus selected from the group consisting of an adenovirus vector, an adeno-associated virus vector, a vaccinia virus vector, a retrovirus vector, and a lentivirus vector.