RNA-GUIDED NUCLEIC ACID MODIFYING ENZYMES AND METHODS OF USE THEREOF

• US 20180346927A1
• Filed: 08/03/2018
• Published: 12/06/2018
• Est. Priority Date: 09/30/2016
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Doudna, Jennifer A., Banfield, Jillian, Burstein, David, Harrington, Lucas Benjamin, Strutt, Steven C.
• Granted Patent: US 10,570,415 B2
• Time in Patent Office: Days
• CPC Class Codes: C07K 19/00 Hybrid peptides , i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/03 containing a transmembrane segment
• CPC Class Codes: C07K 2319/06 containing a lysosomal/endosomal localisation signal
• CPC Class Codes: C07K 2319/09 containing a nuclear localisation signal
• CPC Class Codes: C07K 2319/20 containing a tag with affinity for a non-protein ligand
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: 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
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 15/74 Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 15/88 using microencapsulation, e.g. using amphiphile liposome vesicle
• CPC Class Codes: C12N 15/902 using homologous recombination
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2740/13043 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2740/16043 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2750/14143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 9/1007 : Methyltransferases (general) (2.1.1.)
• CPC Class Codes: C12N 9/1025 : Acyltransferases (2.3)
• CPC Class Codes: C12N 9/22 : Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

The present disclosure provides CasX proteins, nucleic acids encoding the CasX proteins, and modified host cells comprising the CasX proteins and/or nucleic acids encoding same. CasX proteins are useful in a variety of applications, which are provided. The present disclosure provides CasX guide RNAs that bind to and provide sequence specificity to the CasX proteins, nucleic acids encoding the CasX guide RNAs, and modified host cells comprising the CasX guide RNAs and/or nucleic acids encoding same. CasX guide RNAs are useful in a variety of applications, which are provided. The present disclosure provides archaeal Cas9 polypeptides and nucleic acids encoding same, as well as their associated archaeal Cas9 guide RNAs and nucleic acids encoding same.

Claims

1-131. -131. (canceled)

132. A method of modifying a target nucleic acid, the method comprising contacting the target nucleic acid with:

a) a CasX polypeptide; and

b) a CasX guide RNA comprising a guide sequence that hybridizes to a target sequence of the target nucleic acid,

wherein said contacting results in modification of the target nucleic acid by the CasX polypeptide.

133. The method of claim 132, wherein said modification is cleavage of the target nucleic acid.

134. The method of claim 132, wherein the target nucleic acid is selected from:

double stranded DNA, single stranded DNA, RNA, genomic DNA, and extrachromosomal DNA.

135. The method of claim 132, wherein said contacting takes place in vitro outside of a cell.

136. The method of claim 132, wherein said contacting takes place inside of a cell in culture in vitro.

137. The method of claim 132, wherein said contacting takes place inside of a cell in vivo.

138. The method of claim 137, wherein the cell is a eukaryotic cell.

139. The method of claim 138, wherein the cell is selected from:

a plant cell, a fungal cell, a mammalian cell, a reptile cell, an insect cell, an avian cell, a fish cell, a parasite cell, an arthropod cell, a cell of an invertebrate, a cell of a vertebrate, a rodent cell, a mouse cell, a rat cell, a primate cell, a non-human primate cell, and a human cell.

140. The method of claim 136, wherein the cell is a prokaryotic cell.

141. The method of claim 132, wherein said contacting results in genome editing.

142. The method of claim 132, wherein said contacting comprises:

introducing into a cell;

(a) the CasX polypeptide, or a nucleic acid comprising a nucleotide sequence encoding the CasX polypeptide, and (b) the CasX guide RNA, or a nucleic acid comprising a nucleotide sequence encoding the CasX guide RNA.

143. The method of claim 142, wherein said contacting further comprises:

introducing a DNA donor template into the cell.

144. The method of claim 132, wherein the CasX guide RNA is a single guide RNA.

145. The method of claim 132, wherein the CasX guide RNA is a dual guide RNA.

146. The method of claim 132, wherein the CasX guide RNA comprises one or more of:

a non-natural internucleoside linkage, a nucleic acid mimetic, a modified sugar moiety, and a modified nucleobase.

147. The method of claim 132, wherein the CasX guide RNA comprises one or more of:

(i) a non-natural internucleoside linkage selected from a phosphorothioate, an inverted polarity linkage, and an abasic nucleoside linkage;

(ii) a locked nucleic acid (LNA); and

(iii) a modified sugar moiety selected from 2′

-O-methoxyethyl, 2′

-O-methyl, and 2′

-fluoro.

148. The method of claim 132, wherein the CasX guide RNA comprises one or more of:

a peptide nucleic acid (PNA), a morpholino nucleic acid, a cyclohexenyl nucleic acid (CeNA), and/or a locked nucleic acid (LNA).

149. The method of claim 132, wherein the CasX guide RNA comprises one or more modified sugar moieties selected from:

2′

-O-(2-methoxyethyl), 2′

-dimethylaminooxyethoxy, 2′

-dimethylaminoethoxyethoxy, 2′

-O-methyl, and 2′

-fluoro.

150. The method of claim 132, wherein the CasX polypeptide compromises an amino acid sequence having at least 95% identity to the amino acid sequence set forth in SEQ ID NO:

1.

151. The method of claim 132, wherein the CasX polypeptide compromises an amino acid sequence having at least 95% identity to the amino acid sequence set forth in SEQ ID NO:

2.

152. The method of claim 132, wherein the CasX polypeptide compromises an amino acid sequence having at least 95% identity to the amino acid sequence set forth in SEQ ID NO:

3.

153. The method of claim 132, wherein CasX polypeptide comprises one or more nuclear localization signal (NLS).

154. The method of claim 153, wherein the one or more NLS is at the N-terminus of the CasX polypeptide or wherein the one or more NLS is at the C-terminus of the CasX polypeptide.

155. The method of claim 153, wherein CasX polypeptide comprises an NLS at the N-terminus and an NLS at the C-terminus of the CasX polypeptide.

156. The method of claim 132, wherein said contacting comprises:

introducing into a cell;

(a) a nucleic acid comprising a nucleotide sequence encoding the CasX polypeptide, and (b) a nucleic acid comprising a nucleotide sequence encoding the CasX guide RNA.

157. The method of claim 156, wherein the nucleotide sequence encoding the CasX polypeptide is operably linked to a first promoter, and wherein the nucleotide sequence encoding the CasX guide RNA is operably linked to a second promoter.

158. The method of claim 157, wherein the first promoter is a CMV promoter, and wherein the second promoter is a U6 promoter.

159. A composition comprising:

a) a CasX polypeptide, or a nucleic acid molecule encoding the CasX polypeptide; and

b) a CasX guide RNA, or one or more DNA molecules encoding the CasX guide RNA, wherein the CasX guide RNA is a single guide RNA.

160. A CasX fusion polypeptide comprising:

a) a CasX polypeptide; and

b) a heterologous polypeptide fused to the CasX polypeptide.

161. A variant CasX polypeptide, where the variant CasX polypeptide is catalytically inactive.