Granted Patent
B2
US 12,637,691 · App. 18/513,347 · Granted May 26, 2026
RNA-guided nucleic acid modifying enzymes and methods of use thereof
Inventors:
Jennifer A. Doudna (Berkeley, CA); Jillian F. Banfield (Berkeley, CA); David Burstein (Berkeley, CA); Lucas Benjamin Harrington (Berkeley, CA); Steven C. Strutt (Berkeley, CA)
C12N15/8509C07K19/00C12N9/1007C12N9/1025C12N9/22C12N15/102C12N15/113C12N15/52C12N15/62C12N15/74C12N15/85C12N15/86C12N15/88C12N15/902C07K2319/02C07K2319/03C07K2319/06C07K2319/09C07K2319/20C12N2310/20C12N2740/13043C12N2740/16043C12N2750/14143
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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 (31)
1 . A method of modulating transcription from a target DNA, modifying a target DNA, or modifying a protein associated with a target DNA, the method comprising contacting the target DNA with:
a) a CasX fusion polypeptide comprising a CasX polypeptide fused to a heterologous polypeptide; and
b) a CasX guide RNA comprising a guide sequence that hybridizes to a target sequence of the target DNA and binds the CasX fusion polypeptide,
(i) wherein the method is for modifying the target DNA and wherein modifying the target DNA comprises cleavage of the target DNA and wherein the heterologous polypeptide is a nuclease, or
(ii) wherein the method is for modulating transcription from the target DNA,
wherein modulating transcription comprises increasing transcription and the heterologous polypeptide is a transcriptional activator, or
wherein modulating transcription comprises decreasing transcription and the heterologous polypeptide is a transcriptional repressor, or
(iii) wherein the method is for modifying histone associated with the target DNA and wherein the heterologous polypeptide is a histone methyltransferase, a histone demethylase, a histone acetylase transferase, or a histone deacetylase.
2 . The method of claim 1 , wherein the CasX guide RNA is a single guide RNA.
3 . The method of claim 1 , wherein the CasX guide RNA is a dual guide RNA.
4 . The method of claim 1 , wherein the method is for modifying the target DNA and wherein modifying the target DNA comprises cleavage of the target DNA and wherein the heterologous polypeptide is a nuclease.
5 . The method of claim 1 , wherein the target DNA is double stranded DNA, single stranded DNA, genomic DNA, or extrachromosomal DNA.
6 . The method of claim 1 , wherein said contacting takes place in vitro outside of a cell.
7 . The method of claim 1 , wherein said contacting takes place inside of a cell in culture.
8 . The method of claim 1 , wherein said contacting takes place inside of a cell in vivo.
9 . The method of claim 7 , wherein the cell is a eukaryotic cell.
10 . The method of claim 1 , wherein said contacting comprises: introducing into a cell: (a) the CasX fusion polypeptide, or a nucleic acid molecule encoding the CasX fusion polypeptide, and (b) the CasX guide RNA, or a nucleic acid molecule encoding the CasX guide RNA.
11 . The method of claim 10 , wherein said contacting further comprises: introducing a DNA donor template into the cell.
12 . The method of claim 1 , 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.
13 . The method of claim 1 , wherein the CasX polypeptide is a catalytically inactive CasX Polypeptide (dCasX).
14 . The method of claim 1 , wherein the CasX polypeptide comprises one or more mutations at a position corresponding to those selected from: D672, E769, and D935 of SEQ ID NO: 1.
15 . The method of claim 1 , wherein the CasX polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence set forth in SEQ ID NO:1.
16 . The method of claim 1 , wherein the CasX polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence set forth in SEQ ID NO:2.
17 . The method of claim 1 , wherein the CasX polypeptide comprises one or more nuclear localization signal (NLS).
18 . The method of claim 1 , wherein the method is for modulating transcription from the target DNA,
wherein modulating transcription comprises increasing transcription and the heterologous polypeptide is a transcriptional activator, or
wherein modulating transcription comprises decreasing transcription and the heterologous polypeptide is a transcriptional repressor.
19 . The method of claim 1 , wherein the method is for modifying histone associated with the target DNA and wherein the heterologous polypeptide is a histone methyltransferase, a histone demethylase, a histone acetylase transferase, or a histone deacetylase.
20 . The method of claim 4 , wherein the CasX polypeptide is a catalytically inactive CasX polypeptide (dCasX) comprising one or more mutations at a position corresponding to those selected from: D672, E769, and D935 of SEQ ID NO: 1.
21 . The method of claim 18 , wherein modulating transcription comprises increasing transcription, the heterologous polypeptide is a transcriptional activator, and the transcriptional activator is VP16, VP64, VP48, or VP160.
22 . The method of claim 18 , wherein modulating transcription comprises decreasing transcription, the heterologous polypeptide is a transcriptional repressor, and the transcriptional repressor is DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), or Krüppel associated box (KRAB).
Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Jan 31, 2024
From: DOUDNA, JENNIFER A.; BANFIELD, JILLIAN F.; BURSTEIN, DAVID; HARRINGTON, LUCAS BENJAMIN; STRUTT, STEVEN C.
To: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
Reel/Frame 066308/0708 →
Continuity (3)
Continuation
16335516
Provisional Application
62402846
· Sep 30, 2016
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