US 2025/0154513 A1 · Applications of Streptococcus-derived Cas9 nucleases on minimal Adenine-rich PAM targets

Abstract

Applications of a Streptococcus Cas9 ortholog from Streptococcus macacae (Smac Cas9), possessing minimal adenine-rich PAM specificity, include an isolated Streptococcus macacae Cas9 protein or transgene expression thereof, a CRISPR-associated DNA endonuclease with PAM interacting domain amino acid sequences that are at least 80% identical to that of the isolated Streptococcus macacae Cas9 protein, and an isolated, engineered Streptococcus pyogenes Cas9 (Spy Cas9) protein with a PID as either the PID amino acid composition of the isolated Streptococcus macacae Cas9 (Smac Cas9) protein or of a CRISPR-associated DNA endonuclease with PID amino acid sequences that are at least 80% identical to that of the isolated Streptococcus macacae Cas9 protein. A method for altering expression of at least one gene product employs Streptococcus macacae Cas9 endonucleases in complex with guide RNA, for specific recognition and activity on a DNA target immediately upstream of either an “NAA” or “NA” or “NAAN” PAM sequence.

Claims20

Dependent claims

An isolated, engineered Streptococcus pyogenes Cas9 (SpyCas9) protein comprising SEQ ID No:

31 or SEQ ID No. 32, wherein said SpyCas9 is modified to have a Protospacer Adjacent Motif (PAM) Interacting Domain (PID) amino acid sequence with at least 80% identity to the amino acid composition of the PID of an isolated wild-type Streptococcus macacae Cas9 (Smac Cas9) protein comprising SEQ ID No:
33, which replaces the SpyCas9 PID.

A method of altering expression of at least one gene product comprising:

introducing, into a eukaryotic cell containing and expressing a DNA molecule having a target sequence and encoding the gene product, an engineered, non-naturally occurring CRISPR-Cas system comprising one or more vectors comprising:
(a) a regulatory element operable in a eukaryotic cell operably linked to at least one nucleotide sequence encoding a CRISPR system guide RNA that hybridizes with the target sequence;
and
(b) a second regulatory element operable in a eukaryotic cell operably linked to a nucleotide sequence encoding one or more proteins selected from the group consisting of:
(i) an isolated, engineered Streptococcus pyogenes Cas9 (SpyCas9) protein comprising SEQ ID No:
31 or SEQ ID No. 32, wherein said SpyCas9 is modified to have a Protospacer Adjacent Motif (PAM) Interacting Domain (PID) amino acid sequence that is identical to the amino acid composition of the PID of an isolated wild-type Streptococcus macacae Cas9 (Smac Cas9) protein comprising SEQ ID No:
33, which replaces the SpyCas9 PID, or a transgene expression thereof;
and
(ii) an isolated, engineered Streptococcus pyogenes Cas9 (SpyCas9) protein comprising SEQ ID No:
31 or SEQ ID No. 32, wherein said SpyCas9 is modified to have a Protospacer Adjacent Motif (PAM) Interacting Domain (PID) amino acid sequence with at least 80% identity to the amino acid composition of the PID of an isolated wild-type Streptococcus macacae Cas9 (Smac Cas9) protein comprising SEQ ID No:
33, which replaces the SpyCas9 PID, or a transgene expression thereof;
wherein components (a) and (b) are located on same or different vectors of the system, whereby the guide RNA targets the target sequence and one or more of the proteins cleave the DNA molecule, whereby expression of the at least one gene product is altered;
and,
wherein the proteins and the guide RNA do not naturally occur together.

11 more elements

Dependencies are inferred from the claim text.

Facts

Application

18/927,790Filed 25 Oct 2024

Priority

6 May 2018

Published

15 May 2025

Term ends

Not estimatedThe source records don’t support an estimate.

Classification7 codes

C12N 15/102
Processes for the isolation, preparation or purification of DNA or RNA: mutagenizing nucleic acids
C12N 15/74
Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression: vectors or expression systems…
C12N 9/22
Acting on ester bonds: ribonucleases [RNase]; Deoxyribonucleases [DNase]
C12N 9/226
Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes: class 2 CAS enzyme complex, e.g. single CAS protein

Applications of Streptococcus-derived Cas9 nucleases on minimal Adenine-rich PAM targets

Life of this patent

Applications of Streptococcus-derived Cas9 nucleases on minimal Adenine-rich PAM targets

Life of this patent

Abstract

Claims20