Novel crispr enzymes, methods, systems and uses thereof

Patent metadata

Field	Value
Publication number	US-2023279373-A1
Application number	US-202017641356-A
Country	US
Kind code	A1
Filing date	Sep 9, 2020
Priority date	Sep 9, 2019
Publication date	Sep 7, 2023
Grant date	—

Abstract

The present invention provides novel systems, methods and compositions for making and using a recombinantly engineered novel Cas9 optimized for human cells, for nucleic acid targeting and manipulation. The present invention is based on the discovery of a novel Cas9 species from Lachnospira bacterium that was codon-optimized and recombinantly produced for use in human ceils. In some embodiments, the novel Cas9 can be used in a base editor. In some embodiments, the novel engineered Cas9 is used to treat human diseases.

First claim

1 . An engineered, non-naturally occurring Cas9 protein modified from Lachnospira Cas9, having at least 80% identity to

(SEQ ID NO: 1)

MSVNVGLDIGIASVGVAVVDSESGEILEAVSDLFESAEANQNVDRRGFRQSRRLKRRQY

NRIHDFMKLWEEFGFVKPENINLNTVGLRVKSLTEQVTLDELYVILLSELKHRGISYLED

SEEVDGGSEYKEGLRINQRELQSKYPCEIQLERLKIYGRYRGNFTVEIDGEKVGLSNVFT

TGAYRKEIQQLLSIQKTYQSKLTDDFINKYLEIFDRKRQYYVGPGNEKSRTDYGRYTTK

KDAEGNYITDENIFEKLIGKCSIYPEEMRAAGASYTAQEFNLLNDLNNLTIGGRKIEEEEK

RAIIETIKSSKVVNVEKIICKVTGEDAETITGARIDKDDKRIYHSFECYRKLKKALETIEVK

IEEYSREELDELARILTLNTEREGILGELEKSFLDLGEEVIDCVIDFRRKNGPLFSKWQSFS

LRLMNDIIPDMYEQPKEQMTLLTEMGLMKSKKEIFKGMKYIPENVMRDDIYNPVVVRS

VRIAVRALNAVIKKYGEIDKVVIEMPRDRNTEEQKKRIDAENKRNREELPGIEKRILEEY

GIKITSAHYRNHKQLGLKLKLWNEQGGICPYSGKTIDLERLLQNAGDYEVDHIIPLSISLD

DSRNNKVLVYASENQKKGNQTPYAYLSSVQREWGWEQYRHYVLSDLKKKKISSKKIE

NYLFMKDISKIDVVKGFIQRNLNDTRYASKVVLNTLESFFKANEKETKVSVIRGSFTSLM

RKNLKLDKSREESYAHHAVDALLIAYSKMGYDSYHKLQGEFIDFETGEILDSRMWETNL

EPDILKGYLYGRKWSEIRENIKIAESRVKYWHMTNKKCNRSLCNQTLYGTRTYDGKIYQ

IKKIKDIRTPEGLKTFKDLVDKNKGDHLLMARNDPKTYEQILQIYRDYSDAKNPFLQYE

METGDCIRKYSKKHNGSRIVSLKYHDGEVNSCIDVSHKYGFEKGSQKVVLMSLNPYRM

DVYKNCNDGKYYLIGLKQSDIKCEGRHYVIDEEKYAKVLVNEKMIQPGQSRKDLPDLG

YEFVMSFYKNEIIQYEKDGKFYKERFLSRTKPASRNYIETKPVDKPNFEKRHQIGLAKTT

FIRKIRTDILGNEYNCDREKFSSIC.

2 .- 5 . (canceled)

6 . The Cas9 protein of claim 1 , wherein (i) the Cas9 protein has nickase activity, (ii) the Cas9 protein comprises at least one amino acid mutation in PAM Interacting, HNH and/or RuvC domain, (iii) the Cas9 protein further comprises a nuclear localization sequence (NLS) and/or a FLAG, HIS or HA tag, and/or (iv) the Cas9 protein recognizes a PAM sequence comprising 5′-NNGNG-3′.

7 . (canceled)

8 . The Cas9 protein of claim 1 , wherein the amino acid sequence comprises at least one mutation in an amino acid residue, wherein (i) the mutation is D8A H593A, and/or N616A, and/or (ii) the mutation results in an inactive Cas9.

9 .- 11 . (canceled)

12 . An engineered, non-naturally occurring Cas9 fusion protein comprising a Cas9 protein having at least 80% identity to SEQ ID NO: 1, and wherein the Cas9 protein is fused to a histone demethylase, a transcriptional activator, or to a deaminase, wherein the deaminase is a cytosine deaminase or an adenosine deaminase.

13 .- 14 . (canceled)

15 . A nucleic acid encoding the Cas9 protein of claim 1 .

16 .- 19 . (canceled)

20 . A method of cleaving a target nucleic acid in a eukaryotic cell comprising:
contacting the cell with a Cas9 of claim 1 , and an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide comprises a direct repeat sequence and a spacer sequence capable of hybridizing to the target nucleic acid, and wherein the Cas9 protein is capable of binding to the RNA guide and of causing a break in the target nucleic acid sequence complementary to the RNA guide, wherein the break is a single-stranded break or a double-stranded break.

21 .- 22 . (canceled)

23 . A method of modifying a target nucleic acid in a eukaryotic cell comprising:
contacting the cell with a Cas9 of claim 1 , and an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide comprises a direct repeat sequence and a spacer sequence capable of hybridizing to the target nucleic acid, and wherein the Cas9 protein is capable of binding to the RNA guide and editing the target nucleic acid sequence complementary to the RNA guide.

24 . The method of claim 23 , wherein the Cas9 protein is an inactive Cas9 (dCas9).

25 . The method of claim 24 , wherein the dCas9 is fused to a deaminase.

26 . The method of claim 20 or 23 , wherein the RNA guide comprises a crRNA and a tracrRNA.

27 . The method of claim 26 , wherein the crRNA comprises a guide sequence and a direct repeat (DR) sequence of between about 16 and 26 nucleotides long.

28 .- 30 . (canceled)

31 . The method of claim 26 , wherein the crRNA comprises a DR sequence comprising a sequence having at least about 80% identity to AUUUUAGUUCCUGGAUAAUUCAAGUUAGUGUAAAAC (SEQ ID NO: 3), or at least about 80% identity to AUUUUAGUUCCUGGAUAAUUCA (SEQ ID NO: 4); or comprises a sequence of

(SEQ ID NO: 5)

NNNNNNNNNNNNNNNNNNNNAUUUUAGUUCCUGGAUAAUUCA.

32 .- 36 . (canceled)

37 . The method of claim 26 , wherein the tracrRNA comprises a sequence having at least about 80% identity to

(SEQ ID NO: 6)

UGAAUUAUUCAGACCAACUAAAACAAGGCUUUAUGCCGAAAUCAAGGACAC

CUUCGGGUGUCCUUUU1:JU.

38 .- 39 . (canceled)

40 . The method of claim 20 or 23 , wherein the RNA guide comprises a sgRNA, wherein the sgRNA comprises a sca

Assignees

• Beam Therapeutics Inc

Inventors

• Zetsche Bernd
• Barrera Luis

Classifications

• C12N9/22 Primary

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N9/78

  acting on carbon to nitrogen bonds other than peptide bonds (3.5) · CPC title

• C12N15/86

  Viral vectors · CPC title

• C12N15/11

  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} · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title