# US 12,559,774 B2 · CRISPR enzymes and systems

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- Number: US 12,559,774 B2
- Document ID: US12559774B2
- Status: Granted
- Kind: B2
- Application number: 18/345,935
- Filed: 2023-06-30
- Published: 2024-01-18
- Granted: 2026-02-24
- Earliest claimed: 2016-08-17
- Assignees: The Broad Institute, Inc.; Massachusetts Institute of Technology
- Applicants: The Broad Institute, Inc.; Massachusetts Institute of Technology
- Inventors: Feng Zhang; David Arthur Scott; Winston Xia Yan; Sourav Choudhury; Mattias Heidenreich
- Web page: https://patentgrep.com/patent/US12559774B2

## CPC

- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- A61K 9/1271: Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers (liposomes as conjugates {A61K47/6911})
- A61K 9/5184: {Virus capsids or envelopes enclosing drugs (liposomes with additional exogenous lipids A61K9/127)}
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- C12N 15/1089: {Design, preparation, screening or analysis of libraries using computer algorithms}
- 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 15/86: Viral vectors
- C12N 15/907: {in mammalian cells}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2310/32: of the sugar
- C12N 2320/32: Special delivery means, e.g. tissue-specific
- C12N 2740/13042: virus or viral particle as vehicle, e.g. encapsulating small organic molecule
- C12N 2740/13043: viral genome or elements thereof as genetic vector
- C12N 2800/22: Vectors comprising a coding region that has been codon optimised for expression in a respective host
- G16B 20/00: ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
- G16B 30/00: ICT specially adapted for sequence analysis involving nucleotides or amino acids
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY

## Abstract

Embodiments disclosed herein are directed to engineered CRISPR-Cas effector proteins that comprise at least one modification compared to an unmodified CRISPR-Cas effector protein that enhances binding of the CRISPR complex to the binding site and/or alters editing preference as compared to wild type. In certain example embodiments, the CRISPR-Cas effector protein is a Type V effector protein. In certain other example embodiments, the Type V effector protein is Cpf1. Embodiments disclosed herein are directed to viral vectors for delivery of CRISPR-Cas effector proteins, including Cpf1. In certain example embodiments, the vectors are designed so as to allow packaging of the CRISPR-Cas effector protein within a single vector. There is also an increased interest in the design of compact promoters for packing and thus expressing larger transgenes for targeted delivery and tissue-specificity. Thus, in another aspect certain embodiments disclosed herein are directed to delivery vectors, constructs, and methods of delivering larger genes for systemic delivery.

## Claims

### Claim 1

A method for preparing a CRISPR-Cas guide molecule comprising:
selecting a set of candidate therapeutic target sequences for one or more loci in a target population, wherein the candidate therapeutic target sequences do not contain variants occurring above a threshold allele frequency in the target population;
removing any candidate therapeutic target sequences having off-target candidates in haplotypes that occur in at least 0.1% of the target population from the set of candidate therapeutic target sequences to thereby define a final target sequence set; and
preparing one or more guide molecules based on the final therapeutic target sequence set targeting one or more loci associated with a disease or disorder,
wherein the guide molecules are Type II or Type V guide molecules.

### Claim 2 (depends on 1)

The method of claim 1, wherein the candidate target sequences are further selected based on optimization of one or more parameters selected from the group consisting of PAM type, PAM nucleotide content, PAM length, target sequence length, PAM restrictiveness, target cleavage efficiency, and target sequence position within a gene, a locus, or other genomic region.

### Claim 3 (depends on 1)

The method of claim 1, further comprising conducting a sequencing-based double-strand break detection assay wherein off-target candidates, PAM restrictiveness, target cleavage efficiency, and/or effector protein specificity is determined.

### Claim 4 (depends on 1)

The method of claim 1, further comprising obtaining sequencing data from a subject to be treated, wherein the one or more guide molecules are prepared based at least in part on the sequencing data of the subject.

### Claim 5 (depends on 4)

The method of claim 4, wherein the sequencing data is whole genome sequencing data.

### Claim 6 (depends on 1)

The method of claim 1, wherein the guide molecule is a Type II guide molecule.

### Claim 7 (depends on 6)

The method of claim 6, wherein the guide molecule is a Cas9 guide molecule.

### Claim 8 (depends on 1)

The method of claim 1, wherein the guide molecule is a Type V guide molecule.

### Claim 9 (depends on 8)

The method of claim 8, wherein the guide molecule is a Cas12 guide molecule.

### Claim 10 (depends on 1)

The method of claim 1, wherein the one or more loci include a 3-globin or γ-globin gene, or regulatory region controlling expression of the 3-globin or γ-globin gene.

### Claim 11 (depends on 1)

The method of claim 1, wherein one or more loci include one or more transcription factors.

### Claim 12 (depends on 11)

The method of claim 11, wherein the one or more transcription factors is BCL11A.

### Claim 13 (depends on 1)

The method of claim 1, wherein the disease is sickle cell anemia or β-thalassemia.

### Claim 14

A set of CRISPR-Cas guide molecules prepared by a method comprising:
selecting a set of candidate therapeutic target sequences for one or more loci in a target population, wherein the candidate therapeutic target sequences do not contain variants occurring above a threshold allele frequency in the target population;
removing any candidate therapeutic target sequences having off-target candidates in haplotypes that occur in at least 0.1% of the target population from the set of candidate therapeutic target sequences to thereby define a final target sequence set,
preparing the set of guide molecules based on the final therapeutic target sequence set targeting one or more loci associated with a disease or disorder,
wherein the guide molecules are Type II or Type V guide molecules.

### Claim 15 (depends on 14)

The set of CRISPR-Cas guide molecules of claim 14, wherein the method further comprises selecting candidate target sequences based on optimization of one or more parameters selected from PAM type, PAM nucleotide content, PAM length, target sequence length, PAM restrictiveness, target cleavage efficiency, and target sequence position within a gene, a locus, or other genomic region.

### Claim 16 (depends on 14)

The set of claim 14, wherein the method further comprises conducting a sequencing-based double-strand break detection assay wherein off-target candidates, PAM restrictiveness, target cleavage efficiency, and/or effector protein specificity is determined.

### Claim 17 (depends on 14)

The set of claim 14, wherein guide molecules are Cas9 guide molecules.

### Claim 18 (depends on 17)

A CRISPR-Cas system comprising a Cas9 and one or more guide molecules from the set of claim 17.

### Claim 19 (depends on 14)

The set of claim 14, wherein the guide molecules are Cas12 guide molecules.

### Claim 20 (depends on 14)

The set of claim 14, wherein the one or more loci include a β-globin or γ-globin gene, or regulatory region controlling expression of the β-globin or γ-globin gene.

### Claim 21 (depends on 14)

The set of claim 14, wherein one or more loci include one or more transcription factors.

### Claim 22 (depends on 21)

The set of claim 21, wherein the one or more transcription factors is BCL11A.

### Claim 23 (depends on 14)

The set of claim 14, wherein the disease is sickle cell anemia or β-thalassemia.

### Claim 24 (depends on 14)

A CRISPR-Cas system comprising a Cas9 and one or more guide molecules from the set of claim 14.

### Claim 25 (depends on 24)

The system of claim 24, wherein the Cas9 is a nickase.

### Claim 26 (depends on 14)

A CRISPR-Cas system comprising Cas12 and one or more guide molecules from the set of claim 14.

### Claim 27 (depends on 14)

The method of claim 14, wherein the one or more loci includes proprotein convertase subtilisin/kexin type 9 (PCSK9).