Conceptio › Archive › Google Patents

Google Patentsopen access

CRISPR enzymes and systems — The Broad Institute, Inc. (US12305204B2)

The Broad Institute, Inc. · Google Patents

Google Patents · Patents · License: Open Access

Open Source ↗

fengzhang thebroadinstitute

patent, google patents, intellectual property, US12305204B2, The Broad Institute, Inc., Feng Zhang, en, 2025

ABSTRACT

Abstract

In one aspect, embodiments disclosed herein are directed to engineered CRISPRCas effector proteins that comprise at least one modification compared to an unmodified CRISPR-Cas effector protein that enhances binding of the 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 II effector protein. In certain other example embodiments, the Type V effector protein is Cas9 or an orthologs or engineered variant thereof. Example Cas9 proteins suitable for use in the embodiments disclosed herein are discussed in further detail below.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 16/325,892 filed Feb. 15, 2019, which is a National Stage application of International Application No. PCT/US2017/047458, filed Aug. 17, 2017, which claims the benefit of U.S. Provisional Application No. 62/376,372 filed Aug. 17, 2016, and U.S. Provisional Application No. 62/437,031 filed Dec. 20, 2016. The entire contents of the above-identified applications are hereby fully incorporated herein by reference.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numbers MH100706 and MH110049 awarded by the National Institutes of Health. The government has certain rights in the invention.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (âBROD-0971US-CONI_ST26.xmlâ; Size is 96,890 bytes; was created on Oct. 17, 2023) is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention generally relates to systems, methods and compositions related to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and components thereof. The present invention also generally relates to delivery of large payloads and includes novel delivery particles, particularly using lipid and viral particle, and also novel viral capsids, both suitable to deliver large payloads, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), CRISPR protein (e.g., Cas, Cas9), CRISPR-Cas or CRISPR system or CRISPR-Cas complex, components thereof, nucleic acid molecules, e.g., vectors, involving the same and uses of all of the foregoing, amongst other aspects. Additionally, the present invention relates to methods for developing or designing CRISPR-Cas system based therapy or therapeutics.

BACKGROUND OF THE INVENTION

Recent advances in genome sequencing techniques and analysis methods have significantly accelerated the ability to catalog and map genetic factors associated with a diverse range of biological functions and diseases. Precise genome targeting technologies are needed to enable systematic reverse engineering of causal genetic variations by allowing selective perturbation of individual genetic elements, as well as to advance synthetic biology, biotechnological, and medical applications. Although genome-editing techniques such as designer zinc fingers, transcription activator-like effectors (TALEs), or homing meganucleases are available for producing targeted genome perturbations, there remains a need for new genome engineering technologies that employ novel strategies and molecular mechanisms and are affordable, easy to set up, scalable, and amenable to targeting multiple positions within the eukaryotic genome. This would provide a major resource for new applications in genome engineering and biotechnology.

The CRISPR-Cas systems of bacterial and archaeal adaptive immunity show extreme diversity of protein composition and genomic loci architecture. The CRISPR-Cas system loci has more than 50 gene families and there is no strictly universal genes indicating fast evolution and extreme diversity of loci architecture. So far, adopting a multi-pronged approach, there is comprehensive cas gene identification of about 395 profiles for 93 Cas proteins. Classification includes signature gene profiles plus signatures of locus architecture. A new classification of CRISPR-Cas systems is proposed in which these systems are broadly divided into two classes, Class 1 with multisubunit effector complexes and Class 2 with single-subunit effector modules exemplified by the Cas9 protein. Novel effector proteins associated with Class 2 CRISPR-Cas systems may be developed as powerful genome engineering tools and the prediction of putative novel effector proteins and their engineering and optimization is important.

The development of CRISPR-Cas RNA-guided endonucleases for eukaryotic genome editing has sparked intense interest in the use of this technology for therapeutic applications.

Extensive research has led to the identification of different technologies which can address the challenges of safety and efficacy. In order to allow the translation of this genome editing technologies to the clinic. There is a need for the development of an algorithm for developing a CRISPR-Cas based therapeutic, which takes into account the different variables which need to be considered.

In contrast to small molecule therapies, which target highly conserved protein active sites, treatment of disease at the genomic level must contend with significant levels of genetic variation in patient populations. Recently, large scale sequencing datasets from the Exome Aggregation Consortium (ExAC) and 1000 Genomes Project have provided an unprecedented view of the landscape of human genetic variation. This variation can affect both the efficacy of a CRISPR-based therapeutic, by disrupting the target site, and its safety, by generating off-target candidate sites.

Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.

SUMMARY OF THE INVENTION

In certain example embodiments, an engineered CRISPR-Cas effector protein that complexes with a nucleic acid comprising a guide sequence to form a CRISPR complex, and wherein in the CRISPR complex the nucleic acid molecule target one or more polynucleotide loci and the protein comprises at least one modification compared to the unmodified protein that enhances binding of the CRISPR complex to the binding site and/or alters editing preferences as compared to wildtype. The editing preference may relate to indel formation. In certain example embodiments, the at least one modification may increase formation of one or more specific indels at a target locus. The CRISPR-Cas effector protein may be Type II CRISPR-Cas effector protein. In certain example embodiments, the CRISPR-Cas protein is Cas9 or orthologue thereof.

In certain other example embodiments, the invention is directed to vectors for delivery of the CRISPR-Cas system, including vector based systems allowing for encoding of both the effector protein and guide sequence in a single vector.

In certain other example embodiments, the invention relates to methods for developing or designing CRISPR-Cas systems. In an aspect, the present invention relates to methods for developing or designing CRISPR-Cas system based therapy or therapeutics. The present invention in particular relates to methods for improving CRISPR-Cas systems, such as CRISPR-Cas system based therapy or therapeutics. Key characteristics of successful CRISPR-Cas systems, such as CRISPR-Cas system based therapy or therapeutics involve high specificity, high efficacy, and high safety. High specificity and high safety can be achieved among others by reduction of off-target effects.

The methods of the present invention in particular involve optimization of selected parameters or variables associated with the CRISPR-Cas system and/or its functionality, as described herein further elsewhere. Optimization of the CRISPR-Cas system in the methods as described herein may depend on the target(s), such as the therapeutic target or therapeutic targets, the mode or type of CRISPR-Cas system modulation, such as CRISPR-Cas system based therapeutic target(s) modulation, modification, or manipulation, as well as the delivery of the CRISPR-Cas system components. One or more targets may be selected, depending on the genotypic and/or phenotypic outcome. For instance, one or more therapeutic targets may be selected, depending on (genetic) disease etiology or the desired therapeutic outcome. The (therapeutic) target(s) may be a single gene, locus, or other genomic site, or may be multiple genes, loci or other genomic sites. As is known in the art, a single gene, locus, or other genomic site may be targeted more than once, such as by use of multiple gRNAs.

These and other aspects, objects, features, and advantages of the example embodiments will become apparent to those having ordinary skill in the art upon consideration of the following detailed description of illustrated example embodiments. These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

FIG. 1 a - f : Depicts how human genetic variation significantly impacts the efficacy of RNA-guided endonucleases. (a), Schematic illustrating the genomic target, RNA guide, and target variation. (b), Fraction of residues for individual nucleotides containing variation in the ExAC dataset. (c), Fraction of 2-nt PAM motifs altered by variants in the ExAC dataset. (d), Percent of targets variants at different allele frequencies for each CRISPR endonuclease. (e), Cumulative targets containing variants for each enzyme. (f), Fraction of targets containing homozygous variants at different allele frequencies. The mean and standard deviation for all enzymes is shown.

FIG. 2 a - c : Depicts how a selection of platinum targets maximizes population efficacy. (a), Schematic showing target variation within exon 2 of PCSK9-001, with regions containing high coverage in the ExAC dataset indicated (black lines below exons). (b), Frequency of target variation plotted by cut site position for targets spanning the start of PCSK9-001 exon 2, with targets shown in (a) indicated by arrows. The horizontal line at 0.01% separates platinum targets (grey) from targets with high variation (dark grey). The classification for each target is depicted below for each enzyme (grey or dark grey boxes). (c), Classification of targets for each enzyme spanning exons 2-5 of PCSK9-001.

FIG. 3 a - c : Depicts how human genetic variation significantly impacts CRISPR endonuclease therapeutic safety. (a), Schematic illustrating off-target candidates arising due to multiple different haplotypes. SEQ ID NOs 71-75 indicate the on-target sequence (SEQ ID NO: 71) and variants (SEQ ID NOs: 72-75). Bolded nucleotides, except for the PAM site (TGG), indicate variation from the on-target site. (b), Number of off-target candidates for each CRISPR endonuclease at different allele frequencies. (c), Distribution of the number of off-target candidates per platinum target for each CRISPR endonuclease.

FIG. 4 a - d : Depicts how gene- and population-specific variation informs therapeutic design. (a), Distribution of the number of off-target candidates per platinum target for 12 therapeutically relevant genes. (b), Total off-target candidates for platinum targets spanning exons 2-5 of PCSK9-001 are shown for each enzyme. (c), Principal component analysis (PCA) separating 1000 Genomes individuals into super populations based on patient-specific off-target profiles for platinum targets spanning 12 therapeutically relevant genes. PC2 and PC3 are shown. AFR, African; AMR, Ad mixed American; EAS, East Asian; EUR, European; SAS, South Asian. (d), Proposed therapeutic design framework.

FIG. 5 a - e : Left part of each panel, fraction of PAMs altered by variants in the ExAC dataset; center, distribution of PAM-altering variant frequencies; right part of each panel, fraction of homozygous variants by frequency. Data shown for AsCpf1 (a), SpCas9-VQR (b), SpCas9 (c), SaCas9 (d), and SpCas9-VRER (e).

FIG. 6 a - d : Top part of each panel, distribution of target variation for therapeutically relevant genes. Targets with frequencies of variation less than 0.01% (dark grey line) are considered platinum. Bottom part of each panel, fraction of all targets in these genes containing variation. Data shown for AsCpf1 (a), SpCas9-VWR (b), SpCas9-WT (c), SaCas9-WT (d).

FIG. 7 : Separation of 1000 Genomes individuals into super populations based on patient specific off-target profiles for targets spanning 12 therapeutically relevant genes. Principal components 1-5 shown. AFR, African; AMR, Ad mixed American; EAS, East Asian; EUR, European; SAS, South Asian.

FIG. 8 : Separation of 1000 Genomes individuals into populations based on patient specific off-target profiles for targets spanning 12 therapeutically relevant genes. Principle components 1-5 shown. CHB, Han Chinese in Beijing, China; JPT, Japanese in Tokyo, Japan; CHS, Southern Han Chinese; CDX, Chinese Dai in Xishuangbanna, China; KHV, Kinh in Ho Chi Minh City, Vietnam; CEU, Utah Residents (CEPH) with Northern and Western Ancestry; TSI, Toscani in Italia; FIN, Finnish in Finland; GBR, British in England and Scotland; IBS, Iberian Population in Spain; YRI, Yoruba in Ibadan, Nigeria; LWK, Luhya in Webuye, Kenya; GWD, Gambian in Western Divisions in the Gambia; MSL, Mende in Sierra Leone; ESN, Esan in Nigeria; ASW, Americans of African Ancestry in SW USA; ACB, African Caribbeans in Barbados; MXL, Mexican Ancestry from Los Angeles USA; PUR, Puerto Ricans from Puerto Rico; CLM, Colombians from Medellin, Colombia; PEL, Peruvians from Lima, Peru; GIH, Gujarati Indian from Houston, Texas; PJL, Punjabi from Lahore, Pakistan; BEB, Bengali from Bangladesh; STU, Sri Lankan Tamil from the UK; ITU, Indian Telugu from the UK.

FIG. 9 : Separation of 1000 Genomes individuals by sex based on patient specific off-target profiles for targets spanning 12 therapeutically relevant genes. Principle components 1-5 shown.

FIG. 10 : Is a diagram depicting example parameters to be selected and optimized in accordance with certain example embodiments.

FIG. 11 shows illustrations of AAV-CRISPR protein of the invention, wherein Cas9 protein is fused or tethered to VP3, for example at the N-terminus of VP3. Cas9 is attached to some, but not all VP3 subunits to avoid steric blocking of cell entry sites on AAV surface. In the AAV9.Cas9 vector, a Cas9 protein fused or tethered to the C-term of VP1, VP2 or VP3 is depicted.

FIG. 12 A- 12 B show a Western blot confirming expression of Cas9-VP3 fusion proteins in cells transfected with plasmids encoding for Cas9 and Cas9-VP3 fusions (AAVCas9:wt 1:6). ( FIG. 12 A ), Left panel: SYPRO Ruby protein staining of fractions from AAVCas9:wt 1:6. Right panel: Anti-SpCas9 blotting of fractions from AAVCas9:wt 1:6. ( FIG. 12 B ), Left panel: SYPRO Ruby protein staining of fractions from wtAAV9. Right panel: Anti-SpCas9 blotting of fractions from wtAAV9.

FIG. 13 illustrates exterior loops and interior sites in AAV9 VP3 for protein insertion.

FIG. 14 depicts electron micrography of wtAAV. Dark particle centers indication empty particles.

FIG. 15 depicts e

CROSS-REFERENCE TO RELATED APPLICATIONS

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numbers MH100706 and MH110049 awarded by the National Institutes of Health. The government has certain rights in the invention.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (âBROD-0971US-CONI_ST26.xmlâ; Size is 96,890 bytes; was created on Oct. 17, 2023) is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

The development of CRISPR-Cas RNA-guided endonucleases for eukaryotic genome editing has sparked intense interest in the use of this technology for therapeutic applications.

Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.

SUMMARY OF THE INVENTION

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 9 : Separation of 1000 Genomes individuals by sex based on patient specific off-target profiles for targets spanning 12 therapeutically relevant genes. Principle components 1-5 shown.

FIG. 10 : Is a diagram depicting example parameters to be selected and optimized in accordance with certain example embodiments.

FIG. 13 illustrates exterior loops and interior sites in AAV9 VP3 for protein insertion.

FIG. 14 depicts electron micrography of wtAAV. Dark particle centers indication empty particles.

FIG. 15 depicts electron micrography of AAV.Cas9 virus particles comprising 50wtAAV:10AAVCas9.

FIG. 16 depicts electron micrography of AAV.Cas9 virus particles comprising 30wtAAV:30AAVCas9.

FIGS. 17 A- 17 B depicts sortase-mediated protein linkage. ( FIG. 17 A ) schematic of proteins anchored to a cell wall via sortase in Gram-positive bacteria is shown (see, Guimares, et al., Nat. Prot. 2013). ( FIG. 17 B ) linkage of Cas9 to AAV by TEV-sortase method. CRISPR protein modified at its C terminus with the LPXTG (SEQ ID NO: 78) sortase-recognition motif followed by a handle for purification (often His6) is incubated with sortase A. Sortase cleaves the threonine-glycine bond and forms an acyl intermediate with threonine. Addition of TEV-cleaved AAV (âprobeâ) comprising N-terminal glycine residues ligates the AAV to the C terminus of the CRISPR protein (see, Guimares, et al., Nat. Prot. 2013).

FIG. 18 depicts linkage of Cas9 to AAV by split intein reconstitution.

FIG. 19 shows interior packaging of proteins:

Packaging A0060

VP3 only loop3 Cre 1:10

Packaging A0061

VP3 only loop3 Cre 1:1

Packaging A0062

VP3 only loop3 Cas9 1:10

Packaging A0063

VP3 only loop3 Cas9 1:1

Packaging A0064

VP3 only loop4 Cre 1:10

Packaging A0065

VP3 only loop4 Cre 1:1

A0068

VSVG Cas9 gesicle

A0069

VSVG Cre gesicle

A0070

RVG Cas9 gesicle

A0071

RVG Cre gesicle

Packaging A0072

AAV9 loop6 (His)6 1:10

Packaging A0073

AAV9 loop6 (His)6 1:1

Packaging A0074

VP3 only loop4 Cas9 1:10

Packaging A0075

VP3 only loop4 Cas9 1:1

A0084

VSVG-CRE

A0085

DNase treatment

A0086

(+G âS)

A0087

(âG +S)

FIG. 20 shows Interior SunTag-GFP. Western blots detect VP3 (top left) and GFP (bottom left) for native VP3 and VP3-GFP fusion. Electron micrographs show GFP-filled capsid ( 103 ).

FIG. 21 depicts Vesicular stomatitis virus (VSV) and Rabies virus (RV) sources of packaging vesicles.

FIG. 22 shows a schematic for transduction of cells with lentiviral vectors packaged in vesicular stomatitis virus-G (VSVG) vesicles. (Cronin et al., Curr Gene Ther. 5 (4): 387-398 (2005)).

FIG. 23 depicts infection of TLR19 cells with VSVG and RVG vesicles harboring Cas9 and sgRNA inducing frameshift mutations to allow mCherry expression. Cas9 RNP vesicles were synthesized by contransfection of VSVG (or RVG) with eSpCas9(1.1) and GFPg2 plasmid.

The figures herein are for illustrative purposes only and are not necessarily drawn to scale.

DETAILED DESCRIPTION OF THE INVENTION

General Definitions

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of common terms and techniques in molecular biology may be found in Molecular Cloning: A Laboratory Manual, 2 nd edition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4 th edition (2012) (Green and Sambrook); Current Protocols in Molecular Biology (1987) (F. M. Ausubel et al. eds.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (1995) (M. J. MacPherson, B. D. Hames, and G. R. Taylor eds.): Antibodies, A Laboratory Manual (1988) (Harlow and Lane, eds.): Antibodies A Laboratory Manual, 2 nd edition 2013 (E. A. Greenfield ed.); Animal Cell Culture (1987) (R.I. Freshney, ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008 (ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2nd edition (2011).

As used herein, the singular forms âaâ, âanâ, and âtheâ include both singular and plural referents unless the context clearly dictates otherwise.

The term âoptionalâ or âoptionallyâ means that the subsequent described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

The terms âaboutâ or âapproximatelyâ as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of +/â10% or less, +/â5% or less, +/â1% or less, and +/â0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier âaboutâ or âapproximatelyâ refers is itself also specifically, and preferably, disclosed.

Reference throughout this specification to âone embodimentâ, âan embodiment,â âan example embodiment,â means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases âin one embodiment,â âin an embodiment,â or âan example embodimentâ in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

It will be appreciated that the terms Cas enzyme, CRISPR enzyme, CRISPR protein, Cas protein and CRISPR Cas are generally used interchangeably and at all points of reference herein refer by analogy to novel CRISPR effector proteins further described in this application, unless otherwise apparent, such as by specific reference to Cas9. The CRISPR effector proteins described herein are preferably Cas9 effector proteins.

All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference.

Overview

In one aspect, 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 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 II effector protein. In certain other example embodiments, the Type V effector protein is Cas9 or an orthologs or engineered variant thereof. Example Cas9 proteins suitable for use in the embodiments disclosed herein are discussed in further detail below.

In another aspect, embodiments disclosed herein are directed to viral vectors for delivery of CRISPR-Cas effector proteins, including Cas9. 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.

In another aspect, the present invention relates to methods for developing or designing CRISPR-Cas systems. In an aspect, the present invention relates to methods for developing or designing optimized CRISPR-Cas systems a wide range of applications including, but not limited to, therapeutic development, bioproduction, and plant and agricultural applications. In certain based therapy or therapeutics. The present invention in particular relates to methods for improving CRISPR-Cas systems, such as CRISPR-Cas system based therapy or therapeutics. Key characteristics of successful CRISPR-Cas systems, such as CRISPR-Cas system based therapy or therapeutics involve high specificity, high efficacy, and high safety. High specificity and high safety can be achieved among others by reduction of off-target effects. Improved specificity and efficacy likewise may be used to improve applications in plants and bioproduction.

Accordingly, in an aspect, the present invention relates to methods for increasing specificity of CRISPR-Cas systems, such as CRISPR-Cas system based therapy or therapeutics. In a further aspect, the invention relates to methods for increasing efficacy of CRISPR-Cas systems, such as CRISPR-Cas system based therapy or therapeutics. In a further aspect, the invention relates to methods for increasing safety of CRISPR-Cas systems, such as CRISPR-Cas system based therapy or therapeutics. In a further aspect, the present invention relates to methods for increasing specificity, efficacy, and/or safety, preferably all, of CRISPR-Cas systems, such as CRISPR-Cas system based therapy or therapeutics.

In certain embodiments, the CRISPR-Cas system comprises a CRISPR effector as defined herein elsewhere.

CRISPR-Cas system activity, such as CRISPR-Cas system design may involve target disruption, such as target mutation, such as leading to gene knockout. CRISPR-Cas system activity, such as CRISPR-Cas system design may involve replacement of particular target sites, such as leading to target correction. CISPR-Cas system system design may involve removal of particular target sites, such as leading to target deletion. CRISPR-Cas system activity may involve modulation of target site functionality, such as target site activity or accessibility, leading for instance to (transcriptional and/or epigenetic) gene or genomic region activation or gene or genomic region silencing. The skilled person will understand that modulation of target site functionality may involve CRISPR effector mutation (such as for instance generation of a catalytically inactive CRISPR effector) and/or functionalization (such as for instance fusion of the CRISPR effector with a heterologous functional domain, such as a transcriptional activator or repressor), as described herein elsewhere.

Engineered CRISPR-Cas Systems

In general, CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats), also known as SPIDRs (SPacer Interspersed Direct Repeats), constitute a family of DNA loci that are usually specific to a particular bacterial species. The CRISPR locus comprises a distinct class of interspersed short sequence repeats (SSRs) that were recognized in E. coli (Ishino et al., J. Bacteriol., 169:5429-5433 [1987]; and Nakata et al., J. Bacteriol., 171:3553-3556 [1989]), and associated genes. Similar interspersed SSRs have been identified in Haloferax mediterranei, Streptococcus, Anabaena , and Mycobacterium tuberculosis (See, Groenen et al., Mol. Microbiol., 10:1057-1065 [1993]; Hoe et al., Emerg. Infect. Dis., 5:254-263 [1999]; Masepohl et al., Biochim. Biophys. Acta 1307:26-30 [1996]; and Mojica et al., Mol. Microbiol., 17:85-93 [1995]). The CRISPR loci typically differ from other SSRs by the structure of the repeats, which have been termed short regularly spaced repeats (SRSRs) (Janssen et al., OMICS J. Integ. Biol., 6:23-33 [2002]; and Mojica et al., Mol. Microbiol., 36:244-246 [2000]). In general, the repeats are short elements that occur in clusters that are regularly spaced by unique intervening sequences with a substantially constant length (Mojica et al., [2000], supra). Although the repeat sequences are highly conserved between strains, the number of interspersed repeats and the sequences of the spacer regions typically differ from strain to strain (van Embden et al., J. Bacteriol., 182:2393-2401 [2000]). CRISPR loci have been identified in more than 40 prokaryotes (See e.g., Jansen et al., Mol. Microbiol., 43:1565-1575 [2002]; and Mojica et al., [2005]) including, but not limited to Aeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Haloarcula, Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus, Pyrococcus, Picrophilus, Thermoplasma, Corynebacterium, Mycobacterium, Streptomyces, Aquifex Porphyromonas, Chlorobium, Thermus, Bacillus, Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma, Fusobacterium, Azoarcus, Chromobacterium, Neisseria, Nitrosomonas, Desulfovibrio, Geobacter, Myxococcus, Campylobacter, Wolinella, Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus, Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia, Treponema , and Thermotoga.

General Features of Cas9 Effector Protein

The application describes methods for using CRISPR-Cas proteins for polynucleotide editing and modifications. This is exemplified herein with Cas9, whereby a number of Cas9 orthologs or homologs have been identified. It will be apparent to the skilled person that further Cas9 orthologs or homologs can be identified and that any of the functionalities described herein may be engineered into other Cas9 orthologs, including chimeric enzymes comprising fragments from multiple orthologs.

Methods for Identifying New CRISPR-Cas Loci

The Cas9 gene is found in several diverse bacterial genomes, typically in the same locus with cas1, cas2, and cas4 genes and a CRISPR cassette. Furthermore, the Cas9 protein contains a readily identifiable C-terminal region that is homologous to the transposon ORF-B and includes an active RuvC-like nuclease, an arginine-rich region.

For instance, computational methods of identifying novel CRISPR-Cas loci are described in EP3009511 or US2016208243 and may comprise the following steps: detecting all contigs encoding the Cas1 protein; identifying all predicted protein coding genes within 20 KB of the cas1 gene; comparing the identified genes with Cas protein-specific profiles and predicting CRISPR arrays; selecting unclassified candidate CRISPR-Cas loci containing proteins larger than 500 amino acids (>500 aa); analyzing selected candidates using methods such as PSI-BLAST and HHPred to screen for known protein domains, thereby identifying novel Class 2 CRISPR-Cas loci (see also Schmakov et al. 2015, Mol Cell. 60 (3): 385-97). In addition to the above mentioned steps, additional analysis of the candidates may be conducted by searching metagenomics databases for additional homologs. Additionally or alternatively, to expand the search to non-autonomous CRISPR-Cas systems, the same procedure can be performed with the CRISPR array used as the seed.

In one aspect the detecting all contigs encoding the Cas1 protein is performed by GenemarkS which a gene prediction program as further described in âGeneMarkS: a self-training method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions.â John Besemer, Alexandre Lomsadze and Mark Borodovsky, Nucleic Acids Research (2001) 29, pp 2607-2618, herein incorporated by reference.

In one aspect the identifying all predicted protein coding genes is carried out by comparing the identified genes with Cas protein-specific profiles and annotating them according to NCBI Conserved Domain Database (CDD) which is a protein annotation resource that consists of a collection of well-annotated multiple sequence alignment models for ancient domains and full-length proteins. These are available as position-specific score matrices (PSSMs) for fast identification of conserved domains in protein sequences via RPS-BLAST. CDD content includes NCBI-curated domains, which use 3D-structure information to explicitly define domain boundaries and provide insights into sequence/structure/function relationships, as well as domain models imported from a number of external source databases (Pfam, SMART, COG, PRK, TIGRFAM). In a further aspect, CRISPR arrays were predicted using a PILER-CR program which is a public domain software for finding CRISPR repeats as described in âPILER-CR: fast and accurate identification of CRISPR repeatsâ, Edgar, R. C., BMC Bioinformatics, January 20; 8:18 (2007), herein incorporated by reference.

In a further aspect, the case by case analysis is performed using PSI-BLAST (Position-Specific Iterative Basic Local Alignment Search Tool). PSI-BLAST derives a position-specific scoring matrix (PSSM) or profile from the multiple sequence alignment of sequences detected above a given score threshold using protein-protein BLAST. This PSSM is used to further search the database for new matches, and is updated for subsequent iterations with these newly detected sequences. Thus, PSI-BLAST provides a means of detecting distant relationships between proteins.

In another aspect, the case by case analysis is performed using HHpred, a method for sequence database searching and structure prediction that is as easy to use as BLAST or PSI-BLAST and that is at the same time much more sensitive in finding remote homologs. In fact, HHpred's sensitivity is competitive with the most powerful servers for structure prediction currently available. HHpred is the first server that is based on the pairwise comparison of profile hidden Markov models (HMMs). Whereas most conventional sequence search methods search sequence databases such as UniProt or the NR, HHpred searches alignment databases, like Pfam or SMART. This greatly simplifies the list of hits to a number of sequence families instead of a clutter of single sequences. All major publicly available profile and alignment databases are available through HHpred. HHpred accepts a single query sequence or a multiple alignment as input. Within only a few minutes it returns the search results in an easy-to-read format similar to that of PSI-BLAST. Search options include local or global alignment and scoring secondary structure similarity. HHpred can produce pairwise query-template sequence alignments, merged query-template multiple alignments (e.g. for transitive searches), as well as 3D structural models calculated by the MODELLER software from HHpred alignments.

In certain example embodiments, methods for identifying novel CRISPR loci may include comparison to properties and elements of known CRISPR loci. Example methods are disclosed in U.S. Provisional Application No. 62/376,387 filed Aug. 17, 2016 and entitled âMethods for identifying Class 2 CRISPR-Cas systems,â U.S. Provisional Application No. 62/376,383 filed Aug. 17, 2016 and entitled âMethods for Identifying Novel Gene Editing Elements,â and Shmakov et al. âDiversity and evolution of class 2 CRISPR-Cas systems,â Nat Rev Microbiol. 2017 15 (3): 169-182. Finally, methods such as those disclosed above may also be adaptive to identify genomic structures comprising repeating motifs in general as opposed to specific known CRISPR objects such as Cas9.

It should be further recognized that putative novel CRISPR-Cas loci may be further discovered and/or integrated, in particular for relevant nuclease activity, using the methods disclosed in the section below under the header âMethods for determining on/off target activity and selecting suitable sequences/guides.â

Orthologs of Cas9

The terms âorthologueâ (also referred to as âorthologâ herein) and âhomologueâ (also referred to as âhomologâ herein) are well known in the art. By means of further guidance, a âhomologueâ of a protein as used herein is a protein of the same species which performs the same or a similar function as the protein it is a homologue of. Homologous proteins may but need not be structurally related, or are only partially structurally related. An âorthologueâ of a protein as used herein is a protein of a different species which performs the same or a similar function as the protein it is an orthologue of. Orthologous proteins may but need not be structurally related, or are only partially structurally related. Homologs and orthologs may be identified by homology modelling (see, e.g., Greer, Science vol. 228 (1985) 1055, and Blundell et al. Eur J Biochem vol 172 (1988), 513) or âstructural BLASTâ (Dey F, Cliff Zhang Q, Petrey D, Honig B. Toward a âstructural BLASTâ: using structural relationships to infer function. Protein Sci. 2013 April; 22 (4): 359-66. doi: 10.1002/pro.2225.). See also Shmakov et al. (2015) for application in the field of CRISPR-Cas loci. Homologous proteins may but need not be structurally related, or are only partially structurally related.

The Cas9 gene is found in several diverse bacterial genomes, typically in the same locus with cas1, cas2, and cas4 genes and a CRISPR cassette Furthermore, the Cas9 protein contains a readily identifiable C-terminal region that is homologous to the transposon ORF-B and includes an active RuvC-like nuclease, an arginine-rich region.

In particular embodiments, the effector protein is a Cas9 effector protein from an organism from a genus comprising Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum , Sphaerochaeta, Lactobacillus, Eubacterium , or Corynebacte.

In particular embodiments, the effector protein is a Cas9 effector protein from an organism from a genus comprising Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium , Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethylophilus, Porphyromonas, Prevotella , Bacteroidetes, Helcococcus , Letospira, Desulfovibrio, Desulfonatronum , Opitutaceae, Tuberbacillus, Bacillus, Brevibacillus, Methylobacterium , or Acidaminococcus.

In further particular embodiments, the Cas9 effector protein is from an organism selected from S. mutans, S. agalactiae, S. equisimilis, S. sanguinis, S. pneumonia; C. jejuni, C. coli; N. salsuginis, N. tergarcus; S. auricularis, S. carnosus; N. meningitides, N. gonorrhoeae; L. monocytogenes, L. ivanovii; C. botulinum, C. difficile, C. tetani, C. sordellii . In particular embodiments, the effector protein is a Cas9 effector protein from an organism from Streptococcus pyogenes, Staphylococcus aureus , or Streptococcus thermophilus Cas9.

The effector protein may comprise a chimeric effector protein comprising a first fragment from a first effector protein (e.g., a Cas9) ortholog and a second fragment from a second effector (e.g., a Cas9) protein ortholog, and wherein the first and second effector protein orthologs are different. At least one of the first and second effector protein (e.g., a Cas9) orthologs may comprise an effector protein (e.g., a Cas9) from an organism comprising Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium , Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethylophilus, Porphyromonas, Prevotella , Bacteroidetes, Helcococcus , Letospira, Desulfovibrio, Desulfonatronum , Opitutaceae, Tuberbacillus, Bacillus, Brevibacillus, Methylobacterium or Acidaminococcus ; e.g., a chimeric effector protein comprising a first fragment and a second fragment wherein each of the first and second fragments is selected from a Cas9 of an organism comprising Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium , Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium , Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethylophilus, Porphyromonas, Prevotella , Bacteroidetes, Helcococcus , Letospira, Desulfovibrio, Desulfonatronum , Opitutaceae, Tuberbacillus, Bacillus, Brevibacillus, Methylobacterium or Acidaminococcus wherein the first and second fragments are not from the same bacteria; for instance a chimeric effector protein comprising a first fragment and a second fragment wherein each of the first and second fragments is selected from a Cas9 of S. mutans, S. agalactiae, S. equisimilis, S. sanguinis, S. pneumonia; C. jejuni, C. coli; N. salsuginis, N. tergarcus; S. auricularis, S. carnosus; N. meningitides, N. gonorrhoeae; L. monocytogenes, L. ivanovii; C. botulinum, C. difficile, C. tetani, C. sordellii; Francisella tularensis 1, Prevotella albensis , Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus , Peregrinibacteria bacterium GW2011_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17 , Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020 , candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237 , Leptospira inadai , Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens and Porphyromonas macacae , wherein the first and second fragments are not from the same bacteria.

In a more preferred embodiment, the Cas9 is derived from a bacterial species selected from Streptococcus pyogenes, Staphylococcus aureus , or Streptococcus thermophilus Cas9. In certain embodiments, the Cas9p is derived from a bacterial species selected from Francisella tularensis 1 , Prevotella albensis , Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus , Peregrinibacteria bacterium GW2011_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2 44_17 , Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020 , candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai , Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens and Porphyromonas macacae . In certain embodiments, the Cas9p is derived from a bacterial species selected from Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020. In certain embodiments, the effector protein is derived from a subspecies of Francisella tularensis 1, including but not limited to Francisella tularensis subsp. Novicida.

The nucleic acid-targeting system may be derived advantageously from a Type VI CRISPR system. In some embodiments, one or more elements of a nucleic acid-targeting system is derived from a particular organism comprising an endogenous RNA-targeting system. In particular embodiments, the Type VI RNA-targeting Cas enzyme is C2c2. In an embodiment of the invention, there is provided a effector protein which comprises an amino acid sequence having at least 80% sequence homology to the wild-type sequence of any of Leptotrichia shahii C2c2, Lachnospiraceae bacterium MA2020 C2c2, Lachnospiraceae bacterium NK4A179 C2c2, Clostridium aminophilum (DSM 10710)C2c2, Carnobacterium gallinarum (DSM 4847)C2c2 , Paludibacter propionicigenes (WB4)C2c2, Listeria weihenstephanensis (FSL R9-0317)C2c2, Listeriaceae bacterium (FSL M6-0635) C2c2, Listeria newyorkensis (FSL M6-0635)C2c2, Leptotrichia wadei (F0279)C2c2, Rhodobacter capsulatus (SB 1003)C2c2, Rhodobacter capsulatus (R121)C2c2, Rhodobacter capsulatus (DE442)C2c2, Leptotrichia wadei (Lw2)C2c2, or Listeria seeligeri C2c2.

In particular embodiments, the homologue or orthologue of Cas9 as referred to herein has a sequence homology or identity of at least 80%, more preferably at least 85%, even more preferably at least 90%, such as for instance at least 95% with Cas9. In further embodiments, the: homologue or orthologue of Cas9 as referred to herein has a sequence identity of at least 80%, more preferably at least 85%, even more preferably at least 90%, such as for instance at least 95% with the wild type Cas9. Where the Cas9 has one or more mutations (mutated), the homologue or orthologue of said Cas9 as referred to herein has a sequence identity of at least 80%, more preferably at least 85%, even more preferably at least 90%, such as for instance at least 95% with the mutated Cas9.

In an embodiment, the Cas9 protein may be an ortholog of an organism of a genus which includes, but is not limited to Streptococcus sp. or Staphilococcus sp.; in particular embodiments, Cas9 protein may be an ortholog of an organism of a species which includes, but is not limited to Streptococcus pyogenes, Staphylococcus aureus , or Streptococcus thermophilus Cas9. In particular embodiments, the homologue or orthologue of Cas9p as referred to herein has a sequence homology or identity of at least 80%, more preferably at least 85%, even more preferably at least 90%, such as for instance at least 95% with one or more of the Cas9 sequences disclosed herein. In further embodiments, the homologue or orthologue of Cas9 as referred to herein has a sequence identity of at least 80%, more preferably at least 85%, even more preferably at least 90%, such as for instance at least 95% with the wild type SpCas9, SaCas9 or StCas9.

In particular embodiments, the Cas9 protein of the invention has a sequence homology or identity of at least 60%, more particularly at least 70, such as at least 80%, more preferably at least 85%, even more preferably at least 90%, such as for instance at least 95% with SpCas9, SaCas9 or StCas9. In further embodiments, the Cas9 protein as referred to herein has a sequence identity of at least 60%, such as at least 70%, more particularly at least 80%, more preferably at least 85%, even m

CLAIMS

Claims ( 14 )

What is claimed:

1. A method for preparing a Type II or a Type V 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.

2. 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.

3. 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.

4. 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.

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

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

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

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

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

10. The method of claim 1 , wherein the one or more loci are associated with a cancer, a cardiovascular disease, a neurological disease, a trinucleotide repeat expansion disorder, diabetes mellitus.

11. The method of claim 10 , wherein the cancer is a Non-Hodgkin's lymphoma, multiple myeloma, B-cell lymphoma, acute myeloid leukemia.

12. The method of claim 1 , wherein one or more loci are selected from the group consisting of a tumor antigen selected from human telomerase reverse transcriptase (hTERT), survivin, mouse double minute 2 homolog (MDM2), cytochrome P450 1B 1 (CYP1B), HER2/neu, Wilms' tumor gene 1 (WT1), livin, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 16 (MUC16), MUC1, prostate-specific membrane antigen (PSMA), p53, and cyclin (DI).

13. The method of claim 1 , wherein one or more loci are selected from the group consisting of B cell maturation antigen (BCMA), transmembrane activator and CAML Interactor (TACI), B-cell activating factor receptor (BAFF-R), CD19, PD-1, CD38, CD138, CS-1, CD33, CD26, CD30, CD53, CD70, CD92, CD100, CD148, CD150, CD200, CD261, CD262, and CD362.

14. The method of claim 1 , wherein the one or more loci is transthyretin (TTR) or PCSK9.

US18/488,261

2016-08-17

2023-10-17

CRISPR enzymes and systems

Active

US12305204B2

( en )

Priority Applications (2)

Application Number

Priority Date

Filing Date

Title

US18/488,261

US12305204B2

( en )

2016-08-17

2023-10-17

CRISPR enzymes and systems

US19/053,983

US20250223579A1

( en )

2016-08-17

2025-02-14

Novel crispr enzymes and systems

Applications Claiming Priority (5)

Application Number

Priority Date

Filing Date

Title

US201662376372P

2016-08-17

US201662437031P

2016-12-20

PCT/US2017/047458

WO2018035387A1

( en )

2016-08-17

2017-08-17

Novel crispr enzymes and systems

US201916325892A

2019-02-15

US18/488,261

US12305204B2

( en )

2016-08-17

2023-10-17

CRISPR enzymes and systems

Related Parent Applications (2)

Application Number

Title

Priority Date

Filing Date

PCT/US2017/047458

Continuation

WO2018035387A1

( en )

2016-08-17

2017-08-17

Novel crispr enzymes and systems

US16/325,892

Continuation

US20200283743A1

( en )

2016-08-17

2017-08-17

Novel crispr enzymes and systems

Related Child Applications (1)

Application Number

Title

Priority Date

Filing Date

US19/053,983

Continuation

US20250223579A1

( en )

2016-08-17

2025-02-14

Novel crispr enzymes and systems

Publications (2)

Publication Number

Publication Date

US20240124860A1

US20240124860A1 ( en )

2024-04-18

US12305204B2

true

US12305204B2 ( en )

2025-05-20

Family

ID=61197453

Family Applications (3)

Application Number

Title

Priority Date

Filing Date

US16/325,892

Pending

US20200283743A1

( en )

2016-08-17

2017-08-17

Novel crispr enzymes and systems

US18/488,261

Active

US12305204B2

( en )

2016-08-17

2023-10-17

CRISPR enzymes and systems

US19/053,983

Pending

US20250223579A1

( en )

2016-08-17

2025-02-14

Novel crispr enzymes and systems

Family Applications Before (1)

Application Number

Title

Priority Date

Filing Date

US16/325,892

Pending

US20200283743A1

( en )

2016-08-17

2017-08-17

Novel crispr enzymes and systems

Family Applications After (1)

Application Number

Title

Priority Date

Filing Date

US19/053,983

Pending

US20250223579A1

( en )

2016-08-17

2025-02-14

Novel crispr enzymes and systems

Country Status (4)

Country

Link

( 3 )

US20200283743A1

( en )

( 1 )

EP3500670B1

( en )

( 1 )

CN110312799A

( en )

( 1 )

WO2018035387A1

( en )

Families Citing this family (97)

* Cited by examiner, â Cited by third party

Publication number

Priority date

Publication date

Assignee

Title

AU2015330699B2

( en )

2014-10-10

2021-12-02

Editas Medicine, Inc.

Compositions and methods for promoting homology directed repair

WO2016073990A2

( en )

2014-11-07

2016-05-12

Editas Medicine, Inc.

Methods for improving crispr/cas-mediated genome-editing

EP3294896A1

( en )

2015-05-11

2018-03-21

Editas Medicine, Inc.

Optimized crispr/cas9 systems and methods for gene editing in stem cells

KR102796744B1

( en )

2015-06-09

2025-04-15

ìëíì¤ ë©ëì , ì¸ì½í¬ë ì´í°ë

CRISPR/CAS-related methods and compositions for improving transplantation

EP3353296B1

( en )

2015-09-24

2020-11-04

Editas Medicine, Inc.

Use of exonucleases to improve crispr/cas-mediated genome editing

US11597924B2

( en )

2016-03-25

2023-03-07

Editas Medicine, Inc.

Genome editing systems comprising repair-modulating enzyme molecules and methods of their use

WO2017180694A1

( en )

2016-04-13

2017-10-19

Editas Medicine, Inc.

Cas9 fusion molecules gene editing systems, and methods of use thereof

US20190234874A1

( en )

2016-07-19

2019-08-01

Altius Institute For Biomedical Sciences

Methods for fluorescence imaging microscopy

US11078481B1

( en )

2016-08-03

2021-08-03

KSQ Therapeutics, Inc.

Methods for screening for cancer targets

WO2018035387A1

( en )

2016-08-17

2018-02-22

The Broad Institute, Inc.

Novel crispr enzymes and systems

US11078483B1

( en )

2016-09-02

2021-08-03

KSQ Therapeutics, Inc.

Methods for measuring and improving CRISPR reagent function

WO2018064208A1

( en )

2016-09-28

2018-04-05

The Broad Institute, Inc.

Systematic screening and mapping of regulatory elements in non-coding genomic regions, methods, compositions, and applications thereof

US20180128545A1

( en )

2016-11-08

2018-05-10

Berry Metal Company

Modular furnace cooling wall

WO2018119010A1

( en )

2016-12-19

2018-06-28

Editas Medicine, Inc.

Assessing nuclease cleavage

EP3565907B1

( en )

2017-01-06

2022-05-04

Editas Medicine, Inc.

Methods of assessing nuclease cleavage

US12350368B2

( en )

2017-04-14

2025-07-08

The Broad Institute, Inc.

Delivery of large payloads

US11499151B2

( en )

2017-04-28

2022-11-15

Editas Medicine, Inc.

Methods and systems for analyzing guide RNA molecules

WO2018204777A2

( en )

2017-05-05

2018-11-08

The Broad Institute, Inc.

Methods for identification and modification of lncrna associated with target genotypes and phenotypes

AU2018273986A1

( en )

2017-05-25

2019-12-12

The General Hospital Corporation

Bipartite base editor (BBE) architectures and type-II-C-Cas9 zinc finger editing

AU2018280116B2

( en )

2017-06-05

2025-03-27

Research Institute At Nationwide Children's Hospital

Enhanced modified viral capsid proteins

JP7518620B2

( en )

2017-06-09

2024-07-18

ã¨ãã£ã¿ã¹ã»ã¡ãã£ã·ã³ï¼ã¤ã³ã³ã¼ãã¬ã¤ããã

Engineered CAS9 nuclease

SG11201912179SA

( en )

2017-06-15

2020-01-30

Univ California

Targeted non-viral dna insertions

WO2019014564A1

( en )

2017-07-14

2019-01-17

Editas Medicine, Inc.

Systems and methods for targeted integration and genome editing and detection thereof using integrated priming sites

WO2019018700A1

( en )

2017-07-19

2019-01-24

Altius Institute For Biomedical Sciences

Methods and compositions for viral nano-fish

WO2019084552A1

( en )

2017-10-27

2019-05-02

The Regents Of The University Of California

Targeted replacement of endogenous t cell receptors

WO2019102381A1

( en )

2017-11-21

2019-05-31

Casebia Therapeutics Llp

Materials and methods for treatment of autosomal dominant retinitis pigmentosa

US12089930B2

( en )

2018-03-05

2024-09-17

Marquette University

Method and apparatus for non-invasive hemoglobin level prediction

EP3765040A4

( en )

2018-03-13

2021-12-01

Regents of the University of Minnesota

LYMPHOHEMATOPOIETIC ENGINEERING USING CAS9 BASIC EDITORS

WO2019191114A1

( en )

2018-03-27

2019-10-03

The Trustees Of The University Of Pennsylvania

Modified immune cells having enhanced function and methods for screening for same

CA3098427A1

( en )

2018-04-18

2019-10-24

Altius Institute For Biomedical Sciences

Methods for assessing specificity of cell engineering tools

US12312383B2

( en )

2018-04-18

2025-05-27

Altius Institute For Biomedical Sciences

Animal pathogen-derived polypeptides and uses thereof for genetic engineering

WO2019210305A1

( en )

2018-04-27

2019-10-31

The Trustees Of Columbia University In The City Of New York

Methods of inactivating gene editing machineries

US20210324381A1

( en )

2018-04-27

2021-10-21

Seattle Children's Hospital (dba Seattle Children's Research Institute)

Therapeutic genome editing in x-linked hyper igm syndrome

EP3814527B1

( en )

2018-06-26

2023-02-22

Massachusetts Institute of Technology

Crispr effector system based amplification methods, systems, and diagnostics

MX2020013461A

( en )

2018-06-26

2021-04-28

Broad Inst Inc

Crispr double nickase based amplification compositions, systems, and methods.

EP3814370A4

( en )

2018-06-27

2022-06-22

Altius Institute For Biomedical Sciences

Nucleases for genome editing

WO2020006126A1

( en )

2018-06-27

2020-01-02

Altius Institute For Biomedical Sciences

Nucleic acid binding domains and methods of use thereof

EP3814524A4

( en )

2018-06-27

2022-04-27

Altius Institute For Biomedical Sciences

Gapped and tunable repeat units for use in genome editing and gene regulation compositions

AU2019291918B2

( en )

2018-06-29

2025-06-12

Editas Medicine, Inc.

Synthetic guide molecules, compositions and methods relating thereto

CN108795902A

( en )

2018-07-05

2018-11-13

æ·±å³ä¸æºå»å¦ç§ææéå¬å¸

A kind of safe and efficient CRISPR/Cas9 gene editings technology

US10724052B2

( en )

2018-09-07

2020-07-28

Crispr Therapeutics Ag

Universal donor cells

US20230193251A1

( en )

2018-09-19

2023-06-22

The University Of Hong Kong

Improved high-throughput combinatorial genetic modification system and optimized cas9 enzyme variants

BR112021007301A2

( en )

2018-10-18

2021-07-27

Intellia Therapeutics, Inc.

compositions and methods to express factor ix

JP7460178B2

( en )

2018-11-15

2024-04-02

ä¸å½â²è¾²â¼â²æ¥â¼å¤§å¦

CRISPR-Cas12j enzyme and system

US20220098619A1

( en )

2018-12-10

2022-03-31

The Board Of Trustees Of The Leland Stanford Junior University

Anti-crispr-mediated control of genome editing and synthetic circuits in eukaryotic cells

WO2020124050A1

( en )

2018-12-13

2020-06-18

The Broad Institute, Inc.

Tiled assays using crispr-cas based detection

EP3921417A4

( en )

2019-02-04

2022-11-09

The General Hospital Corporation

ADENINE DNA BASE EDITOR VARIANTS WITH REDUCED OFF-TARGET RNA EDITING

US20220136013A1

( en )

2019-02-26

2022-05-05

Emendobio Inc.

Differential knockout of a heterozygous allele of stat1

US20220154258A1

( en )

2019-03-14

2022-05-19

The Broad Institute, Inc.

Crispr effector system based multiplex diagnostics

WO2020191102A1

( en )

2019-03-18

2020-09-24

The Broad Institute, Inc.

Type vii crispr proteins and systems

US12384811B2

( en )

2019-04-05

2025-08-12

Academia Sinica

Sialidase-resistant saccharide and method of making and using the same

US20220213456A1

( en )

2019-04-30

2022-07-07

Emendobio Inc.

Novel omni crispr nucleases

CA3142425A1

( en )

2019-06-01

2020-12-10

Sivec Biotechnologies, Llc

A bacterial platform for delivery of gene-editing systems to eukaryotic cells

CN114258398A

( en )

2019-06-13

2022-03-29

æ»å»é¢å¬å¸

Engineered human endogenous virus-like particles and methods of delivery to cells using the same

US11401530B2

( en )

2019-08-19

2022-08-02

The Catholic University Of America

Bacteriophage-based artificial viruses for human genome remodeling

US20220333208A1

( en )

2019-09-03

2022-10-20

The Broad Institute, Inc.

Crispr effector system based multiplex cancer diagnostics

US11118195B2

( en )

2019-09-05

2021-09-14

Crispr Therapeutics Ag

Universal donor cells

AU2020340622A1

( en )

2019-09-05

2022-03-03

Crispr Therapeutics Ag

Universal donor cells

US11844800B2

( en )

2019-10-30

2023-12-19

Massachusetts Institute Of Technology

Methods and compositions for predicting and preventing relapse of acute lymphoblastic leukemia

CN110975831B

( en )

2019-12-10

2022-09-02

åå°æ»¨å·¥ä¸å¤§å¦

Preparation method of magnetic beta-cyclodextrin phospholipid membrane and experimental method for analyzing and degrading chlorobenzene compounds by using magnetic beta-cyclodextrin phospholipid membrane

JP2023510870A

( en )

2020-01-14

2023-03-15

ã·ã³ã»ã«ã¤ã³ ã¤ã³ã³ã¼ãã¬ã¤ããã

CD122 with altered ICD STAT signaling

WO2021146641A1

( en )

2020-01-17

2021-07-22

The Broad Institute, Inc.

Small type ii-d cas proteins and methods of use thereof

AU2021212189A1

( en )

2020-01-30

2022-08-04

Pairwise Plants Services, Inc.

Compositions, systems, and methods for base diversification

CN111337666B

( en )

2020-02-12

2021-04-02

å±±ä¸å¤§å¦

I-motif recombination mediated FRET probe and application thereof in-situ imaging cancer cell surface protein homodimerization

JPWO2021171688A1

( en )

2020-02-26

2021-09-02

WO2021174441A1

( en )

2020-03-04

2021-09-10

æ·±å³å¤§å¦

Platanus mutant, method for obtaining platanus mutant, and application

EP4121532B1

( en )

2020-03-17

2025-07-30

The Broad Institute, Inc.

Crispr system high throughput diagnostic systems and methods

WO2021224506A1

( en )

2020-05-08

2021-11-11

Universitat Pompeu Fabra

Crispr-cas homology directed repair enhancer

WO2021243289A1

( en )

2020-05-29

2021-12-02

The General Hospital Corporation

Systems and methods for stable and heritable alteration by precision editing (shape)

CN116670154A

( en )

2020-07-24

2023-08-29

æ»å»é¢å¬å¸

Enhanced virus-like particles and methods of using same for delivery to cells

EP4371564B1

( en )

2020-07-29

2026-03-18

The Catholic University Of America

Design of bacteriophage-based artificial viruses for human genome remodeling

EP4232572A4

( en )

2020-10-22

2024-09-25

Duke University

COMPOSITIONS AND METHODS RELATED TO ALZHEIMER'S DISEASE

CN112415198B

( en )

2020-11-20

2022-11-11

åå·å¤§å¦åè¥¿å»é¢

Application of GP1BB detection reagent in preparation of lung cancer screening kit

KR20230146007A

( en )

2020-12-31

2023-10-18

í¬ë¦¬ì¤í¼ íë¼í¨í±ì¤ ìê²

universal donor cells

CN112881352B

( en )

2021-01-07

2022-07-29

éå²åä¸å¤§å¦

Nucleic acid aptamer-quantum dot biosensor for detection and elimination of Salmonella, its preparation method and application

CN112813129B

( en )

2021-02-05

2023-09-08

æ±åå¤§å¦

Method for improving yield of 7-dehydrocholesterol in saccharomycetes by utilizing compartmentalization

JP2024508696A

( en )

2021-02-07

2024-02-28

RNA adeno-associated virus (RAAV) vectors and their uses

US20240200229A1

( en )

2021-04-26

2024-06-20

Gordian Biotechnology, Inc.

Compositions and methods for in vivo screening of therapeutics

WO2022256440A2

( en )

2021-06-01

2022-12-08

Arbor Biotechnologies, Inc.

Gene editing systems comprising a crispr nuclease and uses thereof

WO2022266849A1

( en )

2021-06-22

2022-12-29

ä¸å½ç§å¦é¢èç§å¦ä¸æºè½ææ¯åè¶åæ°ä¸å¿

Screening of novel crispr-cas13 protein and use thereof

JP2024538697A

( en )

2021-10-08

2024-10-23

ãã¤ã¯ããã¼ãï¼ã¤ã³ã¯ï¼

Meta-epigenomics-based disease diagnostics

IL311570A

( en )

2021-10-14

2024-05-01

Arsenal Biosciences Inc

Immune cells having co-expressed shrnas and logic gate systems

WO2023069425A2

( en )

2021-10-18

2023-04-27

Logicbio Therapeutics, Inc.

Gene therapy for the treatment of wilson''s disease

CA3216335A1

( en )

2021-11-24

2023-06-01

Jim Fallon

Systems and methods for an intranasal drug delivery system

CN120712355A

( en )

2023-02-15

2025-09-26

A gene editing method for inhibiting aberrant splicing in the inhibitory microtubule assembly protein 2 (STMN2) transcript

EP4568709A2

( en )

2023-04-08

2025-06-18

Rock Biomedical Inc.

Methods and compositions for targeted delivery by polymersomes

AU2024252371B2

( en )

2023-04-08

2026-02-12

Rock Biomedical Inc.

Methods and compositions for dendritic cell targeting nano-delivery

EP4713441A2

( en )

2023-05-17

2026-03-25

Rock Biomedical Inc.

Mgat1 deficient cells and uses thereof

WO2025019273A1

( en )

2023-07-14

2025-01-23

Epicrispr Biotechnologies, Inc.

Engineered nucleases and guide rna spacer sequences therefor, and compositions, and methods of use thereof

WO2025049877A1

( en )

2023-08-30

2025-03-06

H. Lee Moffitt Cancer Center And Research Institute Inc.

Chemo-sensitive dominant clone for adaptive therapy

WO2025072446A1

( en )

2023-09-27

2025-04-03

Keith Brown

Cell specific activation

WO2025101646A1

( en )

2023-11-10

2025-05-15

Joseph Fenton Lawler

Trans-splicing methods and compositions for generation of single sex offspring

WO2025171210A1

( en )

2024-02-09

2025-08-14

Arbor Biotechnologies, Inc.

Compositions and methods for gene editing via homology-mediated end joining

CN118500404B

( en )

2024-05-09

2025-12-02

A global path planning method for UAV inspection of power transmission lines based on the fast travel method

WO2025245193A1

( en )

2024-05-21

2025-11-27

The University Of North Carolina At Chapel Hill

Methods and compositions for inhibition of mutant gnaq and gna11

WO2026015832A2

( en )

2024-07-12

2026-01-15

Arbor Biotechnologies, Inc.

Reverse transcriptases and gene editing systems comprising such

WO2026015829A2

( en )

2024-07-12

2026-01-15

Arbor Biotechnologies, Inc.

Small reverse transcriptases and gene editing systems comprising such

Citations (66)

* Cited by examiner, â Cited by third party

Publication number

Priority date

Publication date

Assignee

Title

US20030053990A1

( en )

1998-11-10

2003-03-20

University Of North Carolina At Chapel Hill

Virus vectors and methods of making and administering the same

US20030100707A1

( en )

2001-08-10

2003-05-29

Ahram Biosystems Inc.

System for detecting protease

US20050120395A1

( en )

2001-11-01

2005-06-02

Austin Burt

Method for genetically modifying a target population of an organism

US20110104051A1

( en )

2006-03-27

2011-05-05

The Regents Of The University Of California

Chemically Modified Viral Capsids as Targeted Delivery Vectors for Diagnostic and Therapeutic Agents

WO2014018423A2

( en )

2012-07-25

2014-01-30

The Broad Institute, Inc.

Inducible dna binding proteins and genome perturbation tools and applications thereof

US8697359B1

( en )

2012-12-12

2014-04-15

The Broad Institute, Inc.

CRISPR-Cas systems and methods for altering expression of gene products

WO2014093701A1

( en )

2012-12-12

2014-06-19

The Broad Institute, Inc.

Functional genomics using crispr-cas systems, compositions, methods, knock out libraries and applications thereof

WO2014093694A1

( en )

2012-12-12

2014-06-19

The Broad Institute, Inc.

Crispr-cas nickase systems, methods and compositions for sequence manipulation in eukaryotes

WO2014093718A1

( en )

2012-12-12

2014-06-19

The Broad Institute, Inc.

Methods, systems, and apparatus for identifying target sequences for cas enzymes or crispr-cas systems for target sequences and conveying results thereof

WO2014093712A1

( en )

2012-12-12

2014-06-19

The Broad Institute, Inc.

Engineering of systems, methods and optimized guide compositions for sequence manipulation

WO2014093635A1

( en )

2012-12-12

2014-06-19

The Broad Institute, Inc.

Engineering and optimization of improved systems, methods and enzyme compositions for sequence manipulation

WO2014093595A1

( en )

2012-12-12

2014-06-19

The Broad Institute, Inc.

Crispr-cas component systems, methods and compositions for sequence manipulation

WO2014093655A2

( en )

2012-12-12

2014-06-19

The Broad Institute, Inc.

Engineering and optimization of systems, methods and compositions for sequence manipulation with functional domains

WO2014093622A2

( en )

2012-12-12

2014-06-19

The Broad Institute, Inc.

Delivery, engineering and optimization of systems, methods and compositions for sequence manipulation and therapeutic applications

WO2014093709A1

( en )

2012-12-12

2014-06-19

The Broad Institute, Inc.

Methods, models, systems, and apparatus for identifying target sequences for cas enzymes or crispr-cas systems for target sequences and conveying results thereof

US20140287938A1

( en )

2013-03-15

2014-09-25

The Broad Institute, Inc.

Recombinant virus and preparations thereof

US20140359799A1

( en )

2011-12-23

2014-12-04

Case Western Reserve University

Targeted gene modification using hybrid recombinant adeno-associated virus

WO2014204728A1

( en )

2013-06-17

2014-12-24

The Broad Institute Inc.

Delivery, engineering and optimization of systems, methods and compositions for targeting and modeling diseases and disorders of post mitotic cells

WO2014204725A1

( en )

2013-06-17

2014-12-24

The Broad Institute Inc.

Optimized crispr-cas double nickase systems, methods and compositions for sequence manipulation

WO2014204727A1

( en )

2013-06-17

2014-12-24

The Broad Institute Inc.

Functional genomics using crispr-cas systems, compositions methods, screens and applications thereof

WO2014204729A1

( en )

2013-06-17

2014-12-24

The Broad Institute Inc.

Delivery, use and therapeutic applications of the crispr-cas systems and compositions for targeting disorders and diseases using viral components

WO2014204723A1

( en )

2013-06-17

2014-12-24

The Broad Institute Inc.

Oncogenic models based on delivery and use of the crispr-cas systems, vectors and compositions

WO2014204726A1

(<span itemprop="pr

CRISPR enzymes and systems — The Broad Institute, Inc. (US12305204B2)

Related documents