WO2018209712A1 - Ingénierie d'un système crispr/cas minimal sacas9 pour l'édition de gènes et la régulation transcriptionnelle optimisée par un arn guide amélioré - Google Patents

Ingénierie d'un système crispr/cas minimal sacas9 pour l'édition de gènes et la régulation transcriptionnelle optimisée par un arn guide amélioré Download PDF

Info

Publication number
WO2018209712A1
WO2018209712A1 PCT/CN2017/085202 CN2017085202W WO2018209712A1 WO 2018209712 A1 WO2018209712 A1 WO 2018209712A1 CN 2017085202 W CN2017085202 W CN 2017085202W WO 2018209712 A1 WO2018209712 A1 WO 2018209712A1
Authority
WO
WIPO (PCT)
Prior art keywords
mini
sacas9
naturally occurring
split
dsacas9
Prior art date
Application number
PCT/CN2017/085202
Other languages
English (en)
Inventor
Zhen XIE
Dacheng Ma
Shuguang PENG
Original Assignee
Tsinghua University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tsinghua University filed Critical Tsinghua University
Priority to PCT/CN2017/085202 priority Critical patent/WO2018209712A1/fr
Priority to EP17910146.4A priority patent/EP3625338A4/fr
Priority to CN201780091204.2A priority patent/CN110662835B/zh
Priority to US15/619,518 priority patent/US11674128B2/en
Publication of WO2018209712A1 publication Critical patent/WO2018209712A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]

Definitions

  • the instant application is related to the biological arts, in particular the directed modification of genetic material.
  • the claimed invention is related to improvements upon the Cas9 CRISPR-associated protein and related products thereof.
  • the CRISPR-associated protein 9 (Cas9) discovered from Streptococcus pyogenes is a multi-domain protein, which has been widely used in genome editing and transcriptional control in mammalian cells due to its superior modularity and versatility. Delivering synthetic gene circuits in vivo has been limited due to size constraints particularly with smaller delivery systems with a payload capacity nearly equal to an entire Cas9 complex.
  • dCas9 nuclease deactivated Cas9
  • multiple transcriptional regulatory domains can be recruited to the dCas9 by tagging the dCas9 with a repeating peptide scaffold, or by fusing repeating RNA motifs to the cognate gRNA.
  • biomedical applications of the CRISPR/Cas system require the exploration of new platforms for engineering mammalian synthetic circuits that integrate and process multiple endogenous inputs.
  • the application of CRISPR/Cas therapeutic circuits is also challenging due to the restrictive cargo size of existing viral delivery vehicles.
  • the split Cas9 system can be used in general to bypass the packing limit of the viral delivery vehicles and in the claimed invention dCas9 is split and reconstituted in human cells.
  • One of the challenges of therapeutic applications is to find an optimal delivery system that can carry all CRISPR/Cas9 components to the desired organ or cell population for genetic manipulation.
  • Using the CRISPR/Cas system to greatest potential has been greatly limited by its physical size when incorporated into a viral delivery system.
  • the entire cas9 complex is akin to a computer operating system taking up 95%of available memory leaving only a small portion for synthetic biology programming purposes.
  • the claimed invention represents a substantial improvement over existing CAS9 delivery techniques and includes additional enhancements for genetic control and programming.
  • adeno-associated virus offers high gene transfer efficiency and very low immune response.
  • packaging capacity is confined to 4.7kb to 5kb which is problematic when compared with human optimized Cas9 size at over 4.2 kb with promoter sequences reaching over 5kb.
  • intein-mediated split Cas9 inteins function as protein introns and are excised out of a sequence and join the remaining flaking regions (exteins) with a peptide bond without leaving a scar.
  • split site selection particular attention is given to split sites which are surface exposed due to the sterical need for protein splicing.
  • This system allows the coding sequence of Cas9 to be distributed on a dual-vector or multi-vector system and reconstituted post-translationally.
  • the claimed invention expands the reach of synthetic biology by targeting specific diagnostic and therapeutic applications through improvements in genetic circuitry and higher level genetic circuit delivery enhancements.
  • the claimed embodiments of the invention overcome existing size limitations through optimal splitting of Cas9 allowing for higher level synthetic gene circuitry to be accommodated by smaller delivery systems.
  • the presently claimed invention utilizes downsized Staphylococcus aureus Cas9 variants (mini-SaCas9) which retain DNA binding activity by deleting conserved functional domains.
  • mini-SaCas9 downsized Staphylococcus aureus Cas9 variants
  • FokI nuclease domain is fused to the middle of the split mini-SaCas9 to trigger efficient DNA cleavage.
  • the genetic editing system is small enough to be housed within a single AAV containing the mini-SaCas9 fused with a downsized transactivation domain along with an optimized and compact gRNA expression cassette with an efficient transactivation activity.
  • the claimed invention highlights a practical approach to generate an all in one AAV-CRISPR/Cas9 system with different effector domains for in-vivo applications.
  • the 4.2-kb Cas9 from Streptococcus pyogenes (SpCas9) is split and packaged into two separate AAVs along with the guide RNA (gRNA) expression unit, which allows functional reconstitution of full-length SpCas9 in vivo.
  • Another strategy is to search for natural class 2 CRISPR effectors with a diminished size, such as the 3.2-kb SaCas9 and ⁇ 3-kb CasX identified in uncultivated organisms by using metagenomic datasets.
  • the ⁇ 70-bp glutamine tRNA can be used to replace the ⁇ 250-bp RNA polymerase III promoter to drive expression of the tRNA: gRNA fusion transcript that is cleaved by endogenous tRNase Z to produce the active gRNA.
  • CRISPR/Cas9 system has particular utility in biomedical applications in which viral delivery vehicles with a restrictive cargo size are preferred.
  • Foreseen variants include combination of the split Cas9/dCas9 system with rAAV delivery systems, Cas9/dCas9 activity can be controlled to edit and regulate endogenous genes in vivo.
  • Figure 1 is a diagram of EBFP2 transcription activation assay for the compact Cas9 derivatives fused with the VPR domain.
  • Figure 2 is a diagram of dSpCas9, mini-dSpCas9-1 and mini-dSpCas9-2 domain organization and corresponding gene activation efficiency.
  • Figure 3 is a schematic diagram of domain organization of dSaCas9 and its derivatives and results gene activation efficiency.
  • Figure 4 is a diagram of dAsCpf1 and mini-AsCpf1-1 domain organization and corresponding gene activation efficiency.
  • Figure 5 is a schematic representation of optimized gRNAs and the corresponding gene activation efficiency.
  • Figure 6 is a diagram of Glutamine (Glu) tRNA used as the promoter to express the optimized gRNA-2.
  • Figure 7 is a diagram of domain organization of dSaCas9 variants including split dSaCas9 and split mini-dSaCas9-4 fused with the FokI domain.
  • Figure 8 is a diagram of the EYFP reconstitution assay to demonstrate the DNA cleavage efficiency and illustration of DNA cleavage by the dimeric FokI nuclease fused to split dSaCas9 and split minidSaCas9-4.
  • Figure 9 is a graphical representation of DNA cleavage efficiency by the split dSaCas9 variants with a spacer length ranging from 12-bp to 24-bp.
  • Figure 10 is a schematic representation of VPR, VTR1, VTR2 and VTR3 transcription activation domain and their corresponding gene activation efficiency evaluated by using the EBFP2 reporting system.
  • Figure 11 is a schematic representation of the SpyTag and MoonTag repeating array for transcription activation with corresponding gene activation efficiency data.
  • Figure 12 is a schematic representation of transcription activation by using a single AAV loaded with the compact CRISPR/Cas9 system with a graphical illustration of an activation efficiency of EBFP2.
  • Figure 13 is a schematic representation of packaging mini-Cas9, effector domain, gRNA expression cassette and additional parts in a single AAV vector for transcription activation, DNA cleavage and base editing.
  • Figure 14 is a schematic representation of miniSaCas9-3 and Split SaCas9 and the corresponding miniSaCas9-3 and Split SaCas9 activation efficiency.
  • Figure 15 is a genetic sequence illustration of CCR5 gene illustrating spacer region and corresponding gel agarose graphical data.
  • FIG. 1 is a diagram of EBFP2 transcription activation assay for the compact Cas9 derivatives (101) fused with the VPR domain.
  • the constitutively expressed mKate2 is used as a transfection control.
  • the reporting system is utilized in cultured human embryonic kidney 293 (HEK293) cells.
  • Two mini-dSpCas9 are created by respectively deleting the Cterminal region of REC1 domain (REC-C, ⁇ 501-710) and the HNH domain ( ⁇ 777-891) that may be dispensable for DNA binding activity of the nuclease deactivated Cas9 (dCas9) and respectively fused to the VP64-p65-Rta (VPR) transactivation domain.
  • Figure 2 is a diagram of dSpCas9, mini-dSpCas9-1 and mini-dSpCas9-2 domain organization (201) and graphical illustration of corresponding gene activation efficiency (203) .
  • the two mini-dSpCas9: VPR variants retain more than 50%of transactivation capacity compared to the dSpCas9: VPR.
  • FIG. 3 is a schematic diagram of domain organization of dSaCas9 and its derivatives (301) and graphical illustration (303) depicting gene activation efficiency.
  • the wildtype SaCas9 has been engineered to recognize an altered PAM ( “NNNRRT” ) .
  • the disclosed embodiment constructs the mini-SaCas9-1: VPR by replacing the conserved REC-C domain ( ⁇ 234-444) with a “GGGGSGGGG” linker (GS-linker) , which only retained ⁇ 2.5%transactivation activity of the dSaCas9: VPR.
  • the GS-linker is widely used as a flexible linker, it may still distort the SaCas9 structure.
  • an adjacent residue searching (ARS) protocol is used to search for existing structures between discontinued Cas9 fragments.
  • Replacement of the GS-linker with a “KRRRRHR” (R-linker) from the SaCas9 BH domain that appropriately filled in the REC-C deletion gap by using the ARS protocol results in a 11-fold increase in the transactivation capacity of mini-SaCas9-2.
  • a “GSK” linker is used, derived from a putative gene (Accession number, O67859) in Aquifex aeolicus by using the SAR protocol, which fit the deletion gap of the HNH domain ( ⁇ 479-649) .
  • the 2-kb mini-SaCas9-3 is created by deleting both the REC-C domain and the HNH domain only retained 0.6%transactivation activity over the control.
  • FIG. 4 is a diagram of dAsCpf1 and mini-AsCpf1-1 domain organization (401) and graphical illustration (403) of corresponding gene activation efficiency.
  • a reporter reconstitution assay is used where DNA cleavage can trigger the reconstitution of the active enhanced yellow fluorescent protein (EYFP) reporter gene from the inactive form. Either deleting the REC-C or deleting both the REC-C and HNH domains resulted in a background EYFP expression, suggesting that the domain deletion abolished the DNA cleavage activity of mini-SaCas9 variants.
  • EYFP active enhanced yellow fluorescent protein
  • Figure 5 is a schematic representation of optimized gRNAs (501) and the corresponding graphical illustration (503) of gene activation efficiency.
  • An A-U flip or a U-G conversion can be introduced to disrupt the putative RNA Pol III terminator sequences in the first stem loop of the gRNA scaffold to enhance the efficiency of the dCas9-mediated DNA labeling.
  • the putative RNA Pol III terminator sequences are shown in the box, and the A-U and G-C mutations are labeled in shading at the third and fourth positions.
  • Representative scatter plots of the flow cytometry data are also shown on the right.
  • the constitutively expressed mKate2 is used as a transfection control.
  • the transactivation efficiency of miniSaCas9-3 is increased ⁇ 100-fold by introducing either one or two point mutations in the putative RNA Pol III terminator sequences.
  • FIG. 6 is a diagram of Glutamine (Glu) tRNA (601) used as the promoter to express the optimized gRNA-2.
  • Glu Glutamine
  • HEK293 cells are cotransfected with plasmids expressing the mini-SaCas9-3: VPR and gRNA-2 driven by either the Glu RNA or the U6 promoter.
  • Representative flow cytometry scatter plots (603) are shown on the right.
  • the constitutively expressed iRFP is used as a transfection control. Data are shown as the mean ⁇ SEM fold change of EBFP2 fluorescence from three independent replicates measured by using flow cytometer 48 h after transfection into HEK293 cells.
  • ⁇ 50%transactivation activity is obtained when using glutamine tRNA instead of the U6 promoter to drive gRNA expression.
  • Figure 7 is a diagram of domain organization of dSaCas9 variants (701) including split dSaCas9 and split mini-dSaCas9-4 fused with the FokI domain and additionally includes Fok1 fused to the N terminal of dSaCas9 and mini-dSaCas9-4.
  • Figure 8 is a diagram (801) of the EYFP reconstitution assay to demonstrate the DNA cleavage efficiency and illustration (803) of DNA cleavage by the dimeric FokI nuclease fused to split dSaCas9 or split minidSaCas9-4.
  • EYFP reconstitution assay to evaluate the DNA cleavage efficiency of dSaCas9 derivatives fused with FokI along with two truncated gRNAs that respectively containing 18-nt sequences complementary to the target.
  • DNA cleavage activity is not detected when FokI is fused to the N-terminal of mini-SaCas9-2.
  • dSaCas9 is split at residue 733 and fused the FokI after the splitting point with a triplicate G-linker ( “GGGGS” ) .
  • GGGGS triplicate G-linker
  • the first four residues of the C-terminal fragment ( ⁇ 734-737) that might interfere with the reconstitution of two split fragments is removed.
  • split dSaCas9 or split mini-dSaCas9-4 without the HNH domain results in ⁇ 8%to 30%of the EYFP expression level induced by the wild-type SaCas9, with a spacer ranging from 12 bp to 24 bp in the PAM-out orientation.
  • Figure 9 is a graphical representation (901) of DNA cleavage efficiency by the split dSaCas9 variants with a spacer length ranging from 12-bp to 24-bp.
  • Figure 10 is a schematic representation (1001) of VPR, VTR1, VTR2 and VTR3 transcription activation domain and graphical representation (1003) of the corresponding gene activation efficiency evaluated by using the EBFP2 reporting system.
  • compact transcription activators based on dCas9-VPR are engineered.
  • the entire P65 contains a DNA binding domain in the N-terminal, and two transactivation domains (TA1 and TA2) in the C-terminal.
  • TA1 and TA2 transactivation domains in the C-terminal.
  • only the TA2 and the partial TA1 are included in the tripartite VPR domain.
  • VTR1 is constructed by replacing the P65 domain in the VPR with the TA1 and TA2 domains.
  • the P65 domain in the VPR with two repeats of the TA1 domain is constructed and termed VTR2.
  • the VTR1 and VTR2 domains retain 45%and 21%transactivation efficiency of the VPR domain respectively.
  • VTR3 is additionally depicted and is approximately 200 bp shorter than VTR2 and VTR1 making it an optimal illustrative embodiment.
  • Figure 11 is a schematic representation (1101) of the SpyTag and MoonTag repeating array for transcription activation with graphical representation (1103) for corresponding gene activation efficiency data.
  • SpyTag 13-residue peptide tag derived from Streptococcus pyogenes fibronectin-binding protein
  • a repeating peptide array with a smaller size than the SunTag system is constructed by fusing four tandem repeats of SpyTag to the C-terminal of mini-SaCas9-3 and fusing the SpyCatcher with the VPR domain, allowing spontaneous assembly of a VPR transactivation scaffold in cells.
  • the SpyTag system induces the expression of the enhanced blue fluorescent protein 2 (EBFP2) reporter gene to 100-fold compared to the negative control.
  • EBFP2 enhanced blue fluorescent protein 2
  • WP_054278706 from Streptococcus phocae with 60%sequence similarity to the FbaB.
  • this protein is split similarly to SpyTag and SpyCatcher.
  • a similar scaffold system called the MoonTag system is hereby disclosed and implemented by fusing four tandem repeats of the 13-residue MoonTag to the mini-SaCas9-3 and making a hybrid of the MoonCatcher and VPR domains.
  • the MoonTag system was not orthogonal to the SpyTag system, the MoonTag system is 5-fold more efficient to activate the EBFP2 expression.
  • Figure 12 is a schematic representation (1201) of transcription activation by using a single AAV loaded with the compact CRISPR/Cas9 system with a graphical illustration (1203) of an activation efficiency of EBFP2.
  • transcription activation by using a single AAV loaded with the compact CRISPR/Cas9 system that contains the mini-SaCas9-3: VTR1 and U6-driven optimized gRNA-2 is depicted.
  • the plasmid DNAs that encode the EBFP2 reporter gene and the mKate2 control gene are introduced into HEK293 cells by transient transfection, following by the AAV infection.
  • the lower panel shows the activation efficiency of EBFP2 after infection of the AAV encoding the mini-SaCas9-3: VTR1 and the representative microscopic images (1205) .
  • Scale bar in images represents 200 ⁇ m.
  • a single AAV virus is produced that encoded the constitutively expressed mini-SaCas9-3: VTR1 and the optimized gRNA that targeted the TRE promoter.
  • a TREdriven EBFP2 reporter gene is introduced into HEK293 cells by transient transfection.
  • the AAV infection activated the EBFP2 expression up to ⁇ 130-fold over the negative control in the HEK293 cells, which was also more efficient than AAV virus loaded with the mini-SaCas9-3: VP64.
  • Figure 13 is a schematic representation (1301) of packaging mini-Cas9, effector domain, gRNA expression cassette and additional parts in a single AAV vector for transcription activation, DNA cleavage and base editing.
  • a set of compact Cas9 derivatives are engineered by deleting conserved HNH and/or REC-C domains based on the structural information across variant class 2 CRISPR effectors.
  • a novel strategy to engineer the dimeric gRNA-guided nuclease by splitting the mini-dSaCas9 and fusing the FokI domain right after the split point is disclosed, which can increase the on-target DNA cleavage efficiency and potentially reduce the off-target effect because of a closer proximity of dimeric FokI nuclease to the target sequence.
  • a practical approach is disclosed to load the entire CRISPR/Cas system with different effector domains for transactivation, DNA cleavage and base editing into a single AAV virus.
  • Such an all-in-one AAV-CRISPR/Cas9 system will be particularly appealing in biomedical applications that require safe and efficient delivery in vivo.
  • Figure 14 is a schematic representation (1401) of miniSaCas9-3 and Split SaCas9 and graphical data (1403) of the corresponding miniSaCas9-3 and Split SaCas9 activation efficiency.
  • Figure 15 is a genetic sequence illustration (1501) of CCR5 gene illustrating spacer region and gel electrophoresis graphical data (1503) illustrating CCR5 gene relative percentage of cleavage.
  • the terms “comprises, ” “comprising, ” “includes, ” “including, ” “has, ” “having, ” or any other variation thereof, are intended to cover a non-exclusive inclusion.
  • a process, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such process, article, or apparatus.
  • any examples or illustrations given herein are not to be regarded in any way as restrictions on, limits to, or express definitions of any term or terms with which they are utilized. Instead, these examples or illustrations are to be regarded as being described with respect to one particular embodiment and as being illustrative only. Those of ordinary skill in the art will appreciate that any term or terms with which these examples or illustrations are utilized will encompass other embodiments which may or may not be given therewith or elsewhere in the specification and all such embodiments are intended to be included within the scope of that term or terms. Language designating such nonlimiting examples and illustrations includes, but is not limited to: “for example, ” “for instance, ” “e.g., ” and “in one embodiment. ”
  • the claimed invention has industrial applicability in biomedical and industrial biotechnology applications.
  • the improved Cas9 system provides greater gene editing and regulatory control capabilities over traditional Cas9 systems.
  • mini-SaCas9-2 VPR
  • mini-dSaCas9-6N AID
  • VTR3 SEQUENCE

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne la régulation programmable et précise de fonctions Cas9 en utilisant un ensemble de dérivés de Cas9 réduits créés par suppression de domaines HNH et/ou REC-C conservés sur la base des informations structurales à travers des effecteurs CRISPR de classe 2 variants. L'invention concerne également une nouvelle stratégie pour l'ingénierie de la nucléase guidée par gARN dimère par la division du mini-dSaCas9 et la fusion du domaine FokI juste après le point de division pour augmenter l'efficacité de clivage d'ADN sur cible et potentiellement réduire l'effet hors cible en raison d'une proximité plus proche d'une nucléase FokI dimère de la séquence cible. En combinant la cassette d'expression de gARN optimisée et compacte et les dérivés de SaCas9 réduits, l'ensemble du système CRISPR/Cas avec différents domaines effecteurs pour transactivation, clivage d'ADN et édition de base est chargé en un seul virus AAV. Un tel système AAV-CRISPR/Cas9 tout-en-un sera particulièrement attrayant dans des applications biomédicales qui nécessitent une administration sûre et efficace in vivo.
PCT/CN2017/085202 2016-12-12 2017-05-19 Ingénierie d'un système crispr/cas minimal sacas9 pour l'édition de gènes et la régulation transcriptionnelle optimisée par un arn guide amélioré WO2018209712A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
PCT/CN2017/085202 WO2018209712A1 (fr) 2017-05-19 2017-05-19 Ingénierie d'un système crispr/cas minimal sacas9 pour l'édition de gènes et la régulation transcriptionnelle optimisée par un arn guide amélioré
EP17910146.4A EP3625338A4 (fr) 2017-05-19 2017-05-19 Ingénierie d'un système crispr/cas minimal sacas9 pour l'édition de gènes et la régulation transcriptionnelle optimisée par un arn guide amélioré
CN201780091204.2A CN110662835B (zh) 2017-05-19 2017-05-19 工程化改造用于由增强的指导RNA优化的基因编辑和转录调节的最小化SaCas9 CRISPR/Cas系统
US15/619,518 US11674128B2 (en) 2016-12-12 2017-06-11 Engineering of a minimal SaCas9 CRISPR/Cas system for gene editing and transcriptional regulation optimized by enhanced guide RNA

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/085202 WO2018209712A1 (fr) 2017-05-19 2017-05-19 Ingénierie d'un système crispr/cas minimal sacas9 pour l'édition de gènes et la régulation transcriptionnelle optimisée par un arn guide amélioré

Publications (1)

Publication Number Publication Date
WO2018209712A1 true WO2018209712A1 (fr) 2018-11-22

Family

ID=64273211

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/085202 WO2018209712A1 (fr) 2016-12-12 2017-05-19 Ingénierie d'un système crispr/cas minimal sacas9 pour l'édition de gènes et la régulation transcriptionnelle optimisée par un arn guide amélioré

Country Status (3)

Country Link
EP (1) EP3625338A4 (fr)
CN (1) CN110662835B (fr)
WO (1) WO2018209712A1 (fr)

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110423736A (zh) * 2019-08-16 2019-11-08 中国人民解放军陆军军医大学第一附属医院 碱基编辑工具及其应用以及在真核细胞内进行宽窗口和无序列偏好性碱基编辑的方法
CN110551760A (zh) * 2019-08-08 2019-12-10 复旦大学 一种CRISPR/Sa-SeqCas9基因编辑系统和其应用
WO2019235627A1 (fr) 2018-06-08 2019-12-12 株式会社モダリス PROTÉINE Cas9 MODIFIÉE ET UTILISATION DE CELLE-CI
WO2020236967A1 (fr) * 2019-05-20 2020-11-26 The Broad Institute, Inc. Mutant de délétion de crispr-cas aléatoire
US11098297B2 (en) 2017-06-09 2021-08-24 Editas Medicine, Inc. Engineered Cas9 nucleases
US11236313B2 (en) 2016-04-13 2022-02-01 Editas Medicine, Inc. Cas9 fusion molecules, gene editing systems, and methods of use thereof
CN114026226A (zh) * 2019-04-26 2022-02-08 株式会社图尔金 靶特异性crispr突变体
US11390884B2 (en) 2015-05-11 2022-07-19 Editas Medicine, Inc. Optimized CRISPR/cas9 systems and methods for gene editing in stem cells
US11499151B2 (en) 2017-04-28 2022-11-15 Editas Medicine, Inc. Methods and systems for analyzing guide RNA molecules
US11597924B2 (en) 2016-03-25 2023-03-07 Editas Medicine, Inc. Genome editing systems comprising repair-modulating enzyme molecules and methods of their use
US11667911B2 (en) 2015-09-24 2023-06-06 Editas Medicine, Inc. Use of exonucleases to improve CRISPR/CAS-mediated genome editing
US11866726B2 (en) 2017-07-14 2024-01-09 Editas Medicine, Inc. Systems and methods for targeted integration and genome editing and detection thereof using integrated priming sites
US11911415B2 (en) 2015-06-09 2024-02-27 Editas Medicine, Inc. CRISPR/Cas-related methods and compositions for improving transplantation
US11946039B2 (en) 2020-03-31 2024-04-02 Metagenomi, Inc. Class II, type II CRISPR systems
EP3966323A4 (fr) * 2019-05-06 2024-04-24 The Regents Of The University Of Michigan Thérapie ciblée

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112553246A (zh) * 2020-12-08 2021-03-26 安徽省农业科学院水稻研究所 一种基于CRISPR-SaCas9系统的高效基因组编辑载体及其应用
CN113717961B (zh) * 2021-09-10 2023-05-05 成都赛恩吉诺生物科技有限公司 一种融合蛋白及其多核苷酸、碱基编辑器及其在药物制备中的应用
WO2023165598A1 (fr) * 2022-03-04 2023-09-07 益杰立科(上海)生物科技有限公司 Protéine cas, son utilisation et procédé associé
CN116621947B (zh) * 2023-07-18 2023-11-07 北京智源人工智能研究院 一种基于索烃骨架的拓扑蛋白质、制备方法及应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104520429A (zh) * 2012-03-20 2015-04-15 维尔纽斯大学 通过Cas9-crRNA复合物的RNA指导的DNA裂解
WO2016205759A1 (fr) * 2015-06-18 2016-12-22 The Broad Institute Inc. Modification et optimisation de systèmes, de méthodes, d'enzymes et d'échafaudages guides d'orthologues de cas9 et variant pour la manipulation de séquences
WO2016205613A1 (fr) * 2015-06-18 2016-12-22 The Broad Institute Inc. Mutations d'enzyme crispr qui réduisent les effets non ciblés

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2014235794A1 (en) * 2013-03-14 2015-10-22 Caribou Biosciences, Inc. Compositions and methods of nucleic acid-targeting nucleic acids
JP6793547B2 (ja) * 2013-12-12 2020-12-02 ザ・ブロード・インスティテュート・インコーポレイテッド 最適化機能ＣＲＩＳＰＲ−Ｃａｓ系による配列操作のための系、方法および組成物
US20160304893A1 (en) * 2013-12-13 2016-10-20 Cellectis Cas9 nuclease platform for microalgae genome engineering
EP3307762B1 (fr) * 2015-06-12 2021-12-15 The Regents of The University of California Variants de cas9 rapporteurs et leurs procédés d'utilisation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104520429A (zh) * 2012-03-20 2015-04-15 维尔纽斯大学 通过Cas9-crRNA复合物的RNA指导的DNA裂解
WO2016205759A1 (fr) * 2015-06-18 2016-12-22 The Broad Institute Inc. Modification et optimisation de systèmes, de méthodes, d'enzymes et d'échafaudages guides d'orthologues de cas9 et variant pour la manipulation de séquences
WO2016205613A1 (fr) * 2015-06-18 2016-12-22 The Broad Institute Inc. Mutations d'enzyme crispr qui réduisent les effets non ciblés

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KAYA H ET AL.: "A split staphylococcus aureus Cas9 as a compact genome-editing tool in plants", PLANT CELL PHYSIOL., vol. 58, no. 4, 1 April 2017 (2017-04-01), XP055538981, ISSN: 0032-0781 *
See also references of EP3625338A4 *
STERNBERG SH ET AL.: "Conformational control of DNA target cleavage by CRISPR-Cas9", NATURE, vol. 527, no. 7576, 5 November 2015 (2015-11-05), XP055535411, ISSN: 1476-4687 *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11390884B2 (en) 2015-05-11 2022-07-19 Editas Medicine, Inc. Optimized CRISPR/cas9 systems and methods for gene editing in stem cells
US11911415B2 (en) 2015-06-09 2024-02-27 Editas Medicine, Inc. CRISPR/Cas-related methods and compositions for improving transplantation
US11667911B2 (en) 2015-09-24 2023-06-06 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
US11236313B2 (en) 2016-04-13 2022-02-01 Editas Medicine, Inc. Cas9 fusion molecules, gene editing systems, and methods of use thereof
US11499151B2 (en) 2017-04-28 2022-11-15 Editas Medicine, Inc. Methods and systems for analyzing guide RNA molecules
US11098297B2 (en) 2017-06-09 2021-08-24 Editas Medicine, Inc. Engineered Cas9 nucleases
US11866726B2 (en) 2017-07-14 2024-01-09 Editas Medicine, Inc. Systems and methods for targeted integration and genome editing and detection thereof using integrated priming sites
WO2019235627A1 (fr) 2018-06-08 2019-12-12 株式会社モダリス PROTÉINE Cas9 MODIFIÉE ET UTILISATION DE CELLE-CI
KR20210025046A (ko) 2018-06-08 2021-03-08 가부시키가이샤 모달리스 개변된 Cas9 단백질 및 그의 용도
CN114026226A (zh) * 2019-04-26 2022-02-08 株式会社图尔金 靶特异性crispr突变体
EP3966323A4 (fr) * 2019-05-06 2024-04-24 The Regents Of The University Of Michigan Thérapie ciblée
WO2020236967A1 (fr) * 2019-05-20 2020-11-26 The Broad Institute, Inc. Mutant de délétion de crispr-cas aléatoire
CN110551760B (zh) * 2019-08-08 2022-11-18 复旦大学 CRISPR/Sa-SeqCas9基因编辑系统及其应用
CN110551760A (zh) * 2019-08-08 2019-12-10 复旦大学 一种CRISPR/Sa-SeqCas9基因编辑系统和其应用
CN110423736A (zh) * 2019-08-16 2019-11-08 中国人民解放军陆军军医大学第一附属医院 碱基编辑工具及其应用以及在真核细胞内进行宽窗口和无序列偏好性碱基编辑的方法
US11946039B2 (en) 2020-03-31 2024-04-02 Metagenomi, Inc. Class II, type II CRISPR systems

Also Published As

Publication number Publication date
CN110662835A (zh) 2020-01-07
EP3625338A1 (fr) 2020-03-25
CN110662835B (zh) 2023-04-28
EP3625338A4 (fr) 2021-01-20

Similar Documents

Publication Publication Date Title
WO2018209712A1 (fr) Ingénierie d'un système crispr/cas minimal sacas9 pour l'édition de gènes et la régulation transcriptionnelle optimisée par un arn guide amélioré
US11674128B2 (en) Engineering of a minimal SaCas9 CRISPR/Cas system for gene editing and transcriptional regulation optimized by enhanced guide RNA
US11667677B2 (en) Inducible, tunable, and multiplex human gene regulation using CRISPR-Cpf1
US20200354704A1 (en) RNA-Guided Targeting of Genetic and Epigenomic Regulatory Proteins to Specific Genomic Loci
US20220025347A1 (en) Variants of CRISPR from Prevotella and Francisella 1 (Cpf1)
JP7275043B2 (ja) 増大したｈＡＴファミリートランスポゾン媒介遺伝子導入ならびに関連する組成物、システムおよび方法
CN1981047B (zh) Dna克隆载体质粒的动态载体装配方法
EP2879693B1 (fr) Protéines de fusion modifiant l'adn et leurs procédés d'utilisation
JP5374584B2 (ja) 改良タンパク質発現系
Tremblay et al. Transcription activator-like effector proteins induce the expression of the frataxin gene
WO2003008437A2 (fr) Systemes d'expression de proteines et d'acides nucleiques
CN112543808A (zh) 增强的hAT家族转座子介导的基因转移及相关组合物、系统和方法
CN111117985A (zh) 一种拆分Cas9的方法及其应用
WO2020123512A1 (fr) Régulation à médiation anti-crispr d'édition de génome et de circuits synthétiques dans des cellules eucaryotes
CN112708605A (zh) 一个Cas9蛋白拆分得到的蛋白组及其应用
Luke et al. Development of antibiotic-free selection system for safer DNA vaccination
WO2020032057A1 (fr) Nouvel activateur de transcription
US20200325484A1 (en) Enhancing gene expression by linking self-amplifying transcription factor with viral 2A-like peptide
WO2023165597A1 (fr) Compositions et procédés d'édition du génome
WO2021158658A9 (fr) Crispr-cas fractionné pour intégration de signaux biologiques
WO2023235879A1 (fr) Procédés d'édition de génome d'ovocytes
WO2022197693A2 (fr) Système d'expression génique stable dans des lignées cellulaires et méthodes de fabrication et d'utilisation correspondantes
EP2369000A1 (fr) Production de peptides et de protéines par accumulation dans les mitochondries
AU2002354964A1 (en) Protein and nucleic acid expression systems
MXPA98001617A (en) Method and apparatus for specific recombination of si

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17910146

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2017910146

Country of ref document: EP

Effective date: 20191219