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WO2024138481A1 - Système crispr-cas de type i-f2 pour la régulation et le contrôle de la transcription de gènes de cellules eucaryotes, et son utilisation - Google Patents

Système crispr-cas de type i-f2 pour la régulation et le contrôle de la transcription de gènes de cellules eucaryotes, et son utilisation Download PDF

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WO2024138481A1
WO2024138481A1 PCT/CN2022/143090 CN2022143090W WO2024138481A1 WO 2024138481 A1 WO2024138481 A1 WO 2024138481A1 CN 2022143090 W CN2022143090 W CN 2022143090W WO 2024138481 A1 WO2024138481 A1 WO 2024138481A1
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向华
郭京
龚路遥
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Institute of Microbiology of CAS
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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Definitions

  • the Cas6 is selected from any one of A4) to A6):
  • A6 a fusion protein obtained by connecting a tag to the N-terminus and/or C-terminus of A4) or A5);
  • a protein related to Cas5 obtained by substitution and/or deletion and/or addition of amino acid residues in the amino acid sequences shown in A8) and A7) having more than 80% identity with the protein shown in A7);
  • A3 a fusion protein obtained by connecting a tag to the N-terminus and/or C-terminus of A1) or A2);
  • the Cas6 is selected from any one of A4) to A6):
  • a protein related to Cas5 obtained by substitution and/or deletion and/or addition of amino acid residues in the amino acid sequences shown in A8) and A7) having more than 80% identity with the protein shown in A7);
  • the complex also includes a transcriptional regulatory factor, and the transcriptional regulatory factor is connected to Cas7 or Cas6 or Cas5 via a connecting peptide.
  • the Cas7 is selected from any one of A1) to A3):
  • A2 a protein related to Cas7 obtained by substitution and/or deletion and/or addition of amino acid residues of the amino acid sequence shown in A1) having more than 80% identity with the protein shown in A1);
  • A3 a fusion protein obtained by connecting a tag to the N-terminus and/or C-terminus of A1) or A2);
  • the complex also includes a transcriptional regulatory factor, and the transcriptional regulatory factor is connected to Cas7 or Cas6 or Cas5 via a connecting peptide.
  • nucleic acid molecule is any one of the following:
  • the coding sequence is the DNA molecule (VPR) of sequence 2 1108-2640 in the sequence list;
  • the Cas7 is selected from any one of A1) to A3):
  • A9 a fusion protein obtained by connecting a tag to the N-terminus and/or C-terminus of A7) or A8);
  • B2 an expression cassette containing the nucleic acid molecule described in B1);
  • the coding sequence is the DNA molecule (Cas5) of sequence 2 3403-4500 in the sequence list;
  • M2 A method for single-base editing of genes in eukaryotic cells, the method comprising introducing into a recipient eukaryotic cell a gene encoding a complex for regulating eukaryotic cell gene transcription, a gene encoding a crRNA, and a gene encoding a deaminase, to achieve single-base editing of genes in eukaryotic cells; the crRNA targets a target gene in a eukaryotic cell;
  • M3 A method for epigenetic modification of eukaryotic cells, the method comprising introducing a gene encoding the complex for regulating eukaryotic cell gene transcription and a gene encoding crRNA into a recipient eukaryotic cell to achieve epigenetic modification of the eukaryotic cell; the crRNA targets a target gene in the eukaryotic cell;
  • M1, M2) and M3) the complexes regulating eukaryotic cell gene transcription include Cas7, Cas6 and Cas5,
  • the Cas7 is selected from any one of A1) to A3):
  • amino acid sequence of the protein is SEQ ID No. 3, positions 12-347,
  • A2 a protein related to Cas7 obtained by substitution and/or deletion and/or addition of amino acid residues in the amino acid sequence shown in A1), which has more than 80% identity with the protein shown in A1),
  • A3 a fusion protein obtained by connecting a tag to the N-terminus and/or C-terminus of A1) or A2);
  • the Cas6 is selected from any one of A4) to A6):
  • A6 a fusion protein obtained by connecting a tag to the N-terminus and/or C-terminus of A4) or A5);
  • A8 and a protein related to Cas5 obtained by substitution and/or deletion and/or addition of amino acid residues in the amino acid sequence shown in A7), which has more than 80% identity with the protein shown in A7),
  • A9 and a fusion protein obtained by connecting a tag to the N-terminus and/or C-terminus of A7) or A8).
  • the transcriptional regulatory factor is connected to the C-terminus (carboxyl terminus) of Cas7 via a connecting peptide.
  • the amino acid sequence of the connecting peptide in the complex is SEQ ID No. 3, positions 348-367.
  • Transcriptional regulation refers to the process of changing the level of gene expression by changing the transcription rate. Transcriptional regulation can control when transcription occurs and how much RNA is produced. Genes transcribed by RNA polymerase can be regulated by at least five mechanisms: 1 Specific factors change the specificity of RNA polymerase for a specific promoter or a set of promoters, causing RNA polymerase to bind to these promoters more or less (such as the sigma factor used in prokaryotic transcription). 2 Repressor factors bind to non-coding sequences on the DNA chain that are close to or cover the promoter region, preventing RNA polymerase from entering this chain smoothly, thereby hindering gene expression.
  • RNA polymerase place RNA polymerase at the start position of the protein coding sequence and then release the polymerase to transcribe mRNA.
  • Activator factors enhance the interaction between RNA polymerase and specific promoters, promoting gene expression. Activator factors achieve this effect by enhancing the attraction of RNA polymerase to promoters, which is achieved by interacting with RNA polymerase subunits or indirectly by changing DNA structure.
  • 5 Enhancers are sites located on the DNA helix that bind to activating factors to bend the DNA so that specific promoters are directed toward the initiation complex.
  • the transcription regulatory factor may be a transcription activator, a transcription repressor, or a transcription enhancer.
  • the transcriptional regulatory factor is a transcriptional activator VPR, and the VPR is selected from any one of A10) to A12):
  • A11 a protein related to the transcriptional regulatory factor VPR obtained by substitution and/or deletion and/or addition of amino acid residues in the amino acid sequence shown in A11);
  • A12 a fusion protein obtained by connecting a tag to the N-terminus and/or C-terminus of A10) or A11).
  • the universal template of the crRNA expression cassette is the DNA molecule shown in SEQ ID No.1.
  • hU6-MosBbsI is a DNA molecule with a nucleotide sequence of SEQ ID No.1, where positions 1-249 of SEQ ID No.1 are the hU6 promoter sequence, positions 252-279 and 296-323 of SEQ ID No.1 are repeat sequences, and positions 282-293 are two BbsI restriction enzyme recognition sites.
  • the spacer fragment of the target gene replaces the nucleotide sequence between the two repeat sequences of SEQ ID No.1 to obtain a DNA molecule expressing crRNA targeting the target gene.
  • the complex fused with a transcriptional regulatory factor can regulate the transcription level of genes in eukaryotic cells.
  • the complex fused with a deaminase can be used for single-base editing of eukaryotic cell genes.
  • the complex fused with an epigenetically modified protein can be used for epigenetic modification of eukaryotic cell genes.
  • Figure 2 is a diagram showing the experimental results of Example 2 of the present invention, showing the effect of VPR transcription factor gene activation by fusing different subunits.
  • LB medium weigh 10g sodium chloride, 10g tryptone, 5g yeast extract, add distilled water, stir to dissolve, dilute to 1L, and sterilize with high pressure steam at 121°C for 20min. 1.2% agar powder should be added to the solid medium. The final concentration of ampicillin in the LB medium containing ampicillin (Amp) resistance is 100 ⁇ g/mL.
  • the present invention screened out an I-F2 type system derived from the Moraxella osloensis CCUG 350 strain, whose effector proteins include Cas5, Cas6, and Cas7 (the Cas5 and Cas7 proteins of the I-F2 system have no significant homology with other reported Cas proteins).
  • the size of the Cas5 gene is 1101bp
  • the size of the Cas6 gene is 597bp
  • the size of the Cas7 gene is 1011bp, totaling 2709bp.
  • the transcriptional regulatory factor can be a transcriptional activator or a transcriptional repressor, and the transcriptional activator VPR is taken as an example in the embodiments provided in this application
  • a transcriptional regulatory tool with universal applicability and high efficiency in eukaryotic cells is developed, which is also the smallest I-type system for realizing transcriptional regulation in eukaryotic cells.
  • the I-F2 type CRISPR-Cas system of Moraxella osloensis CCUG 350 includes Cas7, Cas6, Cas5 and a transcriptional regulatory factor coupled to the Cas protein.
  • the transcriptional regulatory factor is a transcriptional activator VPR, which is connected to the C-terminus of Cas7.
  • Cas7, Cas6, Cas5 and the transcriptional regulatory factor are expressed in eukaryotic cells in the form of the same transcript, and its coding sequence is SEQ ID No. 2, positions 10-4500.
  • HBB hemoglobin subunit beta NM_000518.5
  • ASCL1 achaete-scute family bHLH transcription factor 1 NM_004316.4
  • ACTC1 actin alpha cardiac muscle 1 NM_005159.5
  • MIAT myocardial infarction associated transcript NR_003491.3
  • NEUROD1 neuronal differentiation 1 NM_002500.5
  • the ligation reaction system (10 ⁇ L) was as follows: 7 ⁇ L annealed synthetic spacer fragment, 1 ⁇ L pcDNA3.1-hU6-MosBbsI, 1 ⁇ L 10 ⁇ T4 DNA ligase Buffer, 1 ⁇ L T4 DNA ligase. React at 25°C for 1h.
  • the ligation product was transformed into EC DH5 ⁇ competent cells and cultured overnight at 37°C. After a single clone grew out, colony PCR was performed using primer pair pcDNA-seq-F/pcDNA-seq-R (Table 2).
  • the PCR-positive colony was inoculated into 15 mL of LB liquid medium containing Amp (final concentration 100 mg/L) resistance and cultured overnight.
  • the plasmid was extracted and sequenced to obtain the pcDNA3.1-hU6-crRNA plasmid (as shown in Figure 1, B).
  • the obtained pcDNA3.1-hU6-crRNA plasmid was named: pcDNA3.1-hU6-crRNA-IL1B.
  • pcDNA3.1-hU6-crRNA-IL1B is a recombinant expression vector obtained by inserting a DNA molecule having a nucleotide sequence of positions 5-36 of primer IL1B-F between two repeat sequences of vector pcDNA3.1-hU6-MosBbsI, while keeping the other nucleotide sequences of vector pcDNA3.1-hU6-MosBbsI unchanged.
  • the human codon-optimized Cas7VPR, Cas6VPR, and Cas5VPR fragments of the I-F2 type system of Moraxella osloensis CCUG 350 strain were synthesized by a reagent company.
  • the fragment between the KpnI and XhoI restriction recognition sites of the backbone vector pcDNA3.1 plasmid was replaced with Cas7VPR.
  • the other nucleotide sequences of the plasmid pcDNA3.1 were kept unchanged to obtain the recombinant expression vector, namely pcDNA3.1-cmv-Cas7VPR.
  • pcDNA3.1-cmv-Cas6VPR and pcDNA3.1-cmv-Cas5VPR refer to pcDNA3.1-cmv-Cas7VPR, the only difference is that Cas7VPR is replaced by Cas6VPR or Cas5VPR, respectively.
  • the nucleotide sequence of the Cas7VPR fragment is shown in Sequence 2 in the sequence table.
  • Positions 1-9 of SEQ ID No. 2 are kozak sequences, positions 10-39 encode penetrating peptide 1, positions 40-1047 encode Cas7, positions 1048-1107 encode connecting peptide 1, positions 1108-2640 encode VPR, positions 2641-2694 encode T2A-1, positions 2695-2724 encode penetrating peptide 2, positions 2725-3318 encode Cas6, positions 3319-3372 encode T2A-2, positions 3373-3402 encode penetrating peptide 3, and positions 3403-4500 encode Cas5.
  • the fragment between the KpnI and XhoI restriction sites of the pcDNA3.1 vector (the small fragment between the KpnI and XhoI restriction sites) was replaced with the DNA molecule whose nucleotide sequence is shown in SEQ ID No. 2, while keeping the other nucleotide sequences of the pcDNA3.1 vector unchanged, to obtain the pcDNA3.1-cmv-Cas7VPR plasmid.
  • the pcDNA3.1-cmv-Cas7VPR plasmid (A in Figure 1) can express three proteins, namely, Cas7-VPR fusion protein, Cas6 and Cas5.
  • the amino acid sequence of Cas7-VPR fusion protein is SEQ ID No.3, wherein positions 1-11 of SEQ ID No.3 are the amino acid sequence of penetrating peptide 1, positions 12-347 are the amino acid sequence of Cas7, positions 348-367 are the amino acid sequence of connecting peptide 1, and positions 368-878 are the amino acid sequence of VPR.
  • the amino acid sequence of Cas6 is positions 1-198 of SEQ ID No.5, and the amino acid sequence of Cas5 is positions 1-366 of SEQ ID No.7.
  • Cas7-VPR fusion protein, Cas6 and Cas5 form a complex to exert transcriptional regulation function.
  • the fragment between the KpnI and XhoI restriction sites of the pcDNA3.1 vector (the small fragment between the KpnI and XhoI restriction sites) was replaced with a DNA molecule having a nucleotide sequence shown in SEQ ID No. 4, while keeping other nucleotide sequences of the pcDNA3.1 vector unchanged, to obtain the pcDNA3.1-cmv-Cas6VPR plasmid.
  • the pcDNA3.1-cmv-Cas5VPR plasmid (D in FIG. 1 ) can express three proteins, namely, Cas7, Cas6, and Cas5-VPR fusion protein, wherein the amino acid sequence of Cas7 is SEQ ID No.3, positions 12-347, the amino acid sequence of Cas6 is SEQ ID No.5, positions 1-198, and the amino acid sequence of Cas5-VPR fusion protein is SEQ ID No.7, wherein positions 1-366 of SEQ ID No.7 are the amino acid sequence of Cas5, positions 367-386 are the amino acid sequence of connecting peptide 1, and positions 387-897 are the amino acid sequence of VPR.
  • Cas7, Cas6, and Cas5-VPR fusion protein form a complex to exert transcriptional regulation function.
  • the repeat sequence of the Mos350 and Mos41 systems is the same, and the same crRNA expression plasmid can be used.
  • the pcDNA3.1-hU6-Mos-IL1B plasmid is the pcDNA3.1-hU6-crRNA-IL1B in Example 2.
  • the Ain-IL1B fragment is formed by annealing Ain-IL1B-F and Ain-IL1B-R
  • the Asp161-IL1B fragment is formed by annealing Asp161-IL1B-F and Asp161-IL1B-R
  • the Asp185-IL1B fragment is formed by annealing Asp185-IL1B-F and Asp185-IL1B-R.
  • the crRNA expression plasmid pcDNA3.1-hU6-crRNA step refers to 2.1 in Example 2, and the annealing primer sequence used is shown in Table 2.
  • pcDNA3.1-hU6-Ain-IL1B, pcDNA3.1-hU6-Asp161-IL1B, and pcDNA3.1-hU6-Asp185-IL1B were constructed.
  • pcDNA3.1-hU6-Ain-IL1B is a recombinant expression vector obtained by replacing the fragment between the two repeat sequences of the vector pcDNA3.1-hU6-AinBbsI with a DNA molecule having the nucleotide sequence of positions 5-36 of Ain-IL1B-F, while keeping the other nucleotide sequences of the vector pcDNA3.1-hU6-AinBbsI unchanged;
  • pcDNA3.1-hU6-Asp185-IL1B is a recombinant expression vector obtained by replacing the fragment between the two repeat sequences of the vector pcDNA3.1-hU6-MosBbsI with a DNA molecule having the nucleotide sequence of positions 5-36 of Asp185-IL1B-F, while keeping the other nucleotide sequences of the vector pcDNA3.1-hU6-MosBbsI unchanged.
  • the Mos41VPR fragment with human codon optimization of the I-F2 type system of Moraxella osloensis KMC41 strain, the AinVPR fragment with human codon optimization of the I-F2 type system of Acinetobacter indicus MMS9-2 strain, the Asp161VPR fragment with human codon optimization of the I-F2 type system of Acinetobacter sp.C16S1 strain, and the Asp161VPR fragment with human codon optimization of the I-F2 type system of Acinetobacter sp.185 strain were synthesized by the reagent company.
  • the nucleotide sequence of the Mos41VPR fragment is shown in sequence 11 in the sequence table.
  • SEQ ID No. 11 positions 1-9 are kozak sequences, positions 10-39 encode penetrating peptide 1, positions 40-990 encode Mos41Cas7, positions 991-1050 encode connecting peptide 1, positions 1051-2583 encode VPR, positions 2584-2637 encode T2A-1, positions 2638-2667 encode penetrating peptide 2, positions 2668-3261 encode Mos41Cas6, positions 3262-3315 encode T2A-2, positions 3316-3345 encode penetrating peptide 3, and positions 3346-4365 encode Mos41Cas5.
  • the pcDNA3.1-cmv-Mos41VPR plasmid can express three proteins, namely Mos41Cas7-VPR, Mos41Cas6 and Mos41Cas5.
  • the amino acid sequence of Mos41Cas7-VPR is SEQ ID No.12, positions 12-859, wherein positions 12-328 of SEQ ID No.12 are the amino acid sequence of Mos41Cas7, positions 329-348 are the amino acid sequence of connecting peptide 1, and positions 349-859 are the amino acid sequence of VPR.
  • the amino acid sequence of Mos41Cas6 is SEQ ID No.12, positions 888-1085
  • the amino acid sequence of Mos41Cas5 is SEQ ID No.12, positions 1114-1453.
  • the nucleotide sequence of the AinVPR fragment is shown in sequence 13 in the sequence table.
  • Positions 1-9 of SEQ ID No. 13 are kozak sequences, positions 10-39 encode penetrating peptide 1, positions 40-1026 encode AinCas7, positions 1027-1086 encode connecting peptide 1, positions 1087-2619 encode VPR, positions 2620-2673 encode T2A-1, positions 2674-2703 encode penetrating peptide 2, positions 2704-3303 encode AinCas6, positions 3304-3357 encode T2A-2, positions 3358-3387 encode penetrating peptide 3, and positions 3389-4419 encode AinCas5.
  • the pcDNA3.1-cmv-AinVPR plasmid can express three proteins, namely AinCas7-VPR, AinCas6 and AinCas5.
  • the amino acid sequence of AinCas7-VPR is SEQ ID No.14, positions 12-871, wherein positions 12-340 of SEQ ID No.14 are the amino acid sequence of AinCas7, positions 341-360 are the amino acid sequence of connecting peptide 1, and positions 361-871 are the amino acid sequence of VPR.
  • the amino acid sequence of AinCas6 is SEQ ID No.14, positions 900-1099
  • the amino acid sequence of AinCas5 is SEQ ID No.14, positions 1128-1470.
  • nucleotide sequence of the Asp161VPR fragment is shown in sequence 15 in the sequence table.
  • positions 1-9 are kozak sequences, positions 10-39 encode penetrating peptide 1, positions 40-990 encode Asp161Cas7, positions 991-1089 encode connecting peptide 1, positions 1090-2622 encode VPR, positions 2623-2676 encode T2A-1, positions 2677-2706 encode penetrating peptide 2, positions 2707-3315 encode Asp161Cas6, positions 3316-3369 encode T2A-2, positions 3370-3399 encode penetrating peptide 3, and positions 3400-4392 encode Asp161Cas5.
  • pcDNA3.1-hU6-crRNA-ASCL is a recombinant expression vector obtained by replacing the fragment between the two repeat sequences of the vector pcDNA3.1-hU6-MosBbsI with a DNA molecule having the nucleotide sequence of positions 5-36 of ASCL-F, while keeping the other nucleotide sequences of the vector pcDNA3.1-hU6-MosBbsI unchanged;
  • FIG. 4 The experimental results are shown in Figure 4, where Target represents the relative expression level of the gene in the experimental group, and Non Target represents the relative expression level of the gene in the control group.
  • the first row of Figure 4 shows the relative expression levels of the HBB gene, MIAT gene, and ACTC gene in the control group and the experimental group from left to right, respectively, and the second row shows the relative expression levels of the ASCL gene and NEUROD gene from left to right.
  • the experimental results in Figure 4 show that the tool has a significant transcriptional activation effect when targeting the target sites upstream of the promoters of multiple different genes, indicating that the tool can be widely used for transcriptional activation of genes in eukaryotic cells.
  • Example 5 Using this tool to simultaneously target multiple sites in the promoter region of the same gene in HEK293T cells to further improve the transcriptional activation level
  • HBB and NEUROD genes of HEK293T cells were selected as target genes.
  • Two target sites of HBB gene were selected, namely T1 and T2.
  • the spacer fragment of T1 was synthesized by annealing HBB-F1 and HBB-R1 in Table 4, and the spacer fragment of T2 was synthesized by annealing HBB-F2 and HBB-R2 in Table 4.
  • Four target sites of NEUROD gene were selected, namely T1, T2, T3 and T4.
  • the spacer fragment of T1 was synthesized by annealing NEUROD-F1 and NEUROD-R1 in Table 4, the spacer fragment of T2 was synthesized by annealing NEUROD-F2 and NEUROD-R2 in Table 4, the spacer fragment of T3 was synthesized by annealing NEUROD-F3 and NEUROD-R3 in Table 4, and the spacer fragment of T4 was synthesized by annealing NEUROD-F4 and NEUROD-R4 in Table 4.
  • pcDNA3.1-hU6-crRNA-HBB-T2 is a recombinant expression vector obtained by replacing the fragment between the two repeat sequences of the vector pcDNA3.1-hU6-MosBbsI with a DNA molecule having the nucleotide sequence of positions 5-36 of HBB-F2, while keeping the other nucleotide sequences of the vector pcDNA3.1-hU6-MosBbsI unchanged;
  • pcDNA3.1-hU6-crRNA-NEUROD-T4 is a recombinant expression vector obtained by replacing the fragment between the two repeat sequences of the vector pcDNA3.1-hU6-MosBbsI with a DNA molecule having a nucleotide sequence of positions 5-36 of NEUROD-F4, while keeping the other nucleotide sequences of the vector pcDNA3.1-hU6-MosBbsI unchanged.

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Abstract

La présente invention concerne un système CRISPR-Cas de type I-F2 permettant de réguler et de contrôler la transcription des gènes des cellules eucaryotes, ainsi que son utilisation, en rapport avec le domaine technique du génie génétique. La présente invention a pour but de proposer une solution technique permettant de réguler et de contrôler efficacement la transcription dans les cellules eucaryotes grâce à un système CRISPR-Cas simplifié. La solution selon la présente invention consiste en un système CRISPR-Cas de type I-F2 permettant de réguler et de contrôler la transcription des gènes des cellules eucaryotes, comprenant une protéine de fusion constituée par Cas7, un facteur de transcription, Cas6 et Cas5, et un ARNc ciblant un gène cible. Le système CRISPR-Cas de type I-F2 retenu peut réaliser la régulation de la transcription et le contrôle d'un gène cible dans les cellules eucaryotes en couplant un facteur de transcription à une protéine Cas.
PCT/CN2022/143090 2022-12-29 2022-12-29 Système crispr-cas de type i-f2 pour la régulation et le contrôle de la transcription de gènes de cellules eucaryotes, et son utilisation Ceased WO2024138481A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025156264A1 (fr) * 2024-01-26 2025-07-31 中国科学院微生物研究所 Complexe effecteur crispr-cas de type i simplifié et son utilisation

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110662835A (zh) * 2017-05-19 2020-01-07 清华大学 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
CN111979240A (zh) * 2020-06-11 2020-11-24 中山大学 一种基于Type I-F CRISPR/Cas的基因表达调控方法和调控系统
WO2022095929A1 (fr) * 2020-11-06 2022-05-12 The University Of Hong Kong Système transférable d'édition du génome de type i-f crispr-cas

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110662835A (zh) * 2017-05-19 2020-01-07 清华大学 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
CN111979240A (zh) * 2020-06-11 2020-11-24 中山大学 一种基于Type I-F CRISPR/Cas的基因表达调控方法和调控系统
WO2022095929A1 (fr) * 2020-11-06 2022-05-12 The University Of Hong Kong Système transférable d'édition du génome de type i-f crispr-cas

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CHEN YUXI, LIU JIAQI, ZHI SHENGYAO, ZHENG QI, MA WENBIN, HUANG JUNJIU, LIU YIZHI, LIU DAN, LIANG PUPING, SONGYANG ZHOU: "Author Correction: Repurposing type I–F CRISPR–Cas system as a transcriptional activation tool in human cells", NATURE COMMUNICATIONS, NATURE PUBLISHING GROUP, UK, vol. 11, no. 1, 9 July 2020 (2020-07-09), UK, pages 3522, XP093187919, ISSN: 2041-1723, DOI: 10.1038/s41467-020-17379-y *
DATABASE Protein 25 October 2021 (2021-10-25), ANONYMOUS: "type I-Fv CRISPR-associated protein Cas5fv [Moraxella osloensis] ", XP093187921, retrieved from NCBI Database accession no. WP_062332054.1 *
DATABASE Protein 25 October 2021 (2021-10-25), ANONYMOUS: "type I-Fv CRISPR-associated protein Cas7fv [Moraxella osloensis]", XP093187923, retrieved from NCBI Database accession no. WP_062332051.1 *
DATABASE Protein 31 May 2019 (2019-05-31), ANONYMOUS: "type I-F CRISPR-associated endoribonuclease Cas6/Csy4 [Moraxella osloensis]", XP093187920, retrieved from NCBI Database accession no. WP_062332057.1 *
GLEDITZSCH, D. ET AL.: "Modulating the Cascade architecture of a minimal Type I-F CRISPR-Cas system", NUCLEIC ACIDS RESEARCH, vol. 44, no. 12, 23 May 2016 (2016-05-23), XP055653405, DOI: 10.1093/nar/gkw469 *
PAUSCH, P. ET AL.: "Structural Variation of Type I-F CRISPR RNA Guided DNA Surveillance", MOLECULAR CELL, vol. 67, 17 August 2017 (2017-08-17), XP085180183, DOI: 10.1016/j.molcel.2017.06.036 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025156264A1 (fr) * 2024-01-26 2025-07-31 中国科学院微生物研究所 Complexe effecteur crispr-cas de type i simplifié et son utilisation

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