Rna-guided gene editing and gene regulation

A gene and target gene technology, applied in the field of genome engineering and genome modification, genome engineering modification and genome modification of genes in muscles, and can solve the problems of inability to deliver macrodystrophin gene sequence, safety concerns, etc.

Active Publication Date: 2016-06-08
DUKE UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Both approaches have serious security concerns
Furthermore, these strategies are limited by the inability to deliver large and complex dystrophin gene sequences

Method used

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  • Rna-guided gene editing and gene regulation
  • Rna-guided gene editing and gene regulation
  • Rna-guided gene editing and gene regulation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0335] Materials and methods

[0336] Cell culture and transfection. HEK293T cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cancer Center Facility (Duke University Cancer Center Facilities), in DMEM supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin at 37 °C, 5% CO 2 down to maintain. According to the manufacturer's instructions, HEK293T cells were transfected with Lipofectamine2000 (Invitrogen). Transfection efficiencies were routinely greater than 80%, as determined by fluorescence microscopy following delivery of control eGFP expression plasmids. Transfect the Cas9 expression plasmid at a mass ratio of 3:1 to individual gRNA expression plasmids or an equal amount of gRNA expression plasmids (composed of equal amounts of a mixture of 4 gRNAs).

[0337] Primary mouse embryonic fibroblasts (PMEF-HL, Millipore, Billerica, MA) were seeded (75,000 / well) in 24-well TCPS plates (BD, FranklinLakes, NJ) in a...

Embodiment 2

[0352] result

[0353] To generate a CRISPR / Cas9-based transcriptional activation system, the catalytic residues (D10A, H840A) of Cas9 were mutated to generate iCas9, which was genetically fused to the C-terminal VP64 acidic transactivation domain ( figure 1 a, b). Robust iCas9-VP64 expression was observed from transfected plasmids in human embryonic kidney (HEK) 293T cells by Western blotting of the N-terminal Flag epitope tag ( image 3 ). The CRISPR system recognizes its target by base pairing of a 20 bp sequence in the gRNA to a complementary DNA target followed by an NGG protospacer adjacent motif (PAM) sequence, where N is any base pair. Combination of synthetic transcription factors targeting endogenous human promoters results in synergistic and robust activation of gene expression. Therefore, within 500 bp of the transcription initiation site IL1RN Four gRNA target sites followed by the NGGPAM sequence were identified in the promoter of the gene ( Figure 4 ,Tab...

Embodiment 3

[0358] CRISPR targeting the dystrophin gene—methods and materials

[0359] plasmid construct . Expression cassettes of S. pyogenes sgRNA and human codon-optimized Cas9 (hCas9) nuclease were used as previously described (Perez-Pinera et al., NatMethods 10:973-976 (2013)). To generate a fluorescent reporter system to enrich CRISPR / Cas9-modified cells, GeneBlock (IDT) was synthesized. It contained a portion of the 3' end of the Cas9 coding sequence fused to the T2A skipping peptide immediately upstream of multiple cloning sites and was subsequently cloned into the hCas9 expression vector. The eGFP reporter gene was then cloned into a T2 vector for co-translation of Cas9 and eGFP proteins from the same expression vector (hCas9-T2A-GFP, SEQ ID NO: 116).

[0360] Cell Culture and Transfection . HEK293T cells were obtained from the American Tissue Collection (ATCC) via the Duke Cell Culture Facility and maintained in DMEM supplemented with 10% calf serum and 1% penicillin / stre...

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Abstract

Disclosed herein are Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / CRISPR-associated (Cas) 9-based system related compositions and methods of using said CRISPR / Cas9-based system related compositions for altering gene expression and genome engineering. Also disclosed herein are compositions and methods of using said compositions for altering gene expression and genome engineering in muscle, such as skeletal muscle and cardiac muscle.

Description

[0001] Cross References to Related Applications [0002] This application claims U.S. Provisional Application No. 61 / 831,481, filed June 5, 2013, U.S. Provisional Application No. 61 / 839,127, filed June 25, 2013, U.S. Provisional Application No. 61 / 904,911, filed November 15, 2013 , US Provisional Application No. 61 / 967,466, filed March 19, 2014, and US Provisional Application No. 61 / 981,575, filed April 18, 2014, all of which are incorporated herein by reference in their entirety. [0003] Statement of Government Interest [0004] This invention was made with government support under Federal Grant Nos. DP2-OD008586 and R01DA036865 awarded by the NIH and CBET-1151035 awarded by the National Science Foundation. The US Government has certain rights in this invention. technical field [0005] The present disclosure relates to the fields of gene expression alteration, genome engineering and genome alteration using genes based on the Clustered Regularly Interspaced Short Palindrom...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/08C12P21/04A61K39/00
CPCA61K48/005C07K14/4708C12N9/22C12N15/907C07K2319/71C12N2740/16043C12N2800/40C12N2840/20A61K38/465C12N9/96C12Y301/00C12N15/85C07K2319/00C12N15/113C12N15/62C12N15/86C12N2310/20C12N2750/14143A61K48/0058
Inventor C.A.格斯巴奇I.B.希尔顿P.佩雷兹皮内拉A.M.卡巴迪P.I.塔科雷D.G.奥斯特劳特J.B.布拉克
Owner DUKE UNIV
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