A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.
Martin Jinek,Krzysztof Chylinski,Krzysztof Chylinski,Ines Fonfara,Michael H. Hauer,Jennifer A. Doudna,Emmanuelle Charpentier +6 more
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TLDR
This study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.Abstract:
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.read more
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Journal ArticleDOI
Development of germ-line-specific CRISPR-Cas9 systems to improve the production of heritable gene modifications in Arabidopsis
Yanfei Mao,Zhengjing Zhang,Zhengyan Feng,Pengliang Wei,Hui Zhang,José Ramón Botella,Jian-Kang Zhu,Jian-Kang Zhu +7 more
TL;DR: The design and characterization of a germ-line-specific Cas9 system (GSC) for Arabidopsis gene modification in male gametocytes is reported, constructed using a SPOROCYTELESS genomic expression cassette, and the results suggest that future application of the described GSC system will facilitate the screening for targeted gene modifications, especially lethal mutations in the T2 population.
Journal ArticleDOI
Refining strategies to translate genome editing to the clinic
TL;DR: The advances made in the gene-editing field in recent years are discussed, and priorities that need to be addressed to expand therapeutic genome editing to further disease entities are specified.
Patent
Rna-guided human genome engineering
TL;DR: In this paper, a method of altering a eukaryotic cell is provided including transfecting the eukarotic cell with a nucleic acid encoding RNA complementary to genomic DNA of the EKG, where the cell expresses the RNA and the enzyme, the RNA binds to complementary genomic DNA and the enzymes cleaves the genomic DNA in a site specific manner.
Journal ArticleDOI
Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing.
Yuichiro Miyaoka,Jennifer R. Berman,Samantha Cooper,Steven J. Mayerl,Amanda H. Chan,Bin Zhang,George Karlin-Neumann,Bruce R. Conklin +7 more
TL;DR: A novel, rapid, digital PCR–based assay that can simultaneously detect one HDR or NHEJ event out of 1,000 copies of the genome will enable mechanistic studies of genome-editing and help improve genome-EDiting technology.
Journal ArticleDOI
CRISPR-Cas9 gene repair of hematopoietic stem cells from patients with X-linked chronic granulomatous disease.
Suk See De Ravin,Linhong Li,Xiaolin Wu,Uimook Choi,Cornell Allen,Sherry Koontz,Janet Lee,Narda Theobald-Whiting,Jessica Chu,Mary Garofalo,Colin L. Sweeney,Lela Kardava,Susan Moir,Angelia Viley,Pachai Natarajan,Ling Su,Douglas B. Kuhns,Kol A. Zarember,Madhusudan Peshwa,Harry L. Malech +19 more
TL;DR: It is reported that CRISPR, a DNA editing technology, corrected blood stem cells from patients with an immunodeficiency disorder (chronic granulomatous disease) caused by mutations in NOX2, and gene repair of CD34+ hematopoietic stem and progenitor cells (HSPCs) may avoid problems associated with gene therapy.
References
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Journal ArticleDOI
CRISPR provides acquired resistance against viruses in prokaryotes
Rodolphe Barrangou,Christophe Fremaux,Hélène Deveau,Melissa Richards,Patrick Boyaval,Sylvain Moineau,Dennis A. Romero,Philippe Horvath +7 more
TL;DR: It is found that, after viral challenge, bacteria integrated new spacers derived from phage genomic sequences, and CRISPR provided resistance against phages, and resistance specificity is determined by spacer-phage sequence similarity.
Journal ArticleDOI
CRISPR RNA maturation by trans -encoded small RNA and host factor RNase III
Elitza Deltcheva,Krzysztof Chylinski,Krzysztof Chylinski,Cynthia M. Sharma,Karine Gonzales,Yanjie Chao,Zaid Ahmed Pirzada,Maria R. Eckert,Jörg Vogel,Emmanuelle Charpentier,Emmanuelle Charpentier +10 more
TL;DR: In this article, tracrRNA, a trans-encoded small RNA with 24-nucleotide complementarity to the repeat regions of crRNA precursor transcripts, is shown to direct the maturation of crRNAs by the activities of the widely conserved endogenous RNase III and the CRISPR-associated Csn1 protein.
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