Genome editing
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DNA-guided DNA interference by a prokaryotic Argonaute

DNA-guided DNA interference by a prokaryotic Argonaute | Genome editing | Scoop.it

Swarts et al. 2014, Nature

 

RNA interference is widely distributed in eukaryotes and has a variety of functions, including antiviral defence and gene regulation1, 2. All RNA interference pathways use small single-stranded RNA (ssRNA) molecules that guide proteins of the Argonaute (Ago) family to complementary ssRNA targets: RNA-guided RNA interference1, 2. The role of prokaryotic Ago variants has remained elusive, although bioinformatics analysis has suggested their involvement in host defence3. Here we demonstrate that Ago of the bacterium Thermus thermophilus (TtAgo) acts as a barrier for the uptake and propagation of foreign DNA. In vivo, TtAgo is loaded with 5′-phosphorylated DNA guides, 13–25 nucleotides in length, that are mostly plasmid derived and have a strong bias for a 5′-end deoxycytidine. These small interfering DNAs guide TtAgo to cleave complementary DNA strands. Hence, despite structural homology to its eukaryotic counterparts, TtAgo functions in host defence by DNA-guided DNA interference.

Bekir Ülker's insight:

Yet again another faccinating discovery!

Please see also

http://www.pnas.org/content/early/2013/12/26/1321032111.full.pdf

Structure-based cleavage mechanism of Thermus
thermophilus Argonaute DNA guide strand-mediated
DNA target cleavage. Sheng et al. Dec 2013, PNAS

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Science Magazine: A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity

Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A., and Charpentier, E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337: 816-821.

 

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.

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Precision genetic modifications: a new era in molecular biology and crop improvement - Online First - Springer

Precision genetic modifications: a new era in molecular biology and crop improvement - Online First - Springer | Genome editing | Scoop.it

Recently, the use of programmable DNA binding proteins such as ZFP/ZFNs, TALE/TALENs and CRISPR/Cas has produced unprecedented advances in gene targeting and genome editing in prokaryotes and eukaryotes. These advances allow researchers to specifically alter genes, reprogram epigenetic marks, generate site-specific deletions and potentially cure diseases. Unlike previous methods, these precision genetic modification techniques (PGMs) are specific, efficient, easy to use, and economical. Here we discuss the capabilities and pitfalls of PGMs and highlight the recent, exciting applications of PGMs in molecular biology and crop genetic engineering. Further improvement of the efficiency and precision of PGM techniques will enable researchers to precisely alter gene expression and biological/chemical pathways, probe gene function, modify epigenetic marks and improve crops by increasing yield, quality and tolerance to limiting biotic and abiotic stress conditions. 

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Genome editing in Arabidopsis and N. benthamina plants using CAS9 and guide RNA

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