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From 2013.igem.org
Tandem Promoter
In nature, the repetitive sequences including interspersed and tandem repetitive elements usually exist in eukaryotic genomes , even in promoter sequence. Tandem repetitive sequences in eukaryotic genomes are involved in various regulation mechanisms of gene transcription and expression. Many tandem repetitive sequences and different promoters arranged in tandem are also conserved in prokaryotes.
In one study, Scientists designed and developed the promoter clusters consisted of the same promoter arranged in tandem repeats in E.coli. By the analysis the expression of report gene(Here they use GFP, by detecting the fluorescence).
Tis figure shows that the transcript strength has a drastically increase with the increase of number of tandem promoters, especially when the number is less than 6.
CRISPR/Cas system
Bacteria and archaea have evolved RNA mediated adaptive defense systems called CRISPR/Cas (clustered regularly interspaced short palindromic repeats) that protect hosts from invading viruses and plasmids . These defense systems rely on small RNAs for sequence-specific detection and silencing of foreign nucleic acids.
CRISPR/Cas systems are composed of cas genes organized in operon and CRISPR array consisting of genome-targeting sequences, which is called spacers, interspersed with identical repeats .
CRISPR/Cas-mediated immunity occurs in three steps. In the adaptive phase, bacteria and archaea harboring one or more CRISPR loci respond to viral or plasmid challenge by integrating short fragments of foreign sequence (protospacers) into the host chromosome at the proximal end of the CRISPR array . In the expression and interference phases, transcription of the repeat spacer element into precursor CRISPR RNA (pre-crRNA) molecules followed by enzymatic cleavage yields the short crRNAs that can pair with complementary protospacer sequences of invading viral or plasmid targets . Target recognition by crRNAs directs the silencing of the foreign sequences by means of Cas proteins, DNA endonuclease, that function in complex with the crRNAs.
There are three types of CRISPR/Cas systems. The type I and III systems share some overarching features: specialized Cas endonucleases process the pre-crRNAs, and once mature, each crRNA assembles into a large multi-Cas protein complex capable of recognizing and cleaving nucleic acids complementary to the crRNA.
In contrast, type II systems process precrRNAs by a different mechanism in which a trans-activating crRNA (tracrRNA) complementary to the repeat sequences in pre-crRNA triggers
processing by the double-stranded RNA specific ribonuclease RNase III in the presence of the Cas9 protein . Cas9 is a DNA endonuclease, thought to be the sole protein responsible for crRNA-guided silencing of foreign DNA.
And the highly specific targeting is leaded by guide crRNA，binding at the complementary DNA sequence, usually 20nt DNA(N20).
This targeting also requires the presence of a conserved sequence motif,,an NGG, downstream of the target DNA, known as the protospacer-adjacent motif (PAM).
Recently, the programmable nuclease activity of S. pyogenes Cas9 are uesed to direct genome editing,because of its relevant simple device.:
CRISPR/Cas systems are composed of cas genes organized in operon and CRISPR array consisting of genome-targeting sequences, which is called spacers, interspersed with identical repeats .
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1.First, examination of the likely secondary structure of the tracrRNA:crRNA duplex suggested the possibility that the features required for site-specific Cas9-catalyzed DNA cleavage could be captured in a single chimeric RNA，which is called guide RNA(gRNA), by fusing the 3’ end of crRNA to the 5’ end of tracrRNA, leaving a GAAA loop. This chimeric gRNA highly simplifies the manipulated CRISPR/Cas system. -
2.The dCas9 mutant (noncleaving), which contained two silencing mutations of the RuvC1 and HNH nuclease domains (D10A and H840A), when coexpressed with a gRNA, generates a DNA recognition complex that can specifically interfere with transcriptional elongation, RNA polymerase(RNAP) binding, or transcription factor binding，witch would repress expression the target gene. -
3.In addition, a fusion between the omega subunit of the RNAP and a dCas9 directed to bind upstream promoter regions can achieve programmable transcription activation by recruiting RNAP.
