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Technologii inzerční mutageneze lze využít i u živočichů. Zda se využívají 
např. transpozony odvozené z Drosophily (transpozon Minos, viz schéma 
vlevo nahoře (Klinakis et al., 2000). V tomto případě bylo nutne provést 
kotransfekci s tzv. helper plasmiem, kódujícím transponázu (neautonomní 
transpozon). Neo kóduje rezistenci k neomycinu, šipky ukazují směr 
transkripce řízený přislušnými promotory, pA  je polyadenylační signál, ori 
je počátek replikace viru SV40, S‐P je promotor téhož viru. Pro identifikaci 
inzercí „in frame“ se zasaženými geny lze využít transpozony, obsahující 
fůzi akceptorových míst sestřihu s ORF reportérového genu, např. lacZ‐neo 
(bez AUG kodonu). Tento přístup umožňuje identifikovat inzerce do 
aktivních genů prostřednictvím selekce inzerčních mutantů na rezistenci k 
neomycinu, resp. vykazující β‐galaktozidázovou aktivitu (Klinakis et al., 
2000).
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It has been found that dsRNA might be either an intermediate or a trigger in PTGS.
In the first case, dsRNA is formed by the action of RNA‐dependent RNA polymerases (RdRPs), which use specific transcripts as a
template. It is still not clear, how these transcripts are recognized, but it might be e.g. abundant RNA that is a result of viral
amplification or transcription of foreign DNA.
It is not clear, how the foreign DNA might be recognized, possibly, lack of bound proteins on the foreign “naked” DNA and its
subsequent “signature” (e.g. by specific methylation pattern) during packing of the foreign DNA into the chromatin structure might
be involved.
The highly abundant transcripts might be recruited to the RdRPs by the defects in the RNA processing, e.g. lack of polyadenylation.
In the case when dsRNA is a direct trigger, there are two major RNA molecules involved in the process: Short interference RNA
(siRNA) and micro RNA (miRNA), both encoded by the endogenous DNA.
These two functionally similar molecules differ in their origin:
siRNAs are dominantly product of the cleavage of the long dsRNA that are produced by the action of cellular or viral RdRPs.
However, there are also endogenous genes, e.g. short hairpin RNAs (shRNAs) allowing production of the siRNA (see the figure).
miRNAs are involved in the developmental‐specific regulations and are product of transcription of endogenous genes encoding for
small dsRNAs with specific structure (see the figure).
In addition to siRNAs, there are trans‐acting siRNAs (tasiRNAs) that are a special class of siRNAs that appear to function in
development (much like miRNAs) but have a unique mode of origin involving components of both miRNA and siRNA pathways.
Developmental regulations via miRNAs are more often used in animals then in plants.
The dsRNAs of all origins and pre miRNAs are cleaved by DICER or DICER‐like (DCL) enzyme complexes with RNAse activity, leading to
production of siRNAs and miRNA, respectively.
These small RNAs are of 21‐24 bp long and bind either to RNA-induced transcriptional silencing complex (RITS) or RNAinduced
silencing komplex (RISC).
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In siRNA and miRNA biogenesis, DICER or DICER-like (DCL) proteins
cleave long dsRNA or foldback (hairpin) RNA into ~ 21 – 25 nt fragments.
Dicer’s structure allows it to measure the RNA it is cleaving. Like a cook
who “dices” a carrot, DICER chops RNA into uniformly-sized pieces.
Note the two strands of the RNA molecule. The cleavage sites are indicated by 
yellow arrows. 
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ARGONAUTE proteins bind small RNAs and their targets and it is an important
part of both RITS and RISC complexes.
ARGONAUTE proteins are named after the argonaute1 mutant of Arabidopsis;
ago1 has thin radial leaves and was named for the octopus Argonauta which it
resembles (see the figure).
ARGONAUTE proteins were originally described as being important for plant
development and for germline stem‐cell division in Drosophila melanogaster.
ARGONAUTE proteins are classified into three paralogous groups: Argonaute‐like
proteins, which are similar to Arabidopsis thaliana AGO1; Piwi‐like proteins, which are
closely related to D. melanogaster PIWI (P‐element induced wimpy testis); and the
recently identified Caenorhabditis elegans‐specific group 3 Argonautes.
Members of a new family of proteins that are involved in RNA silencing mediated by
Argonaute‐like and Piwi‐like proteins are present in bacteria, archaea and eukaryotes,
which implies that both groups of proteins have an ancient origin.
The number of Argonaute genes that are present in different species varies. There are 8
Argonaute genes in humans (4 Argonaute‐like and 4 Piwi‐like), 5 in the D. melanogaster
genome (2 Argonaute‐like and 3 Piwi‐like), 10 Argonaute‐like in A. thaliana, only 1
Argonaute‐like in Schizosaccharomyces pombe and at least 26 Argonaute genes in C.
elegans (5 Argonaute‐like, 3 Piwi‐like and 18 group 3 Argonautes).
http://youdpreferanargonaute.com/2009/06/
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MicroRNAs are encoded by MIR genes, fold into hairpin structures that are 
recognized and cleaved by DCL (Dicer‐like) proteins. 
In summary, siRNAs-mediates silencing via post-transcriptional and
transcriptional gene silencing, while miRNAs -mediate slicing of mRNA
and translational repression.
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In 2006, Andrwe Z. Fire and Craig C. Mello were honored by the Nobel prize “for
their discovery of RNA interference - gene silencing by double-stranded
RNA“.
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CRISPR–Cas9‐mediated DNA interference in bacterial adaptive immunity. A 
typical CRISPR locus in a type II CRISPR–Cas system comprises an array of 
repetitive sequences (repeats, brown diamonds) interspaced by short stretches 
of nonrepetitive sequences (spacers, colored boxes), as well as a set of CRISPR‐
associated (cas) genes (colored arrows). Preceding the cas operon is the trans‐
activating CRISPR RNA (tracrRNA) gene, which encodes a unique noncoding RNA 
with homology to the repeat sequences. Upon phage infection, a new spacer 
(dark green) derived from the invasive genetic elements is incorporated into the 
CRISPR array by the acquisition machinery (Cas1, Cas2, and Csn2). Once 
integrated, the new spacer is cotranscribed with all other spacers into a long 
precursor CRISPR RNA (pre‐crRNA) containing repeats (brown lines) and spacers 
(dark green, blue, light green, and yellow lines). The tracrRNA is transcribed 
separately and then anneals to the pre‐crRNA repeats for crRNA maturation by 
RNase III cleavage. Further trimming of the 5’ end of the crRNA ( gray 
arrowheads) by unknown nucleases reduces the length of the guide sequence to 
20 nt. During interference, the mature crRNA–tracrRNA structure engages Cas9 
endonuclease and further directs it to cleave foreign DNA containing a 20‐nt 
crRNA complementary sequence preceding the PAM sequence.  Asterisks denote 
conserved, key residues for Cas9‐mediated DNA cleavage activity. Abbreviations: 
Arg, arginine‐rich bridge helix; crRNA, CRISPR RNA; CTD, C‐terminal domain; nt,
nucleotide; NUC, nuclease lobe; PAM, protospacer adjacent motif; REC, 
recognition lobe; tracrRNA, trans‐activating CRISPR RNA.
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The mechanism of CRISPR–Cas9–mediated genome engineering. The synthetic 
sgRNA or crRNA–tracrRNA structure directs a Cas9 endonuclease to almost 
arbitrary DNA sequence in the genome through a user‐defined 20‐nt guide RNA 
sequence and further guides Cas9 to introduce a double‐strand break (DSB) in 
targeted genomic DNA. The DSB generated by two distinct Cas9 nuclease 
domains is repaired by host‐mediated DNA repair mechanisms. In the absence of 
a repair template, the prevalent error‐prone nonhomologous end joining (NHEJ) 
pathway is activated and causes random insertions and deletions (indels) or even 
substitutions at the DSB site, frequently resulting in the disruption of gene 
function. In the presence of a donor template containing a sequence of interest 
flanked by homology arms, the error‐free homology directed repair (HDR) 
pathway can be initiated to create desired mutations through homologous 
recombination, which provides the basis for performing precise gene 
modification, such as gene knock‐in, deletion, correction, or mutagenesis. 
CRISPR–Cas9 RNA‐guided DNA targeting can be uncoupled from cleavage activity 
by mutating the catalytic residues in the HNH and RuvC nuclease domains, 
making it a versatile platform for many other applications beyond genome 
editing. Abbreviations: crRNA, CRISPR RNA; nt, nucleotide; PAM, protospacer 
adjacent motif; sgRNA, single‐guide RNA; tracrRNA, trans‐activating CRISPR RNA.
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