CG020 Genomika
Přednáška 5
RNA interference a editování genomu
Jan Hejátko
Funkční genomika a proteomika rostlin,
Středoevropský technologický institut (CEITEC)
a
Národní centrum pro výzkum biomolekul,
Přírodovědecká fakulta,
Masarykova univerzita, Brno
hejatko@sci.muni.cz, www.ceitec.eu
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 Umlčování genů pomocí RNA interference
 Mechanismus RNAi
Osnova
 Editace genomu
 Princip editace genomu prostřednictvím místně specifických
nukleáz (Site Directed Nucleases, SDNs)
 Zinc-Finger Nucleases (ZFNs)
 Transcription Activator-Like Effectors (TALENs)
 Clustered Regularly Interspaced Short Palindromic
Repeats/Cas9 (CRISPR/Cas9)
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 Umlčování genů pomocí RNA interference
 Mechanismus RNAi
Osnova
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 Molekulární podstata posttranskripčního umlčování genů (PTGS)
 RNAi objevena u rostlin, později u Coenorhabditis
elegans
 U rostlin identifikována jako „sense effect“ v systémové
negativní regulaci genové aktivity
RNA interference
Analysis of GUS expression of supertransformed rice callus. Transgenic rice
tissue containing a single Gus transgene supertransformed with UbiDGus[s],
UbiDGus[ays], UbiDGus[iyr], DGus[iyr].
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Umlčování exprese vnesením další kopie
genu pro biosyntézu flavonoidů
van der Krol et al., Plant Cell (1990)
p35S::DFR
Flowers on petunia VR plants transformed with the dihydroflavonol-4-reductase
(DFR) sense gene construct VIP178 showed either an unaffected flower
pigmentation (top left) or a reduction in pigment synthesis. Shown from top left to
bottom right: 178-1, 178-14, 178-6, 178-16, 178-10, and 178-15. On transformant
178-16, flower pigmentation varies from fully pigmented to white. Transformant
178-15 shows an ectopic expression pattern, resulting in a white ring at the edge
of corolla tissue.
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Systémový efekt na regulaci exprese GFP
 Nicotiana benthamiana
exprimující GFP
 Retransformace
jednoho z listů
konstruktem pro
expresi GFP
 Absence GFP je
viditelná jako červená
fluorescence chlorofylu
Voinnet and Baulcombe, Nature (1997)
We studied Nicotiana benthamiana plants carrying a jellyfish green fluorescent
protein (GFP) transgene5. We infiltrated leaves with strains of Agrobacterium
tumefaciens carrying a GFP reporter gene.Intact GFP transgenic plant infiltrated
18 days previously in a lower leaf (arrow) showing the progression of GFP-
silencing.
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 Molekulární podstata posttranskripčního umlčování genů (PTGS)
 RNAi objevena u rostlin a později u Coenorhabditis
elegans
 U rostlin identifikována jako „sense effect“ v systémové
negativní regulaci genové aktivity
 umlčování bylo indukováno jak sense tak antisense RNA
(pravď. kontaminace obou při in vitro transkripci)
 dsRNA indukovala umlčování cca 10-100x účinněji
RNA interference
Analysis of GUS expression of supertransformed rice callus. Transgenic rice
tissue containing a single Gus transgene supertransformed with UbiDGus[s],
UbiDGus[ays], UbiDGus[iyr], DGus[iyr].
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Waterhaus et al., PNAS (1998)
Posttranskripční umlčování u rostlin je
zprostředkováno dsRNA
Kalusy rýže nesoucí konstrukt pro expresi uidA (GUS), který způsobuje modré
zbarvení (1. řádek) byly retransformovány konstrukty pro expresi uidA v sense (2.
řádek), anti-sense (3. řádek) a přímé a obrácené repetici (4. resp. 5. řádek).
Všimněte si silné represe zbarvení a tedy i exprese uidA genu v případě
retransformace konstruktem vedoucím k tvorbě dsRNA (obrácené repetice, 5.
řádek).
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 Molekulární podstata posttranskripčního umlčování genů (PTGS)
 Umlčování genové exprese prostřednictvím dsRNA je závislé
na vlastních genech
 vyhledávání pomocí přímé genetiky
RNA interference
RNAi rnai
Mello and Conte, Nature (2004)
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 Molekulární podstata posttranskripčního umlčování genů (PTGS)
 je to přirozený mechanismus regulace genové exprese u
všech eukaryot
 podstatou je tvorba dsRNA, která může být spuštěna
několika způsoby:
 přítomnost cizí „aberantní“ DNA
 specifické transgeny obsahující obrácené repetice částí cDNA
 transkripce vlastních genů pro shRNA (short hairpin RNA) nebo
miRNA (micro RNA, endogenní „vlásenková“ RNA)
 dsRNA je procesována enzymovým komplexem (DICER),
což vede k tvorbě siRNA (short interference RNA), která
se pak váže buď na enzymový komplex RITS (RNAInduced
Transcriptional Silencing complex) nebo RISC
(RNA-Induced Silencing Complex)
 RISC zprostředkovává buď degradaci mRNA (v případě
úplné similarity siRNA a cílové mRNA) nebo vede pouze k
zastavení translace (v případě neúplné homologie jako je
tomu např. v případě miRNA
 RITS zprostředkovává reorganizaci genomové DNA
(tvorba heterochromatinu a inhibice transkripce)
RNA interference
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RNA-dependent RNA polymerase
short hairpin RNA
micro RNA
Mechanism of RNA interference
+ tasiRNAs (trans-acting siRNA)
21-25 bp
Mello and Conte, Nature (2004)
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
RNA-induced silencing komplex (RISC).
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From MacRae, I.J., Zhou, K., Li, F., Repic, A., Brooks, A.N., Cande, W.., Adams, P.D., and Doudna, J.A. (2006)
Structural basis for double-stranded RNA processing by Dicer. Science 311: 195 -198. Reprinted with permission from
AAAS. Photo credit: Heidi
Dicer and Dicer-like proteins
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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Reprinted by permission from Macmillan Publishers Ltd: EMBO J. Bohmert, K., Camus, I., Bellini, C., Bouchez, D., Caboche, M., and Benning, C. (1998)
AGO1 defines a novel locus of Arabidopsis controlling leaf development. EMBO J. 17: 170–180. Copyright 1998; Reprinted from Song, J.-J., Smith,
S.K., Hannon, G.J., and Joshua-Tor, L. (2004) Crystal structure of Argonaute and its implications for RISC slicer activity. Science 305: 1434 – 1437.
with permission of AAAS.
Argonauta argo
argonaut pelagickýago1
Argonaute proteins
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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MIR gene
RNA Pol
AAAn
AGO AAAn
RNA Pol
mRNA
AGO
AGO
RNA Pol
AGO
AGO
AAAn
siRNA
miRNA
post-transcriptional gene silencingtranscriptional gene silencing
transcriptional slicing
translational repression
binding to DNA
binding to specific
transcripts
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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The Nobel Prize in Physiology or Medicine 2006
Andrew Z. Fire Craig C. Mello
USA USA
Stanford University
School of Medicine
Stanford, CA, USA
University of
Massachusetts Medical
School
Worcester, MA, USA
b. 1959 b. 1960
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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The Nobel Prize in Physiology or Medicine 2006
Andrew Z. Fire Craig C. Mello
USA USA
Stanford University
School of Medicine
Stanford, CA, USA
University of
Massachusetts Medical
School
Worcester, MA, USA
b. 1959 b. 1960
David Baulcombe
UK
?
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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 Umlčování genů pomocí RNA interference
 Mechanismus RNAi
Osnova
 Editace genomu
 Princip editace genomu prostřednictvím místně specifických
nukleáz (Site Directed Nucleases, SDNs)
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Editace genomu pomocí
SDNs
Pandey et al, Journal of Genetic Syndromes & Gene Therapy (2011)
CRISPR–Cas9, TALEN and ZFN mediated genome editing. (A) ZFN-fok1, TALEN
domain and Cas9ꞏsgRNA-induced DSBs can be repaired by either NHEJ or by
HDR pathways. NHEJ mediated repair is highly efficient but error-prone process,
which causes small insertions and/or deletions (indels) at the cleave site. HDR
requires a donor DNA homologous template to repair the cleavage site and this
process can be used to introduce specific point mutations, correction of mutation
or to knock-in of corrected DNA sequences at cleavage site.
Abbreviations CRISPR: Clustered Regularly Interspaced Short Palindromic
Repeats; HDR: Homology Directed Repair; NHEJ: Non- Homologous End
Joining; TALENs: Transcription Activator-Like Effectors; ZFN: Zinc Finger
Nucleases.
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 Umlčování genů pomocí RNA interference
 Mechanismus RNAi
Osnova
 Editace genomu
 Princip editace genomu prostřednictvím místně specifických
nukleáz (Site Directed Nucleases, SDNs)
 Zinc-Finger Nucleases (ZFNs)
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 Každý zinkový „prst“ je schopen rozpoznat nukleotidový triplet
 Nukleázová doména funguje jako heterodimer – možnost
zvýšení specifity navržením sady „prstů“ rozpoznávajícíh 9 bp
z každé strany cílové sekvence
 Nevýhody
 Špatně se „programuje“
 Omezená specifita
Zinc-Finger Nucleases -
ZFNs
 Místně specifické endonukleázy, schopné rozpoznat
cílovou sekvenci prostřednictvím sady „zinkových prstů“
K zásadním nevýhodám ZFNs poatří omezená specifita – některé ‚prsty“
rozpoznávají více tripletů, pro některé trilplety naopak nejsou známy „prsty“
žádné.
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Zinc-Finger Nucleases
Carroll, Science (2011)
Wikipedia
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 Umlčování genů pomocí RNA interference
 Mechanismus RNAi
Osnova
 Editace genomu
 Princip editace genomu prostřednictvím místně specifických
nukleáz (Site Directed Nucleases, SDNs)
 Zinc-Finger Nucleases (ZFNs)
 Transcription Activator-Like Effectors (TALENs)
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Transcription Activator-Like
Effectors - TALENs
 Proteiny odvozené od sekvenčně specifických
transkripčních aktivátorů
 Identifikovány (zatím pouze) u rostlinných patogenních bakterií
Xanthomonas sp. jako bakteriální efektory, schopné regulovat
transkripci cílových genů rostlin
 Sekvenční specifita určena aminokyselinovou sekvencí DNA
vazebných repetic
 Lze využít k různým typům modifikací
 Nevýhody
 Špatně se „programuje“
 Omezená specifita
Specifita některých RVDs je omezena na na in vitro podmínky, RVDs na 5’ konci
vazebného motivu přispívají ke specifitě více než ty na 3’ konci (možnost vzniku
„mismatches“ na 3’ konci, atd.).
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TALENs, původ
Fichtner et al. Planta (2014)
Discovery of Xanthomonas TAL effectors and the proposed mode of action of
AvrBs3. AvrBs3 TAL effector protein is secreted into the plant cell via a Type III
secretion system. The internal natural nuclear localization signal of AvrBs3 leads
to import to the nucleus, where this TALE searches for the base pair sequence
recognised by the internal RVD structure of the DNA binding region. Upon binding
of the TAL effector to its recognised EBE-box (Effector Binding Element), also
known as upa-box, transcription is initiated, leading to physiological effects in the
infected plant cell such as hypertrophy.
Plant resistance to Xanthomonas derives from resistance (R) genes having a
similar EBE-box and mimicking the natural TALE target site. This leads to
enhanced expression of R genes upon infection.
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TALENs, určení specifity
Fichtner et al. Planta (2014)
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TALENs, využití
Bogdanove and Voytas, Science (2011)
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 Umlčování genů pomocí RNA interference
 Mechanismus RNAi
Osnova
 Editace genomu
 Princip editace genomu prostřednictvím místně specifických
nukleáz (Site Directed Nucleases, SDNs)
 Zinc-Finger Nucleases (ZFNs)
 Transcription Activator-Like Effectors (TALENs)
 Clustered Regularly Interspaced Short Palindromic
Repeats/Cas9 (CRISPR/Cas9)
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Clustered Regularly Interspaced
Short Palindromic Repeats/Cas9 -
CRISPR/Cas9
 Objevena jako součást imunitního systému bakterií
 Principem je cílené začlenění cizorodé DNA (typicky fágové
DNA) do specifických oblastí genomu bakterií
 Po přepisu genů pro trans-activating CRISPR RNA (tracrRNA) a
oblasti se začleněnými částmi cizorodé DNA a následném
procesování vzniklé RNA dojde k tvorbě komplexu crRNA–
tracrRNA
 crRNA–tracrRNA váže Cas9 nukleázu a navádí ji na
komplementární (cizorodou/fágovou) DNA, kterou pak Cas9
štěpí
 crRNA–tracrRNA je v cíleném editování genomu nahrazována
single guide RNA (sgRNA nebo také gRNA)
 Výhody
 Snadno se „programuje“
 Značná specifita
 Možná celá řada dalších aplikací
Specifita některých RVDs je omezena na na in vitro podmínky, RVDs na 5’ konci
vazebného motivu přispívají ke specifitě více než ty na 3’ konci (možnost vzniku
„mismatches“ na 3’ konci, atd.).
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 Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR/Cas9 - Mechanism
Jiang and Doudna, Cell (2017)
trans-activating CRISPR RNA
CRISPR-associated (Cas) genes
CRISPR RNA
20 bp of guide sequence
preceding the Protospacer
Adjacent Motif
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 CRISPRassociated
(cas) genes (colored arrows). Preceding the cas operon is the transactivating
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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CRISPR/Cas9 – Genome
Editing
Jiang and Doudna, Cell (2017) (single guide RNA)
The mechanism of CRISPR–Cas9–mediated genome engineering. The synthetic
single guide (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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CRISPR/Cas9 – Nobel Prize
in 20..2x?
Francisco Mojica Emmanuelle Charpentier Jenifer Doudna
Martin Jinek Jinek et al, Science (2012)
2020!
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 Editování genomu
 Sekvenčně-specifické modifikace genomu s
velkou přesností
 Umožňuje jak vznik náhodných mutací v daném
lokusu, tak
 Cílené vkládání definovaných sekvencí – ideální
nástroj pro cílené modifikace genomu včetně
genové terapie
 CRISPR/Cas9 otevřel cestu snadné, rychlé a
zejména přesné editaci genomu a dalším
odvozeným modifikacím s velkým aplikačním
potenciálem
 RNAi
 Přirozený způsob regulace genové exprese,
vyžadujcí vlastní geny a vysvětlující přítomnost
velkého množství DNA nekódujcí proteiny
 Možno využít pro cílenou regulaci genové
exprese
Základní koncepty
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Diskuse