CG920 Genomics
Lesson 5
RNA Interference and Genome Editing
Jan Hejátko
Functional Genomics and Proteomics of Plants,
CEITEC - Central European Institute of Technology
And
National Centre for Bimolecular Research,
Faculty of Science,
Masaryk University, Brno
hejatko@sci.muni.cz, www.ceitec.eu
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 Knocking-down the genes using RNA interference
 Mechanism of RNAi
Outline
 Genome Editing
 Principle of genome editing using 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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 Knocking-down the genes using RNA interference
 Mechanism of RNAi
Outline
 Molecular mechanism of post-transcriptional gene silencing
(PTGS)
 RNAi discovered in plants, later in Coenorhabditis
elegans
 In plants identified as „sense effect“ in systemic negative
regulation of gene activity
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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Silencing the Expression via Introducing
Additional Gene Copy for Flavonoid
Biosynthesis
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.
Systemic effect in the regulation of GFP
expression
 Nicotiana benthamiana
expressing GFP
 Retransformation of one
of the leaves by construct
for GFP expression
 Absence of GFP can be
seen as a red chlorophyll
fluorescence
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.
 Molecular mechanism of post-transcriptional gene silencing
(PTGS)
 RNAi discovered in plants, later in Coenorhabditis
elegans
 In plants identified as „sense effect“ in systemic negative
regulation of gene aktivity
 Gene silencing induced via both sense and anti-sense RNA
 dsRNA induced gene silencing approx. 100x more efficiently
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)
Post-Transcriptional Silencing in Plants is
mediated via 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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 Molecular basis of posttranscriptional gene silencing (PTGS)
 dsRNA induction is dependent on own genes
 gene identification using forward genetics
RNA interference
RNAi rnai
Mello and Conte, Nature (2004)
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 Molecular basis of posttranscriptional gene silencing (PTGS)
 RNAi found in Coenorhabditis elegans and in plants
 It is a natural mechanism of regulation of gene expression
in all eukaryotes
 The principle is creating dsRNA, which can be triggered in
several ways:
 By presence of foreign „aberrant“ DNA
 Specific transgenes containing inverted repeats of the cDNA parts
 Transcription of own genes for shRNA (short hairpin RNA) or miRNA
(micro RNA, endogenous hairpin RNA)
 dsRNA is processed by enzyme complex (DICER), which
leads to the formation of siRNA (short interference RNA),
which is then bound to enzyme complex RITS (RNAinduced
transcriptional silencing complex) or RISC (RNAinduced
silencing komplex)
 RISC mediates either degradation of mRNA (in case of
full similarity of siRNA and the target mRNA) or leads only
to termination of translation (in case of incomplete
homology, e.g. as in the case of miRNA)
 RITS mediates reorganization of genomic DNA
(heterochromatin formation and inhibition of transcription)
RNA interference
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RNA-dependent RNA polymerase
short hairpin RNA
micro RNA
Mechanism of RNA interference
+ tasiRNAs
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 argoago1
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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 Knocking-down the genes using RNA interference
 Mechanism of RNAi
Outline
 Genome Editing
 Principle of genome editing using Site Directed Nucleases,
(SDNs)
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Genome Editing via 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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 Knocking-down the genes using RNA interference
 Mechanism of RNAi
Outline
 Genome Editing
 Principle of genome editing using Site Directed Nucleases,
(SDNs)
 Zinc-Finger Nucleases (ZFNs)
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 Each zinc „finger“ is recognizing nucleotide triplet
 Nuclease domain acts as heterodimer – possiblity to enhance
the specificity by designing the set of „fingers“ recognizing 9
bp on both sides of the target sequence
 Shortcomings
 Difficult to “program”
 Delimited specificity
Zinc-Finger Nucleases -
ZFNs
 Sequence-specific endonucleases recognizing the target
sequence via set of “zinc fingers”
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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 Knocking-down the genes using RNA interference
 Mechanism of RNAi
Outline
 Genome Editing
 Principle of genome editing using Site Directed Nucleases,
(SDNs)
 Zinc-Finger Nucleases (ZFNs)
 Transcription Activator-Like Effectors (TALENs)
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Transcription Activator-Like
Effectors - TALENs
 Proteins derived from sequence-specific transcription
activators
 Identifified (so far only) in plant pathogenic bacteria
Xanthomonas sp. as bacterial effectors, able to control the
transcription of target genes in plants
 Sekvenční specificity determined by aminoacid sequence of
DNA –binding repeats
 Possible to use for various modification types
 Shortcomings
 Difficult to “program”
 Delimited specificity
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, The Origin
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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Fichtner et al. Planta (2014)
TALENs, Specificity
Determination
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TALENs, Applications
Bogdanove and Voytas, Science (2011)
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 Knocking-down the genes using RNA interference
 Mechanism of RNAi
Outline
 Genome Editing
 Principle of genome editing using 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
 Discovered as a mechanism of bacterial immune system
 The principle is targeted insertion of foreign DNA (typically
phage DNA) into specific bactrial genomu loci
 Transcription of trans-activating CRISPR RNA (tracrRNA) and
the region with inserted foreign DNA followed by RNA processing
allows formation of crRNA–tracrRNA complex
 crRNA–tracrRNA binds Cas9 nuclease, targeting it to
complementary (foreign/phage) DNA, that is then digested
 crRNA–tracrRNA is in the targeted genome editing replaced by a
single guide RNA (sgRNA or gRNA)
 Advanatges
 Easy to „program“
 High specificity
 Number of further applications possible
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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 Genome editing
 Sequence-specific high-precision genome
modifications
 Allows generation of both random mutations in a
specific locus, as well as
 introgression/replacement of defined sequence in
the target locus, including gene therapy
 CRISPR/Cas9 paved the way for easy, fast and
accurate genome editing and further derived
modifications
 RNAi
 Natural mechanism controlling gene expression,
partially explaining existence of large amount of
non-coding DNA in various genomes
 Possible use as a tool for specific gene
expression control
Key concepts
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Discussion