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
 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)
 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
Silencing the Expression via Introducing
Additional Gene Copy for Flavonoid
Biosynthesis
van der Krol et al., Plant Cell (1990)
p35S::DFR
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)
 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
Waterhaus et al., PNAS (1998)
Post-Transcriptional Silencing in Plants is
mediated via dsRNA
9
 Molecular basis of posttranscriptional gene silencing (PTGS)
 dsRNA induction is dependent on its own genes – gene
searching
RNA interference
RNAi rnai
Mello and Conte, Nature (2004)
10
 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
11
RNA-dependent RNA polymerase
short hairpin RNA
micro RNA
Mechanism of RNA interference
+ tasiRNAs
21-25 bp
Mello and Conte, Nature (2004)
12
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
13
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
14
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
15
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
16
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
?
17
 Knocking-down the genes using RNA interference
 Mechanism of RNAi
Outline
 Genome Editing
 Principle of genome editing using Site Directed Nucleases,
(SDNs)
18
Genome Editing via SDNs
Pandey et al, Journal of Genetic Syndromes & Gene Therapy (2011)
19
 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)
20
 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”
21
Zinc-Finger Nucleases
Carroll, Science (2011)
Wikipedia
22
 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)
23
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
24
TALENs, The Origin
Fichtner et al. Planta (2014)
25
Fichtner et al. Planta (2014)
TALENs, Specificity
Determination
26
TALENs, Applications
Bogdanove and Voytas, Science (2011)
27
 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)
28
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
29
 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
30
CRISPR/Cas9 – Genome
Editing
Jiang and Doudna, Cell (2017) (single guide RNA)
31
CRISPR/Cas9 – Nobel Prize
in 20..2x?
Francisco Mojica Emmanuelle Charpentier Jenifer Doudna
Martin Jinek Jinek et al, Science (2012)
2020!
32
 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
33
Discussion