BIO 363 – GENETICA MOLECULAR – I SEMESTRE 2019

i ng
dit
e e
om
en
G

Mo
lecu
lar
“sc
isso
r s”

Ledford H. Nature, 2015, 522(7554):20-4.

Sergio Marshall
 Molecular “scissors”
Targeted DNA-cutting enzyme (nuclease)
Recognizing specific site in genome (DNA sequence) by
a DNA-binding domain
Cutting domain

Site-specific nucleases (SSNs), Site-directed nucleases

(SDNs), Programmable nucleases, Designer nucleases....

Zinc Finger Nucleases ~2005 (ZFN)

TALENs ~2009
CRISPR-Cas ~2012
2
 Natural repair mechanisms

Double strand break (DSB)

repair by the cell

Non-homologous end homologous

joining (NHEJ): recombination
imprecise

repair
template

mutation precise repair

 NHEJ and HDR
DNA

+donor DNA

DNA sequence disrupted

DNA sequence replaced

NHEJ: Non-homologous end joining HDR: homology directed repair

 Genome editing
Precise alterations to the genome

Single base pair changes Insertion Knock-out

rAAV CRISPR TALENs

 What is genome editing?
 A genetic engineering approach in which DNA is inserted,
removed or replaced at a precise location within the genome.
 Engineered nucleases.
 Recombination-based approaches

Viral genome editing: rAAV (Adeno Associated viral Vector)

Nuclease based genome editing:

 Zinc-finger nucleases
 Meganucleases
 TALENs
 CRISPRs
 Nuclease-based genome editing

CRISPRs

CRISPR-Cas system – an form of acquired immunity found in bacteria.

The guide RNA directs the Cas9 protein to a target site.

Creating a guide RNA is very simple.

Cleavage
Guide RNA

Cas9 Protein
 Nuclease-based genome editing

TALENs and CRISPRs

Nuclease-induced DSB

NHEJ-mediated repair HDR-repair

Donor Template Donor Template

Insertion or deletion mutations Single nucleotide alterations Precise sequence insertion

 Nuclease-based genome editing

Engineered nucleases which will cut at a desired position in the genome

TALENs - transcription activator-like effector nucleases

Xanthomonas bacteria express TAL arrays to bind and activate host promoters

TAL array is a series of DNA binding domains assembled to recognise a specific

sequence

33-34 amino acid sequence – only 12th and 13th residue vary – and determine
nucleotide binding.

We can construct these arrays, add a nuclease and use for genome editing

Fok1

TGAGGAGGCGGCAACGGCGGGCGCCGGGGCGGCGGGCCCCGGGGCGAGCA
ACTCCTCCGCCGTTGCCGCCCGCGGCCCCGCCGCCCGGGGCCCCGCTCGT
Fok1

Cleavage
 Zinc Finger Nucleases (ZFN)

 ZFN derived from family of transcription factors recognizing specific

sequences in gene promoters
 Peptide units of which the 3D structure is stabilized by zinc ions leading
to substructures looking like “fingers” (ZFs)
 “Fingers” (ZFs) can be assembled to recognize a specific 3-bp DNA
sequence, an array of these can target a longer sequence in the genome
 For cutting this targeted sequence, a nuclease (FokI) is added (ZFN)
 ZFN and TALEN
ZFN: Zinc finger
nuclease

Jeffrey C Miller, et al. 2007, Nature Biotechnology, 25, 778 – 785.

TALEN: transcription activator–like effector-based

nuclease

Elizabeth P. , 2012, Science

11
 CRISPR-cas

 What is CRISPR-cas9 system?

 Clustered regularly interspaced short
palindromic repeats.
 Segments of prokaryotic DNA containing
repetitive base sequences.
  These play a key role in a bacterial defence
system.
  Form the basis of a genome editing technology
known as CRISPR -Cas9.
 The system allows permanent modification of
genes within organisms.
  CRISPRs are found in approximately 40% of
sequenced bacterial genomes and 90% of
sequenced archaea.
12
short palindromic sequences

RECONOCER
ANA

13
 Identification of CRISPR

Spacer

Clustered Regularly-Interspaced Short Palindromic Repeats

CRISPR
14
 SISTEMA CRISPR/CAS COMO MECANISMO DE DEFENSA

 CRISPR un acronimo para Clustered, Regularly Interspaced, Short,

Palindromic Repeats)–Cas (CRISPR-associated)
 Sistema Inmune adaptativo procarionte para bacterias y archaea
COMPONENTES PRINCIPALES DE SISTEMA CRISPR

El sistema CRISPR/Cas consiste en:

La Proteína nucleasa Cas
Dos elementos individuales de
RNA:

• crRNA (CRISPR RNA). RNA

complementario
• tracrRNA (trans-activating
crRNA).
• Al complejo entre ambos se le
llama sgRNA
• DNA genómico foráneo el cual
contiene una secuencia corta
llamada protospacer adjacent
motif (PAM), la cual consiste en 3
nucleótidos NGG.
Introduction to CRISPR

http://www.addgene.org/CRISPR/guide/
 CRISPR/cas requires:
– the Cas9 protein
– an engineered small guide RNA (sgRNA) with a PAM
sequence upstream of target complementary sequence
– A designed hairpin to mimic the CRISPR RNA and trans-
acting RNA complex.
– Base-pairing between the sgRNA and target DNA causes
double-strand breaks due to the endonuclease activity of
Cas9

http://www.addgene.org/CRISPR/guide/
Qi et al. Cell 2013
 DNA target does not need to be unique and can appear in
multiple locations, all of which will be targeted by the Cas9
nuclease for cleavage.

 Can be on a plasmid or integrated into the bacterial genome.

 The sgRNA can bind on either strand of DNA and the Cas9
will cleave both strands (double strand break, DSB). The
DSB results in the silencing of that DNA sequence.

 Changing the target specificity of the RNA-protein complex

does not require protein engineering but only the design of
the short crRNA guide

 Directing the specificity of dual-RNA:Cas9 with several

different crRNAs allows for the introduction of multiple
mutations at the same time.
Jiang et al. Nature Biotech, 2013
FASES DE INMUNIDAD POR CRISPR-Cas

1) Adaptación: Adquisición de una

espaciador.
2) Transcripción y maduración del
crRNA.
3) Interferencia: Identificación del
target y corte.
FASE DE ADAPTACIÓN

Selección del protospacer y

Generación del spacer

Integración del spacer al

CRISPR array y síntesis de
una nueva repetición

Cas 1 y Cas 2 forman un

complejo Este trasporta el
futuro spacer y lo integra a
CRISPR.

Cas 1 es requerida para el

corte de CRISPR array

La naturaleza palindrómica

Repeteciones palindrómicas
 : FASE DE TRANSCRIPCIÓN Y MADURACIÓN DE crRNA

 Transcripción del loci de CRISPR- cas para generar un complejo guía de RNA-
proteína.
 Se transcribe el locus de CRISPR y el tracrRNA. Luego Cas9 une el tracrRNA con el
transcrito primario e inmaduro de CRISPR.
 Este es cortado por una RNAasa III quedando de forma madura como crRNAs. El
dúo maduro de crRNA:tracrRNA permanence unido a Cas9 como un hetero-
duplex. .
 FASE DE INTERFERENCIA

crRNA del hetero-duplex de

CAS9:RNA se une a la
secuencia complementaria de
la secuencia del DNA foráneo.
Cas9 adopta un estado
conformacional-mente activo
y corta ambas hebras del DNA
en el target, protegiendo a la
célula.
CRISPR/Cas9 system
 Identification of cas genes

Addison V. Wright, et al. 2016, Cell

Cas, CRISPR associated proteins

25
CRISPR BLAST

26
 RNAi-like CRISPR-Cas system (CASS)

Pourcel C, et al., Microbiology, 2005, 151(Part 3):653-663.

Alexander B, et al. Microbiology, 2005, 151(Part 8):2551-2561.

Makarova K S, et al. Biology Direct, 2006, 1(1):1-26. 27

CRISPR confers adaptive immunity

28
 Programming CRISPR

Complementary to:
RNA and coding strand of DNA
Template strand

Cas3, one of the Cas proteins in E. coli K12 CRISPR/cas system

Cascade, casABCDE, CRISPR-associated complex for antiviral defense

First case of directly programming CRISPR-based immunity—a flu shot for bacteria.

Brouns S J J, et al., Science, 2008, 321(5891):960-964.

29
 CRISPR Targets DNA

Self-splicing intron

Plasmid Plasmid

“From a practical standpoint, … it’s possible to manipulate genomic DNA”

- Marraffini and Sontheimer

Marraffini L A and Sontheimer E J. Science, 2009, 322(5909): 1843–1845.

30
 Discovery of tracrRNA

tracrRNA, trans-activating CRISPR RNA

31
 Flexibility in CRISPR processing systems

Susan G. 2011, Nature, 471(7340):588-589. 32

 Structures of Cas proteins

Csy4 bound to RNA substrate CasA The CASCADE complex

Martin J, et al. 2014, Science, (6176):1215-1215; Wiedenheft B, et al., 2009, Structure, 17(6):904-912; Haurwitz R E, et al. 2010, Science, 329(5997):1355-8.
33
34
Flexibility in CRISPR processing systems

35
 Each Cas9 nuclease domain cleaves one DNA strand

Cas9 uses two nuclease domains to cleave the two strands in the target DNA

36
Cleavage sites in target DNA PAM, protospacer adjacent motif

Minimal regions of functional tracrRNA and crRNA

37
 Secondary structure of Dual-tracrRNA: crRNA

S. pyogenes

L. innocua

N. meningitidis

Martin Jinek, etc., 2012, Science (Suppl.)

38
Cas9 can be programmed with sgRNA

39
 Crystal Structure of Cas9/gRNA Complex and Target DNA

Hiroshi N., et al., Feb, 2014, Cell, 156(5):935–949 40

 Structures of Cas proteins

Martin J, et al. Mar, 2014, Science, (6176):1215-1215;

41
 PAM recognition regulates Cas9 nuclease activity

Sternberg S H, et al. March, 2014, Nature, 507(7490):62-67.

 Twenty-Year story of CRISPR

Eric S. Lander, 2016, Cell

43
 Genome Editing in Mammalian Cells

I: Genome modification

II: Genomic deletion

Le C, F Ann R, David C, et al. 2013, Science, (6121):819-823.

44
 Models generated by CRISPR/Cas9 system

Dumpier nematodes Zebrafish embryos Fruit flies

Monkey Rice

Pennisi E. 2013. Science, (6148):833-6.

45
 “Off-target" effect

High efficiency (48.3%)

Low efficiency (14.3%)

Liang, P., et al., 2015, Protein Cell, 6(5): 363-372. (Received March 30, 2015 Accepted April 1, 2015)
 Cpf1, a Single RNA-Guided Endonuclease

Cpf1, CRISPR from Prevotella and Francisella 1

Cpf1 makes staggered cuts

Cpf1 cuts at a distal site
T-rich PAM
Cpf1 may be easier to deliver to cells

Zetsche B, et al., Cell, Oct. 2015, 163(3):759-771

Fagerlund R D, et al., Genome Biology, 2015, 16(1):1-3
47
Pop in and out
 Utilization of CRISPR/Cas9 system

Hsu, P. D., et al., 2014, Cell 157(6): 1262-1278. 49

 Future Directions
Gene Therapy
Genetic liver disease caused by SNP in Fah gene
Single SNP results in skipping of exon 8, unstable protein – loss of function

Adult mice -> injection of CRISPR.

1/250 hepatocytes modified by this approach -> selective pressure -> healthy mouse.

Genetic Disorders -> Correction of mutations

Other potential gene therapy -> HIV “receptor” CCR5 in T-Cells.