This document describes a novel gene targeting approach called aptamer-guided gene targeting (AGT) that uses DNA aptamers selected through capillary electrophoresis systematic evolution of ligands by exponential enrichment to bind site-specific DNA binding proteins and guide donor DNA to specific genetic loci for correction. The approach was shown to increase gene targeting efficiency up to 32-fold in yeast and 16-fold in human cells. It also discusses the potential to develop aptamers for other genome editing tools like CRISPR/Cas9.

1. Genome editing & targetting
tools
Presented by : S.Rasoulinejad
2015 - spring

2. Aptamer-guided gene targeting
• Here we have developed a novel gene targeting approach, in
which we bind a site-specific DNA binding protein by a DNA
aptamer to target a donor molecule to a specific genetic locus
for correction
 (aptamer-guided gene targeting [AGT])
 But what is DNA aptamer and how we can reach it?!

3. Goals:
• This work is designed as a proof-of-principle concept or
prototype to show that by binding a protein that guides the
correcting donor DNA into proximity to its genetic target via an
aptamer,
• AGT the efficiency of gene targeting increases

4. AGT?!
• The AGT approach increases the efficiency of gene targeting
by guiding the exogenous donor DNA into the vicinity of the
site targeted for genetic modification.
• By tethering the exogenous donor DNA to the site-specific
homing endonuclease I-SceI, which recognizes an 18-bp
sequence and generates a DSB
 DNA aptamers were selected for binding to the I-SceI protein
using a variant of capillary electrophoresis systematic
evolution of ligands by exponential enrichment (CE-SELEX)
called ‘Non-SELEX’

5. AGT?!
• By synthesizing a DNA oligonucleotide that contained :
 the I-SceI aptamer sequence
 as well as homology to repair the I-SceI DSB and
 correct a target gene,
 we were able to increase gene targeting frequencies up to 32-
fold in yeast, and up to 16-fold in human cells.

6. What has been done step by step:
 74-mer oligonucleotides containing a central 36-nt variable region
 Was loaded with I-SceI protein on CE with LIF.
 Two rounds of selection were done to obtain DNA aptamers to I-
SceI
 After sequencing the 11 strongest binding aptamers, these
candidate aptamers were further characterized using EMSA gels
to confirm their binding capacity to I-SceI.(showed consistent and
reproducible binding to I-SceI)

7. Non-SELEX Selection of Aptamers

11. EMSA Gel ? Electrophoretic Mobility
Shift Assay
 EMSA Gel ? is a common affinity electrophoresis technique
used to study protein–DNA or protein–RNA interactions.
• DNA moves through the gel faster when not bound to protein
• A reduction in electrophoretic mobility shows that a complex
is formed between DNA and protein

12. 5 basic steps are in conventional EMSA protocol
Preparation of
purified or crude
protein sample
Preparation of nucleic
acid
Binding reactions
Non-denaturing
gel
electrophoresis
Detection of the
outcome

13. EMSA Gel?!

14. Using EMSA to determine kd
I. A fixed concentration of DNA is titrated with
excess protein
II. Bound and free DNA are separated using
EMSA
III. Measure density of bands, Kd=protein
concentration when 50% of DNA is free

15. The I-SceI aptamer stimulates gene correction in
yeast
• using bifunctional single-stranded DNA oligonucleotides containing the
aptamer region I-SceI aptamer at one end
• the donor repairing sequence at the other end
• First, we determined on which end (5̒̒ or 3 ) the I-SceI aptamer should be
positioned in the bifunctional molecule to obtain more effective
stimulation of gene targeting.
 bifunctional oligonucleotides (P1-A7-P2.TRP5.40 for the aptamer with
primers at the 5 end of the bifunctional oligonucleotide and TRP5.40.P1-
A7-P2 which contained 40 bases of homology to correct a disrupted TRP5
gene in yeast

16. I-SceI aptamer stimulates gene targeting in human cells

17. Some results:
 in both yeast (up to 32-fold) and human cells (up to 16-fold).
 I-SceI aptamer in AGT, the donor molecule is brought in the
vicinity of its target site, and this may not only increase HR
with the desired locus but also potentially reduce the
likelihood of random integration
 The novelty and uniqueness of our AGT system lay the
foundation for generating and exploiting other aptamers for
gene targeting.

18.  It may be possible to obtain aptamers for other site-specific
homing endonucleases, or for more modular nucleases, like
zinc-finger nucleases (ZFNs)
 transcription activator-like effector nucleases (TALENs) or the
Cas9 nuclease of the clustered regularly interspaced short
palindromic repeat (CRISPR) system
 Furthermore, the potential aptamer targets to stimulate gene
correction in cells are not limited to endonucleases but could
be any protein that facilitates the targeting process, such as
transcription factors, HR proteins or even NHEJ proteins.

19. But what is CRISPR System?!
• Introduction:
• The clustered, regularly interspaced short palindromic repeat
(CRISPR)/CRISPR-associated (Cas) protein system is an
adaptive RNA-mediated immune system in approximately
40% of bacteria and ~ 90% of Achaea.
• The CRISPR/Cas system can be reprogrammed to reject
invading bacteriophages and conjugative plasmids.
 It is classified into three types (I, II, and III) based on the
sequence and structure of the Cas protein

20. • The crRNA-guided surveillance complexes in types I and III
need multiple Cas subunits
• type II requires only Cas9
• The type II system as a reduced system has been studied
primarily in Streptococcus and Neisseria
• A protospacer within an N21GG (or N20NGG) format is widely
used for S. pyogenes Cas9 targeting. This protospacer contains
a 20-nt base-pairing region immediately followed by a PAM
(NGG).
• Other Cas9 orthologs requiring longer PAM sequences would
reduce our choices on targetable sites in a given gene or
genome.

21. • Protospacer-adjacent motifs= PAM
• protospacer-adjacent motifs(PAMs): are short
conserved nucleotide stretches next to the
protospacers, such as NGG, NGGNG, NAAR, and
NNAGAAW,are absolutely necessary for Cas9 binding
and cleavage.

22. • The native type II system requires at least three crucial
components:
• RNA-guided Cas9 nuclease, crRNA, and a partially
complementary trans-acting crRNA (tracrRNA)
• tracrRNA:is a non protein coading RNA for maturation and
subsequent DNA cleavage

23. Cas9 nuclease.
• Cas9 (formerly known as Csn1 or Csx12)
• is able to cleave double-stranded DNA (dsDNA) in a sequence
specific manner
• Cas9 is a large multidomain protein with two nuclease
domains
 RuvC-like nuclease domain near the amino terminus
 HNH (or McrA) nuclease domain in the middle

24. APPLICATION OF TYPE II CRISPR/Cas SYSTEM
• In general, current applications of type II systems
can be classified into three categories:
 native Cas9-mediated genome editing,
 Cas9 nickase mediated genome editing,
 and inactivated Cas9-mediated transcriptional
control

25. Rate:
• Construction of a mammalian expression system with codon-
optimized Cas9 and gRNA generated targeting rates:
 2 to 25% in various human cells
 36 to 48% in mouse embryonic stem cells
 Co-transformation of a gRNA plasmid and editing template DNA
into a yeast cell constitutively expressing Cas9 generated a near
100% donor DNA recombination frequency at the target loci
 Co-injection of Cas9 mRNA and gRNA transcripts into mouse
zygotes generated mutants with an efficiency of 80%

26. Cas9 nickase-mediated genome editing
• gRNA-guided Cas9n with a RuvC or HNH mutation has the ability
to create a nick instead of a DSB at the target site
• Introduction of a double nick using a pair of gRNA-directed Cas9ns
targeting the opposite strands of the target site has been
successfully applied to generate DSBs and NHEJ-induced
mutations.*(non homologous end joining (NHEJ))
 Interestingly, this paired nicking significantly reduced off-target
cleavages by 50- to 1,500-fold in human cells, but without
sacrificing on-target cleavage efficiency**

27. Cas9 nickase-mediated genome editing
• creating a pair of double nicks at two sites by four
customized gRNAs successfully deleted genomic
fragments of up to 6 kb in HEK 293FT cells
 multiplex nicking created by Cas9n has the
ability to create high-precision genome
editing.