Presented by : S.Rasoulinejad Genome editing & targetting tools Supervisor: S.L.Mousavi Presented by : S.Rasoulinejad 2015 - spring

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?! DNA aptamers are sequences of DNA that are able to bind to a specific target with high affinity because of their unique secondary structure

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

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’ we achieved targeted delivery of exogenous donor DNA to the site of the I-SceI DSB in different genomic locations in yeast and human cells, facilitating gene correction by the donor DNA.

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. The hypothesis is that by delivering targeting DNA within proximity to the site of cleavage, gene targeting frequencies can be increased

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)

Non-SELEX Selection of Aptamers

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

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

EMSA Gel?!

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

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

I-SceI aptamer stimulates gene targeting in human cells . (A) Bifunctional-targeting oligonucleotides containing the A7 aptamer at the 50 end along with a region of homology to restore the function of a defective gene of interest are transformed/transfected into the cell. The I-SceI endonuclease is produced from the chromosome (yeast) or from a transfected expression vector (humans). (B) The A7 aptamer then binds to the I-SceI protein, either in the cytoplasm (shown here) or in the nucleus C) I-SceI drives the bifunctional oligonucleotide to the targeted locus containing the I-SceI site, and (D) generates a DSB at the I-SceI site. (E) Resection of the 50 ends of the DSB gives rise to single-stranded 30 DNA tails. (F) The 30 tail of the bifunctional oligonucleotide anneals to its complementary DNA sequence on the targeted DNA, and after the non-homologous sequence is clipped, (G) DNA synthesis proceeds on the template sequence. (H) After unwinding of the bifunctional oligonucleotide, a second annealing step occurs between the extended 30 end and the other 30 end generated from the DSB. (I) Further processing, gap-filling DNA synthesis, and subsequent ligation complete repair and modification of the target locus.

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.

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.

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

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.

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.

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

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

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

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%

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**

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.