Gene editing is the process in which modified single stranded DNA oligonucleotides (ODNs) are used to correct single base mutations in genes. Using an ODN that targets the non-transcribed (NT) strand, has been shown to yield higher correction efficiencies than an ODN that targets the transcribed (T) strand. Previous studies have shown that DNA damage, repair and cell cycle checkpoint proteins inhibit the gene editing reaction. Scientists have been able to analyze this inhibition using cell line models and by the use of flow cytometry. Two such proteins are mutS homolog2 (MSH2), a mismatch repair (MMR) protein, and p53, a genome surveillance protein. Previous studies have also shown that the use of siRNA to temporarily knockdown these proteins in other mammalian cell lines, can lead to an increase in correction efficiency. Permanent knockdown of MSH2 and p53 in cells is an unfavorable option because research has linked this to a predisposition to cancer. These data support the introduction of siRNA into our mammalian model system that can lead to increased correction efficiency, which if successful, can lead to the treatment of single base mutations in the human genome.
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Genetically based diseases are life threatening and they require lifelong treatment. There are currently over 30,000 single base pair mutations like cystic fibrosis, Down syndrome (in human beings), hemophilia, sickle cell anemia etc. many of which offer insufficient treatments, sometimes no treatment option is available . Gene therapy is one of the advanced therapeutic methods aiming essentially to cure the disease at gene level and to reverse the disease phenotype. Previously researchers have used viral vectors, but have not been very successful because the viral vectors lack specificity with regard to integration sites and there was a high incidence of integration near proto-ontogenetic sites, which result in leukemia in majority of patients. Viral vectors also can trigger immunogenic response that can be fatal. On top of these serious risks, random integration of a full-length gene could result in abnormal gene expression, if the promoter is not endogenous.
Gene editing or targeted nucleotide exchange arose as an alternative strategy that corrects the underlying disease causing mutation in the endogenous gene, so that gene expression pattern remains unchanged. Gene editing became popular in the world of research as an alternative method to treat genetic disorders and to get rid of life-long drug treatments . Currently, Targeted nucleotide exchange (TNE) reaction utilizes a short single strand of DNA, an oligonucleotide (ODN) that pairs with the targeted gene . Previously scientists have used mammalian cells, HCT116 (Human colon tumor cell line), as model cell line in gene editing. These cell lines were cultured and harvested at different conditions, ODNs were introduced into the cells through electroporation, and the results were analyzed using a combination of cell cycle and flow cytometry analyses to know the efficiency of correction or treatment. While studying the mechanism of TNE, recent studies have developed an interest in the function of MutS homolog2 (MSH2), a mismatch repair protein  and p53, a genome surveillance protein. MSH2 has been shown to interfere with the recombination between the sequences in TNE reaction through anti-recombinase activity , while P53 has known to cause suppression of homologous recombination (HR).
Gene editing is the repair of single base mutation in mammalian genes that are directed by short single-stranded DNA oligonucleotides (ssODN). Previously researchers have used HCT116-19 (clone19) cell line that has non-sense mutation at position + 67 resulting in nonfunctional eGFP (enhanced green fluorescent protein). To correct the single base changes in chromosomal DNA, gene editing is employed by using a mutated eGFP ssODNs with sequence of 5'-GAAGCACTGCACGCCGGAGGTCA GGGT-3'; and targeting sequence is 5'GAAGCACTGCACGCCATATGTCAGGGT-3' . Several configurations of synthetic oligonucleotides have been used in gene editing including short and long unmodified DNA molecules, chimerical 2'O-Me-RNA-DNA hairpin oligonucleotides . The editing oligonucleotides are specially designed to restore the fluorescence of the eGFP. In such system, editing is easily evaluated, by visual inspection where corrected cells are identified as green cells under fluorescent microscope and, quantitatively by flow cytometry analyses .
Currently gene editing utilizes a short strand of DNA oligonucleotide (ssODN) that pairs with the target gene. Most commonly used ODN in mammalian system is approximately 72 nucleotide in length with a single and centrally located mismatch. It has a modification with three terminal phosphor thioate linkages on both ends of ODN in place of phophodiester back bone, to protect from nuclease degradation . SsODNs of this fashion have been known to increase the correction efficiencies of 1-2%. ODN-induced gene editing is not necessitated in viral vector delivery systems. ODN delivery in to cells is performed either with the aid of cationic liposomes that coat DNA or with electroporation, which essentially permeabilizes the membrane allowing ODN to enter in to cells.
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Mechanism of ODN-induced gene editing: Research utilizing many cell model systems with different reporter genes has suggested four possible mechanisms. There is evidence that all the four mechanisms support each other, but they may not be mutually exclusive. Initial step in gene editing reaction is; ODN pairing to its target site which is heavily influenced by homologous recombination protein, Rad51 [6, 7]. Each of the four mechanisms initiates ODN pairing to its target DNA causing subsequent D-loop formation . The mechanisms differ based on how the D-loop, a reaction intermediate, is resolved to produce a corrected gene. In direct repair mechanism, [Fig1] D-loop serves as cellular beacon of DNA that can accelerate repair proteins to resolve the mismatch. The non-complimentary pairing resolved by ODN directs the conversion of mutant endogenous base. When ODN leaves the target site, cell is left with a mismatch between newly corrected DNA base and endogenous mutant complimentary base. Another DNA repair step resolves this mismatch by correcting the complimentary base to wild type gene status .
Another possible mechanism in gene editing reaction involves ODN pairing to the target site while the gene being transcribed actively. As RNA polymerase complex occupies the transcribed strand, non-transcribed strand remains unbound providing an opportunity for ODN to bind its complimentary site. The RNA polymerase can switch from transcribed strand to ODN and then back to transcribed strand, creating a corrected mRNA without altering DNA sequences. This would lead to functional protein production, creating a phenotypic change, which will be observed without genotypic change. This mechanism can also explain the strand bias, where in most cases there is higher correction efficiencies in case of ODN compliments to non-transcribed strand than to transcribed strand . ODN may be directly incorporated in to target gene (Fig1) . The direct integration model depend on the formation of D-loop and subsequent replacement of D-loop with ODN.
Researchers have made some manipulations to obtain higher correction efficiencies in gene editing. Such manipulations of cell cycle have determined that progression of S-phase is extremely important for higher correction efficiencies. Targeting cells in mid S-phase leads to elevated corrections, whereas targeting cells in early or in late S-phase have diminished correction efficiencies. Progression of S-phase can be achieved by adding either aphidicoline or dodeoxy cytidine (ddc) to cells, 24hours prior to targeting with ODN.
Mismatch repair pathway (MMR) was involved in this DNA repair event, which is inhibitory to the ODN-induced gene editing reaction in mammalian cells . MSH2, a mismatch repair protein and p53, a genome surveillance protein inhibits gene editing and ultimately leads to reduced correction efficiency. In DNA repair pathway, MSH2 and p53 combines with ODN and inhibits ODN pairing to target DNA.
Cell sorting: Single green fluorescent gene edited cells were sorted on a FACS (Fluorescent Activated Cell Sorting) analyses. Events recorded based on Forward scatter/side scatter, live/dead.
Fig 1: Pathways of gene editing
Small interfering RNA (siRNA) is one of the latest additions to the repertoire of specific gene silencing agents. These molecules silence the specific gene expression. RNA interference or RNA silencing occurs in wide variety of eukaryotic organisms. It is triggered by dsRNA precursors, which vary in length and origin . Short inhibited RNAs of 21-28 nucleotide length were formed from dsRNA. According to their origin or function, three types of naturally occurring small RNAs have been described, interfering RNA(RNAi), micro RNA(miRNA), repeat associated short inhibited RNA(rasiRNA). By using RNA-templated RNA, polymerization or hybridization of overlapping transcripts dsRNA can be produced. Such dsRNA give rise to siRNA, which guide the mRNA degradation. Finally, artificial introduction of dsRNA or siRNA is adopted as a tool for suppression of specific proteins expression. RNA silencing mechanism is first discovered as an antiviral mechanism to protect organisms against RNA viruses.
Structure of siRNA: siRNA molecules are short double stranded RNA duplexes with symmetric 2-3 nucleotide 3'overhangs the 5' phosphate and 3'hydroxylgroups (Fig 2).
Processing of double stranded RNA (dsRNA) precursors: The maturation of small RNAs is a stepwise process catalyzed by dsRNA-specific RNAse-III-type endonucleases, termed as Drosha and Dicer; contains catalytic RNAse III and double stranded RNA binding domain (dsRBDs) (Fig3). Processing of double stranded RNA by Dicer yields RNA duplexes of about 21 nucleotide in length. Several organisms contain more than one Dicer gene, DCR1, DCR2. DCR 2 is associated with the dsRBD that contains a protein R2D2 . The processing of dsRNAs to siRNAs by the DCR-2/R2D2 heterodimer is ATP-dependent and requires a functional RNA helicase domain in DCR2 .
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RNA inducing silencing complex (RISC) activation and catalysis: The active components of RISC are endonucleases called Argonaute proteins. These proteins cleave the target mRNA strand complementary to their bound siRNA .The siRNA will be unbound into two ssRNA named the passenger strand and the guide strand. The guide strand binds the Argonaute protein and directs gene silencing. Passenger strand Called Anti-guide strand will be degraded during RISC activation . The strand selection, as the guide tends to be the one whose 5' end is complementary to the Argonaute protein. R2D2 protein serves as differentiating factor by binding to 5' end of the passenger strand and makes it unavailable for the binding of Argonaute proteins. The separation of two strands is an ATP-dependent process and performed directly by the protein components of RISC , .
RNA-induced transcriptional silencing (RITS): Components of RNA interference pathway are also used in maintenance of the organization and the structure of their genomics. For example, modification of histones and associated induction of heterochromatin formation serves to suppress the genes pre-transcriptionally . This process is referred as RNA-inducing transcriptional complex (RITS), and is carried out by a complex of proteins called RITS complex. In yeast, the RITS complex contains a chromodomain protein Chp1, which spreads the heterochromatin regions. Indeed, deletion of these genes in yeast disrupts the histone methylation and centromere formation . In maintenance of existing Heterochromatin regions, RITS forms a complex with siRNAs complementary to local genes and stably binds local methylated histones, acting co-transcriptionally to degrade any nascent pre-mRNA transcripts that initiated by RNA polymerase (Fig 4).
Concentration of siRNA: 1nM concentration of siRNA has been shown to be effective in exhibiting RNAi. However, some reports have used a high concentration of 25nM of high quality siRNAs.
Optimal cell density for transfection: This is one of the variables that have to be optimized and then maintained. A good starting point for transfection is to use 60-70% confluent cells.
Gene Knockdown: The RNA interference pathway has been exploited in gene editing studies to know the function of genes in cell cultures and in model organisms. Double stranded RNA is synthesized with a sequence complementary to a particular gene and introduced into a cell. Within the cell, this dsRNA activates RNAi pathway. Using this method researcher made a tremendous improvement in suppression of targeted gene. RNAi not permanently abolish the expression of targeted genes . Depending on the experimental model system, long strand exogenous RNA cleaved by a dicer or shortRNAs serve as siRNAs. Shorter RNAs are commonly used in mammalian cells because long double strands induce the mammalian interferon response, a form of innate immunity .
Knock down of DNA damage proteins using siRNA: MSH2 and p53 expression can temporarily be knocked down by using siRNA. These proteins are involved in mismatch recognition and tumor suppression so permanent knock down will predispose the cells for cancer. Cells should be transfected with siRNA 24 hours prior to protein extraction. After 24 hours, suppression of endogenous protein p53 in the protein extracted from transfected cells can be observed through western blotting.
Structure of siRNA
Fig 2: siRNA with 3' hydroxyl and 5'phosphate groups
The RNAi pathway
RISC (RNA inducing silencing complex)
Degradation of mRNA
Fig 3: Long dsRNA molecules are cleaved to produce siRNA by Dicer enzyme (a dsRNA specific RNAse-lll type). SiRNA molecules are then incorporated RNA-inducing silencing complex (RISC). The duplex siRNA is unwound leaving the anti-sense strand to guide RISC to degrade the complementary mRNA.
DsRNA ← viral replication precursor miRNA
â†™ ↓ â†˜
RITS RISC RISC
â†™ â†˜ â†˜
DNA histone methylation mRNAdegradation
Fig 4: The enzyme Dicer catalyzes the cleavage of dsRNA, to form small interfering RNA. These processed siRNA and miRNA incorporated into RISC and RITS to cause mRNA degradation and histone methylation respectively.
p53 also known as tumor protein 53 or protein 53, is a tumor suppressor gene that is encoded by TP53gene [22, 23]. p53 is important in multi cellular organisms where it regulates cell cycle and thus functions as tumor suppressor, which involved in preventing cancer. Referring to role in conserving stability by preventing genome mutation, p53 is described as guardian of the genome. The name p53 relates to its molecular mass as it runs a 53-kilodalton (kDa) protein on SDS-PAGE .
P53 encoded by gene TP53 located on short arm of chromosome 17. Human p53 has 393 amino acids and has seven domains each has specific function . N-terminal transcription activation domain also known as activation domain (AD1) activates transcription factors. Activation domain 2(AD2) is important for apoptotic activity. Proline rich domain is important for apoptotic activity. Central-DNA binding core domain (DBD) contains one zinc atom and several arginine core amino acids. Homo-oligomerization domain (OD) regulates terminization, which is essential for the activity of p53. C-terminal involved in down regulation of DNA binding of the central domain.
Normal functions of p53:
p53 has many anti cancer mechanisms and plays an important role in apoptosis, genetic stability and inhibition of angiogenesis. p53 works through many mechanisms in its anti-cancer role. It activates DNA repair proteins when DNA has damage. It can induce growth arrest by holding the cell cycle at G1/S phase, if p53 holds the cell cycle at this stage DNA damage proteins will have much time and the cell will be allowed to continue cell cycle. If DNA damage is irreparable, p53 can initiate apoptosis (Fig: 6).
Activated p53 binds with DNA and activates the expression of several genes encoding for p21. p21 is important in cell cycle transition as a stop signal for cell division. A tumor p53 will no longer bind to DNA, there will be no p21 production and uncontrollable cell division leads to tumor formation. If TP53 gene is damaged tumor suppression function will be lost. Loss of p53 can result in genomic instability that creates aneuploidy phenotype .
Effects of p53 on gene editing: Apart from tumor suppression and cell cycle checkpoints activation, p53 is also involved in homologous recombination (HR) in gene editing and inhibits the HR proteins like Rad51, which is important for homologous recombination . Furthermore, p53 is recruited to stalled replications to prevent uncontrolled homologous exchanges . p53 acts as antagonist and prevents the initiation of oligonucleotide-directed gene repair, reducing the level of eGFP positive cells and ultimately leads to reduction in correction efficiency.
p53 expression knockdown: In gene editing p53 expression can be temporarily knocked down by using siRNA. siRNA can be introduced in to cells prior to protein extraction; cell extracts are prepared, proteins are separated on a 10%SDS-PAGE gel and electroblotted to a nitrocellulose membrane. The membrane incubated with p53, a mouse monoclonal antibody against total p53, and secondary is goat anti-mouse horseradish peroxidase (HRP) conjugated antibody followed by chemilumniscent detection. Actin expression can be used as an internal control to normalize protein levels and can be used using beta actin primary followed by anti-goat HRP. Suppression of p53 can be observed .
Fig. 5: Structure of p53 protein.
Cell cycle abnormalities
Mdm2+p53→ Activated p53
Cell cycle arrest apoptosis
DNA repair Death and elimination of damaged cells
Cell cycle restart
Cellular and genetic stability
Fig 6: P53 pathway
The official name of MSH2 is 'mutS homolog 2'a mismatch repair protein. MSH2 provides an instruction for making a protein that plays an important role in DNA repair. This protein fixes mistakes that were made when DNA replicated in preparation for cell division. MSH2 protein combines with another two proteins MSH6 and MSH3 forming an active protein complex that identifies places on DNA where mistakes have been made during DNA replication.
Location of MSH2: MSH2 is located on short arm (p) of chromosome 2 at position21. (Fig7)
Effects of MSH2 on gene editing: MSH2 plays a suppressive role in gene editing by precluding the oligonucleotide (ODN) annealing to the target gene. Furthermore, MSH2 has an anti-recombinase activity during homologous recombination (HR) and interact with several proteins involved in the regulation of cell cycle . MSH2 inhibits the recombination between sequences during HR. MSH2 combines with the ODN and prevents its pairing to target DNA.
MSH2 is involved in gene editing of yeast cells but has different reactions. In yeast correction efficiency is decreased in the absence of MSH2 unlike that of mammalian cells where correction efficiency is increased in the absence of MSH2. Therefore, researchers speculate the role of MSH2 in yeast is more direct and active where it involves only in mismatch recognition without effecting HR, whereas in mammalian cells, MSH2 regulates the frequency of repair by inhibiting the reaction through its anti-recombinase activity .
Changes in MSH2: Several mutations in MSH2 predispose people to colorectal cancer. Mutations in MSH2 may cause the production of inactivated MSH2 protein that fails to perform its normal function. When MSH2 is absent, the number of repairs that were left unrepaired increased substantially. If the cell continues to divide, mistakes accumulate in DNA and cell becomes unable to function normally and leads to formation of tumors in colon or in other part of the body. People with mutations in MSH2 have an increased risk of developing many other types of cancer including cancers of endometrium, liver, gall bladder, and skin tumors.
Knockdown of MSH2 by siRNA: Transient knock down of MSH2 is very important to increase correction efficiency in gene editing. Permanent knockdown of MSH2 will prevent its normal function and predispose cells to cancer. siRNA having specific sequence against MSH2 mRNA can be used to down regulate MSH2. Wild-type cells (containing eGFP mutant) transfected with siRNA, allow siRNA to act for 24 hours and knockdown the expression of MSH2. Protein samples from these cells and subjected to western blot to check reduced MSH2 levels. MSH2 suppressed cells can be used in gene editing and targeted with ODN. Correction efficiency can be analyzed through flow cytometry and fluorescent activated cell sorting.
Fig 7: Location of MSH2
Molecular Location on chromosome 2: base pairs 47,630,262 to 47,710,359
Hypothesis: If MSH2 and p53 levels are knocked down temporarily using siRNA, then the gene editing reaction will be less inhibited, therefore increasing correction efficiency or treatment efficiency.
Summary: DNA damage proteins MSH2 and P53 in spite of their normal functions like mismatch recognition and tumor suppression, these proteins have known to be significantly reducing the correction efficiency through their anti-recombinase activity and inhibitory action to homologous recombination respectively. With the use of siRNA temporary knockdown of their expression is possible, which if succeeded provides a superior method of treating genetic disorders.