Gene Editing Techniques
1. Gene Editing – General Background
Gene editing entails inserting, deleting and replace the DNA in a genome of an organism utilizing engineered nucleases. Sections of the natural gene are replaced and completed using a synthetic DNA chain. Also, the natural repair process gets rid of the mismatches and gaps in the DNA. On the other hand, gene disruption is a technique by which the DNA fragment is utilized to replace a genome sequence with a chosen marker gene like the kanavanine resistance. DNA –binding proteins are proteins that consist of DNA-binding domains and hence have affinity for single and double stranded DNA. The paper is aimed at summarizing three gene editing methods including zinc finger nucleases, TALENS and CRISPR-Cas9.
Genetic editing allows genetic modification through the introduction of a double-strand break (DSB) in a certain genomic target sequence and then the desired modifications are made during DNA break repair. The double-strand break that is induced by the zinc finger nuclease is a designed sequence specific endonuclease that can be tailored to cleave of the target DNA. The endonuclease should show different qualities to be effective for genomic engineering. First, it should recognize the long target sequence and have adequate adaptability to retarget the user-defined sequences. The Zinc finger clease satisfies the specifications by connecting the DNA-binding domain of diverse class of eukaryotic transcription factors( Zinc finger proteins) with the nuclease domain of the FOK1 restriction enzyme. The zinc finger mix the favorable qualities of the DNA binding specificity and flexibility of zinc finger proteins and cleave activity that is robust and limited due to the lack of a binding event.
1. a. Definition of Gene Editing
Gene editing is a form of genetic engineering wherein a DNA is either inserted or removed from within a genome by employing artificially contrived nucleases. The process of gene editing essentially involves modifying the nucleotide order of genomes.
1. b. Definition of Gene Disruption
In medical terms, gene disruption is defined as the use of a combination of both the in vitro as well as in vivo to replace an easily chosen mutant gene for the purpose of creating a wild-type gene eventually.
1. c. DNA Binding Proteins
DNA binding proteins are those proteins which comprise of domains that are DNA binding enabled. Such proteins have a specific or typical similarity to either a single-stranded or a double-stranded DNA. Two of the most popular such DNA binding proteins that have been identified through rigorous research are the helix-turn-helix and zinc finer structural motifs.
1. d. Objectives of the review – Summarize three gene editing techniques
Recent technological advancements in the field of genome editing through the use of programmable nucleases have considerably enhanced the ability to make specific changes to the genomes of the eukaryotic cells. Genome editing techniques are already revolutionizing the field of biotechnology while also the ability to interpret the role played by genetics in disease by enabling the formation of more precise cellular and animal models of the pathological processes.
A predominantly enticing use of programmable nucleases is the ability to directly modify various genetic mutations in the tissues and cells that are affected, for treating the diseases that are intractable to conventional therapies. A few such genome editing techniques are gene editing, gene disruption as well as DNA binding proteins.
Gene editing is already tinkering with mankind as it is being extensively used for treating many diseases that are difficult to be cured through conventional therapies. The gene-editing technique is already being used extensively in the field of chemical research and is also believed to hold the ability to alter human DNA with extraordinary ease in the next few years to come. A limited number of experiments have been already approved by the British regulators, especially in relation to human embryos. This technology has the capacity to treat diseases like cystic fibrosis or even sickle cell while also being able to resuscitate extinct species.
Another similar advancement in the field of genome editing is gene disruption, which is also alternatively called gene knockout. This is a genetic technique wherein one of the genes in a living being is knocked out, precisely non-functional. The reason behind using gene disruption and making one of the genes non-functional is to study and understand about specific genes, which would have been sequenced, yet that has either a totally unknown or incomplete information about its function.
The third technology that is becoming highly popular these days is the Zinc finger nucleases (ZFN), which is basically a DNA binding protein. In order to be beneficial in the field of genome engineering, an endonuclease must display an unusual grouping of qualities like for instance, “specific recognition of long target sequences (ideally, long enough for unique occurrence in a eukaryotic genome) coupled with sufficient adaptability for retargeting to user-defined sequences.” The architecture of a ZFN is in complete conformity with the above listed specifications, as the ZFNs link the DNA-binding domain of an adaptable class of “eukaryotic transcription aspects namely the zinc finger proteins (ZFPs) with the nuclease domain of the FokI restriction enzyme.”
2. Zinc Finger Nucleases (ZFN)
2. a. ZFN – Conceptual Background
Species that possess genomes which can be experimentally manipulated govern our ability to examine the pivotal role that genes play biology as well as disease. In creatures like yeast and mice, the capacity to precisely include or erase genetic information facilitates an matchless level of accuracy and meticulousness in studying the functions of genes, and eventually this leads to increased understanding and information about biological mechanisms in the above mentioned species when compared to any other species that fall within the respective taxonomic category.
Genome editing is a technique that enables the extension or enhancement of the capabilities to not just cells as well as also to the entire entities from actually any species. This sort of editing essentially makes way for accurate and specific genetic modification by inducting a double-strand break (DSB) in a particular genome sequence of target, which further involves production of desired alterations during the course of the DNA break repair that ensues. The induction of the DBS happens with the use of ZNFs (zinc finger nucleases).
ZFNs can be defined as a “designed, sequence specific endonuclease that can be customized to cleave a user-chosen DNA target. Since the most recent comprehensive review of the subject.” In order to be beneficial in the field of genome engineering, an endonuclease must display an unusual grouping of qualities like for instance, “specific recognition of long target sequences (ideally, long enough for unique occurrence in a eukaryotic genome) coupled with sufficient adaptability for retargeting to user-defined sequences.”
Protein structure and structure of DNA binding domain
ZFNs are structured in such a way that they combine the positive qualities of both the components, namely, the DNA binding specificity as well as flexibility of the zinc-finger proteins (ZFPs) and a cleavage activity which is strong but controlled when a specific binding event lacks. Yet, the ZFNs retain their functional modularity. Ultimately, both the DNA-binding as well as the catalytic domains can possibly be improved when separated, and this is believed to simply retargeting and support the endeavors of improvement.
Use of ZFNs in gene disruption and gene editing
Gene disruption is possibly one of the simplest forms of genome editing, Gene disruption takes the advantage of flaws that are introduced during the process of DNA repair for disrupting as well as also abolishing the task of a specific gene or a genomic territory. ZFNs are highly helpful and are also extensively used in gene disruption. Below is an example:
In order to disrupt a gene D. melanogaster, the ZFNs that target exonic sequences can be passed through the help of mrNA injection into the early embryo of a fly. The end result of this process is that roughly about 10% of the progeny that is produced by adult flies gets mutated for the specific gene that has been targeted.
2. b. Development of Technique
Conventionally, in order to successfully create a gene disruption animal is purely based on the availability of an embryonic stem cell lines. Till now, such a possibility has only been established in creatures like mouse and rate. Recent developments in the ZFN technology has enabled editing genomes of virtually any animal. Domesticated animals like rabbits, pig and silkworm have also been subject to gene knockout, by adoption of the ZFN technology. Other model animals like frog, zebra-fish, fruit fly, mouse and rate have also been used.
Selecting high affinity sites
Modular assembly method was used in yellow cat fish by employing ZFNs. The nucleotide sequence in the first exon had codon of yellow fish namely the mstn gene, which became the input for identifying and targeting sites as well as the corresponding three zinc-finger left array and three zinc-finger right array with the help of ZiFit software. “S elected potential target sites in the yellow catfish mstn gene output from the software were ACCCACTGCCGTGACGGAGGAGCGt (nt+113—+138) (with GNN score 0.59 by 0.59 and affinity score 3.77 by 4.77) and gATTTCTCTGGGCTTCGTGGTGGCTt (+21—+46) (with GNN score 0.59 by 0.00 and affinity score 6.79 by N/A). The fingers in the output from the same modular sources (SGMO or Barbas)” were selected to make the 3-zinc-finger arrays of two pairs of ZFNs correspondingly
Advantages and disadvantages
In the contemporary approach towards genetic modification, the commonality is the use of engineered non-specific nucleases, which are fused to DNA-binding domains. Such binding domains are cleverly designed for provision of target specificity and the introduction of nucleases which put in double stranded DNA creates the appropriate breaks in the neighboring sequence. Random mutations can be introduced by healing the double-stranded breaks using blunt and non-homologous end joining. Homologous DNA repair can be achieved by introducing an engineered DNA with homology on both sides of the DNA break. This encodes specific mutation.
ZNFs were among the first genome editing nucleases. These are most common DNA-binding domains, especially in eukaryotes. These typically have about 30 amino acid modules, which interact with nuclear tide triplets. The ZNFs have been specifically designed to recognize complete 64 possible tri-nucleotide combinations, stringing together, and various zinc finger moieties. The ZNFs that can recognize any DNA triplets can be created.
The primary advantage of ZFNs is that they take extremely short time to generate knockouts in rats, approximately 6 months to be precise. Moreover, the targeted gene disruption is highly efficient through ZFNs and the ES cell is also applicable to other species as well. One of the primary disadvantages is the screening and assembly of these molecules is quite challenging. In addition, there is a possibility of the background mutations around the targeted genome masking the prototypes aimed for knockout.
Uses of ZFNs in aquaculture genome editing
Recently, a latest technology in relation to gene targeting has been identified with the use of engineered ZFNs that was applied to a fish species. Yet, the successful application of the same has not yet been found out in species that have slow paced development, for instance Salmonidae. Yano et, al., present their finding of the successful application of ZFN for gene disruption in a rainbow trout (Oncorhynchus mykiss). Three pairs of ZFN mRNA that were targeted to specific regions of the sdY gene were injected into rainbow trout eggs that were fertilized. Further, sperm from a 1-year-old male parental generation one or P1, which was carrying a ZFN-induced mutation in their germline was further employed for the production of F1 non-mosaic animals. In these F1 populations, 14 different mutations were characterized in the sdY gene, which even comprised one mutation resulting to the obliteration of leucine 43 (L43) and 13 mutations at other targeted “sites having different effects on the SdY protein, i.e., amino acid insertions, deletions, and frameshift mutations producing premature stop codons in the mRNA.”
3. TALENS
3. a. Background
Transcription activator-like effecter nuclease (TALEN) technology takes advantage of the artificial restriction enzymes that are produced by connecting the TAL effector DNA-binding domain to a DNA cleavage domain. Restriction enzymes cut the DNA strand at a certain sequence. TALEN can be easily engineered to bind any expected DNA sequence.
Origin
TALEN originated from a study on the Xanthomonas genus. The bacteria affect different plants including rice, tomatoes, pepper and cause economic losses in the agricultural sector and hence the motivation for studying them. The bacteria produced effectors proteins known as transcription activator-like effectors (TALEs) to the cytoplasm. The protein affected the cell processes and increased their vulnerability to Xanthomonas genus. Further research was done to determine the mechanisms of action and it showed that the protein able to bind with the DNA and activate the expression of the target genes by imitating the eukaryotic transcription factors.
TALE proteins consist of a central domain that facilitates DNA binding and a nuclear localization signal. They also have a domain that induces the transcription of the target genes. His protein’s ability to bind to the DNA was examined in 2007 and two groups of investigators studied the code to establish whether the TALE protein recognized the target DNA. The DNA-binding domain comprised of monomers and each binds a single nucleotide in the target nucleotide sequence.
Monomers are repeats of 34 amino acid residues and two are situated at the 12 and 13 and they are highly different. The amino acids recognize the nucleotide. The code is degenerative and some of the repeat variable diresidue bind to different nucleotides with varying efficiencies. The molecule of the target DNA has a similar nucleotide, thyimidine that impacts the binding efficiency. The final tandem repeat binds the nucleotide at the 3’ end of the recognition area comprising of 20 amino acid residues. TALES attracted the attention of researchers globally because of its simplicity and the first studies on the development of chimeric TALEN nucleases were initiated. Hence, the sequence encoding the DNA-binding domain of TALEs was inserted into the plasmid vector utilized to develop ZFN. This led to the production of genetic constructs that expressed artificial chimeric nucleases that contained the DNA-binding domain and catalytic domain of restriction endonuclease FOKI.
Uses in Species Genome Editing
TALENs are used in species genome editing as evidenced in tilapia, rats and silkworms. Li et al. (2013) believe that TALENs are an important approach fir targeted genome editing and they have been shown to be effective in different organisms. The researchers noted that TALENS can activate somatic mutations in Nile Tilapia. They developed six pairs of TALENS to target genes linked to sex differentiation.
The genes included dmrt1, foxl2, cyp19a1a, gsdf, igf3 and nrob 1b. The genes led to mutations with maximum efficacy of almost 81% at the chosen loci (Li et al., 2013). The investigators analyzed the effects of dmrt1 and foxl2 mutation on sex differentiation, gonadal phenotype and gene expression using histology, real-time PCR and immunohustochemistry (Li et al., 2013). In dmrt1-lacjing tests, phenotyoes of considerable testicular regression such as deformed efferent ducts were noted. Dmtr1 disruption in XY fish led to elevated foxl2 and cyp19a1a expression, 11-ketotestotes.
Natural Function of Protein
“The standard architecture of a TALEN uses the requisitely dimeric catalytic domain (CD) of the Type IIS restriction enzyme FokI.” TALEN activity eventually would need two DNA recognition territories that flank an unspecific central spacer zone, which is efficient in DNA cleavage and also has interdependence with spacer length as well as the TALE scaffold construction. The extremely redundant nature of the underlying DNA sequence coding for engineered TALEs has made required dedicated synthesis techniques. In addition, TALEN half’s’ are approximately three times bigger than the canonical designer nucleases, and have a total protein complex that is typically greater than 1,800 amino acids.
3. b. Development of Technique – use in gene editing and gene disruption
Zhang et. al. were the first set of scientists to develop a method to create sequence specific TALENs in cultured human cells. Since that time, TALENs have also been constructed in other organisms like rats, zebra fish, yeast, and round worms among others. TALENs have not been able to induce targeted disruptions in silkworms. They also have not been able to create sophisticated genetic modifications other than mere disruptions in organisms. Genetic manipulations enable study of model insects as also exploratory studies and control of insect vectors.
Genetically modified insect generation is a reality in certain key model systems over the last decade or so. Most of these have been based upon transposon vectors. Scientific and public concerns have been raised against transposon vectors that were derived from genetically altered organisms because there are several limitations like random insertion, possible instability and even reduced carrying capacity. Improvement of transposon vector system has been continuous. The utilization of specific recombinases as well as establishment of gene targeting strategies, in union with application of ZNFs has greatly made genome editing sophisticated. However, it still remains a large challenge in insect species. It is estimated that the use of TALENs would lead to overcoming limitations of genetic modification technologies.
Advantages and disadvantages of TALENs
A key advantage of TALENs, similar to the ZFNs is that they are not restrained in terms of mutagenesis in mouse embryonic stem cells. ZNF as well as TALEN modifications have been administered in species like zebrafish, nematodes, rats, and even in monarch butterflies.
The specific use of TALENs to overcome current challenges of genetic modification technologies is seen as a great application. In several cases of manipulating the genome, it is expected that several hundred base pairs could be deleted. This is one of the biggest disadvantage. Adopting ZFNs with single-stranded oligodeoxy nucleotides has enabled targeted deletions of targeted pairs of chromosomes in human beings. Such simplicity and efficiency of TALENs for complicated genome manipulation provides several new possibilities of genetic manipulation in insect species.
3. d. Uses in specific genome editing
TALENs are extremely powerful aides in terms of targeted genome editing as they have been proved to be highly efficient in various types of species. Li, et.al. presented the results of their study of Antagonistic roles of Dmrt1 and Foxl2 in sex differentiation via estrogen production in tilapia as demonstrated by TALENs. This particular study reported TALENs are capable of inducing somatic mutations in Nile tilapia, a very important species for the global aquaculture, with dependably high efficacy.
“Six pairs of TALENs were constructed to target genes related to sex differentiation, including dmrt1, foxl2, cyp19a1a, gsdf, igf3, and nrob1b, and all resulted in indel mutations with maximum efficiencies of up to 81% at the targeted loci. Effects of dmrt1 and foxl2 mutation on gonadal phenotype, sex differentiation, and related gene expression were analyzed by histology, immunohistochemistry, and real-time PCR.” In addition, disruption of Cyp19a1a in the XX fish also resulted to partial reversal of sex with the expression of Dmrt1 and Cyp11b2. In conclusion, this study proved TALENs to be a highly effective tool that are highly helpful in targeted gene editing in tilapia genome. Notably, Foxl2 as well as Dmrt1 play hostile roles in the differentiation of the sex in Nile tilapia through the regulation of cyp19a1a expression along with estrogen production.
CRISPR-Cas9
Background
CRISPR are sections of the prokaryotic DNA that gave short repetition base sequences. The Cas system / CRISPR is a prokaryotic immune system that provides resistance to foreign genetic materials like phages and plasmids. It also provides a kind of acquired immunity. The spacer identifies and cut the genetic materials in the same way as the RNA interference. CRISPR/Cas9 is a gene detection method that targets and modifies the DNA. The CRISPR system was discovered two years after discovering the TALEN protein. CRISPR are non-coding RNA and CAS proteins.
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