Introduction to Gene Editing
Question:
Discuss About The Proceedings National Academy Of Sciences?
Gene editing or genome editing is the psychology that allows modification in the DNA code of an organism. The alteration can be done by the addition, removal or alteration of the genetic materials at specific locations of the genome. A recent method known as the CRISPR Cas9 (abbreviation for clustered regularly interspaced short palindrome repeats-CRISPR and CRISPR-associated protein 9-Cas9). This technology was adapted from the bacterial system of gene editing that allows them to ‘remember’ viral genome by creating arrays of genetic codes called the CRISPR arrays. Once the virus whose genetic code has been added to the CRISPR array of the bacterial DNA enters the bacterial cell, cas9 enzymes are then released to cut the DNA to disable the virus. This system is used by researches to create short RNA strands that has a guide sequence, and binds to a particular DNA code in the genome as well as cas9 enzyme. The RNA sequence then recognizes the specific DNA sequence while the cas9 enzyme cuts the region. Enzymes like cpf1 can also be used instead of Cas9 for cutting the DNA fragments. Once the specific sequence is cut from the genome, it can be repaired with the correct code through the DNA repair process or replaced with a customized DNA code (ghr.nlm.nih.gov 2018; Mali et al. 2013). This can be useful to detect and correct aberrations in the gene that is often characteristics of diseases like cancer (Isola et al. 1995). This technology is currently getting a lot of interest in the research on treatment for human diseases like cystic fibrosis, hemophilia, sickle cell anemia, cancer and HIV (Schwank et al. 2013; Park et al. 2015; Hsu et al. 2014).
(Discussion about the methods of the study. Note: this will be a review of a secondary research)
The study by Hsu et al. (2014) analyzed the use of CRISPR cas9 genome editing technology through secondary analysis of literature. Their studies discuss how CRISPR technology can be used to design programmable nucleases, allow precise editing of the genome, the structural architecture of Cas9, how this technique can be used in an eukaryotic cell, how their recognition fidelity can be improved, and how the technology can be used in research, medicine and biotechnology. The secondary research allows analysis of various studies done on the topic to bring together a comprehensive understanding (Kothari 2004).
Studies have shown that targeted double stranded breaks in the DNA can help genome editing through homologous recombination (HR). Specific locus specific homologous recombination (HR) has also been demonstrated using designer nucleases made from zinc finger proteins (ZFP). Additionally, due to a lack of a template for repair for exogenous homology, double stranded breaks can introduce deletions or insertions through non-homologous pathways of joining of the ends (Hsu et al. 2014). Modifiable DNA binding proteins like: zinc finger nuclease, transcription activator-like effectors (TALEs) and cas9 have been identified that can identify specific DNA sequences (Cong et al. 2013; Mali et al. 2013). The guide sequence in CRISPR array corresponds to the genomic sequence of the phage, thereby providing antiviral resistance. Replacement of this sequence can then be made with a sequence of interest, and thereby targeted by the cas9 nuclease. This customizable DNA binding domain can allow modulation, as well as recruit desired changes like transcriptional activation of a specific genetic locus (Mendenhall et al. 2013).
Understanding CRISPR Cas9 Genome Editing Technology
Studies by Sapranauskas et al. (2011) showed that type II CRISPR system can be transferred, and transplanting the CRISPR locus from Streptococcus thermophilus to Eschericia coli allowed the reconstitution of the CRISPR interface in the recipient cell. Studies by Gasiunas et al. (2012) also showed that purified cas9 guided by guide RNA can be used in vitro to target DNA sequences. Furthermore, RNA guide sequence is made by combining trans-activating crRNA with target guide sequence and tracrRNA (Jinek et al. 2012). This marketing type II CRISPR systems can allow genome editing of mammalian cells, and multiple guide RNA to target multiple genetic codes simultaneously(Cong et al. 2013; Mali et al. 2013; Sander et al. 2014).
Electromagnetic reconstructions of cas9 enzyme of Streptococcus pyogenes shows a significant reshuffling with apo-cas9 (not bound to nucleic acid) and cas9 (in a conjunction with crRNA and tracr RNA) that forms a core path for RNA-DNA hybrid formation (Jinek et al. 2014). Research by Nishimasu et al. (2014) pointed that the cas9 domain consisted of alpha helical recognition (REC) lobe as well as a nuclease lobe. This suggests Spcas9 unbound to guide RNA or target DNA has autoinhibited conformation and the active site of the enzyme is blocked. The guide RNA acts as scaffold surrounding which the folding of cas9 can occur to organize the domains (Nishimasu et al. 2014).
Case 9 is related exclusively to the type II CRISPR locus and functions as a typical type II gene. The type II locus is further categorized into 3 subtypes: IIa, IIb and IIc (Chylinski et al. 2013). This loci generally consists of cas9, cas1, cas2 gene apart from the CRISPR array and tracrRNA, however, IIC locus contains minimal cas genes, and IIA and IIB contains additional genes like csn2 and cas4 (Chylinski et al. 2013). However, even with the evident diversity of genetic assembly, cas9 have structurally similar domains (Fonfara et al. 2013). The type II CRISPR have been identified only in bacterial systems, not like in the case of type I and III, which are found in bacteria and archaea (Chylinski et al. 2013).
Molecule imaging studies shows that the complex of cas9-crRNA-tracrRNA associates with protospacer-adjacent motif first (PAM) at the 3’ end of the target DNA thereby dictating the search method of cas9, and discriminate self versus non self, and facilitate the formation of cleavage conformation of the enzyme (Shah et al. 2013). This binding triggers the nuclease activity of the enzyme, and is evidenced by its domain flexibility (Sternberg et al. 2014; Nishimasu et al. 2014). The PAM has a high specificity within each ortholog even among same species. The range of targets of the cas9 toolkit however can be expanded by the inclusion of additional PAM and allow orthogonal genome editing (Chylinski et al. 2013; Fonfara et al. 2013). However, the specific need of PAM is modified by replacing the PAM interacting domain from one species to another as shown by Nishimasu et al. 2014.
Cas9 derived from Streptococcus pyogenes (SpCas9) was used successfully for editing the genome of various types of cells like bacteria, pig human cell lines, mouse, roundworm, zebra fish, yeast, fruit fly, rat, monkeys, and even common crops (Sander and Joung 2014). Studies by Niu et al. (2014) showed the use of SpCas9 to induce multiplex mutations in monkeys. CrisprCas9 can also act in parallel to target and cleave multiple sequences (Garneau et al. 2010). This ability can be harnessed to indi8ce multiple perturbations, allowing multiplex editing in mammalian cells (Hsu et al. 2014).
Applications of CRISPR Cas9 Genome Editing Technology
Genome editing by cas9 have allowed accelerated the generation of transgenetic models, expanding the scope of biological studies beyond traditional animal models. The psychology also helps to understand the causative functions of variations in genes in the disease aetiology, through the reiteration the mutations in genetic pool (Sander and Joung 2014). Novel transgenic animal models can also be developed with mutations introduced at specific locus or corrected by in-vivo or ex vivo gene correction techniques (Niu et al. 2014; Schwank et al. 2013). Considering that genetic aberrations can lead to cancers, such technology can be useful for cancer research.
The ability to change several target genomes parallel to each other, a genome wide functional screen can be developed that can help to find genetic codes that underlie specific charactersistsics of interest (Hsu et al. 2014). Studies by Wang et al. (2014) and Shalem et al. (2014) showed thousands of genetic elements can be altered parallel, by the insertion of sgRNA directed towards all genes, along with cas9 or in a cell that already produces cas9 enzyme in human cell lines. This can help to create perturbations in non coding genetic elements as well as dissect the function of complex genetic elements by tiled micro deletions of genes. It can therefore be used to map large uncharacterized regions of chromosomes (Hsu et al. 2014). This can be another useful technique to identify genes that can lead to diseases.
The machinery of RNA polymerase can be modulated sterically through the binding of dCas9 molecules to the DNA elements. This property can be used to convert cas9 into a transcriptional activator (Qi et al. 2013). A decent transcriptional upregulation may be attained by targeting cas9 activators in each sgRNA for a promoter gene (Konerman et a. 2013). Such aspects can be used to understand the modulation of the transcription machinery, and study how transcription factors control the expression of genes (Hsu et al. 2014).
Highly complex epigenetic states create a highly dynamic landscape of complex genomic functions. Epigenetic machinery that alters histones acts as transcriptional regulators and play vital role in biological function. Specific genomic loci introduced through different enzymes were able to induce methylation of the DNA or acetylation of the histones (Hsu et al. 2014). This can be a useful mechanism to study the epigenetic factors associated with diseases. This can also be used to understand the epigenetic modifications that shape the regulatory networks of genomes. However, careful characterization of the off target functions and crosstalk between the different genomic domains shill needs to be characterized in detail (Hsu et al. 2014).
Studies show that the function of the genes is controlled by the spatial positioning of the structural as well as functional components in a cell, and this system can be either suppressed or amplified dynamically. Genomic loci that are far apart can be brought close to each other thereby allowing long range trans interactions. This allows a robust way of visualizing the DNA of live cells by analysing the interplay between genes through dynamic states of the chromatin. Labeling of DNA with cas9 enable capturing of live processes and is a good alternative to DNA FSH (Chen et al. 2013). This can be used to understand the processes of diseased as well as normal cells to understand the differences in the cell physiologies and metabolic pathways (Hsu et al. 2014_.
Secondary Research Analysis of CRISPR Cas9 Genome Editing Technology
Study of the structure of cas9 molecule, can help to to split it into two component subunits and then aid their reassembly through light inducible heterodimeric domains, this can allow systemic control of cas 9 in patients as well as animal models (Hsu et al. 2014). This can allow precise tuning of the cas9 molecule to induce specific changes in the genome of the test cell.
Conclusion
CRISPR cas 9 in a novel technology that allows genomic editing with high fidelity and specificity. This technology was derived from the bacterial system that provided them protection against viral pathogens. The system works by incorporating the viral DNA into the bacterial genomic locus called the CRISPR array. The CRISPR array also codes for specific enzymes like cas9 that recognizes specific regions of the DNA, based on the guide RNA codes. Genomic editing is done by synthesizing a guide RNA code complementary of DNA target sequence, and joining the cas9 molecule to the guide sequence. Once the RNA sequence hybridizes with the DNA target, the cas9 enzyme cuts the DNA segment. Later, the missing fragment can be replaced by any genomic locus of choice. This technology has been studied extensively for the treatment of several genetic diseases and has shown promising results in animal test and human cell lines. The ability of CRISPR cas9 to induce multiple genetic changes simultaneously, modulate transcription mechanisms and analyze epigenetic modifications of genes, has made it possible to study both simple and complex genomic functions. This has a vital implication in the understanding of how different disease changes the genetic as well as epigenetic functions. The technology also provides a breakthrough way of visualizing live cells, and can be used in the study of cellular functions in greater detail.
References:
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