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The genome editing tools and CRISPR technologies have reached the current exciting stage following decades of foundational research and development by a huge team of researchers. This review will present a brief history and the significant advances in the field of genome editing and significant genome-engineering tools. However, the review will primarily discuss the CRISPR technology.

Its applications outside genome editing, such as targeted gene control, epigenetic modification, chromatin manipulation, and live cell chromatin imaging, will be particularly emphasized. Finally, it will address the current and future impact of these tools in science, medicine, and biotechnology. The genomes of the eukaryotic organism are made up of billions of DNA bases. The capacity to modify these DNA bases at exactly predetermined positions is of immense value not only for molecular biology but also for medicine and biotechnology.

Thus, the introduction of desired modifications into genomes, i.e., genome editing, has been a long-desired objective in molecular biology. To this end, the discovery of restriction enzymes that naturally protect bacteria against phages in the late 1970s was a breakthrough that energized the era of recombinant DNA technology. For the first time, scientists acquired the ability to manipulate DNA in test tubes.

Even though such endeavors pioneered numerous discoveries in genetics and molecular biology, the capability to specifically modify DNA in living eukaryotic cells arrived a few decades later. In the mid to late 1980s, gene research, transcriptome, proteome, genome link, nebula dna several breakthroughs emerged that were aimed towards this end. Targeted gene disruption studies were first performed in eukaryotic yeast cells4 and soon followed by Capecchi and Smithies with their work on mammalian cells. Their study demonstrated that mammalian cells are capable of incorporating an exogenous copy of DNA into their genome through a process called homologous recombination.

Targeted nucleases for genome editing

Scientists sought alternative methods to overcome the limitations. The first breakthroughs were when they realized that an increase in targeted genome integration occurs by several orders of magnitude following the introduction of a double-strand break at a target site. Thus, many research groups made efforts to create various strategies to introduce targeted DSBs. During the first experiments, researchers utilized rare endonuclease-cutting enzymes such as the 18-bp cutter I-Sei to create specific DSBs in the mouse genome10.

Although such mega nucleases, which target long pieces of 14–40 bp DNA, increased the genome editing efficiency, gene library, neogen genomics, genomapp, genome dna, sgrna library the method was subject to two main drawbacks. Initially, despite the existence of hundreds of natural occurrence mega nucleases, each of them has a unique recognition sequence. Therefore, the chances of identifying a mega nuclease that can target a specific locus remained low. Secondly, and most importantly, most induced DSBs are repaired by the error-prone, non-homologous end-joining DNA repair pathway. Thus, not only might the exogenously added DNA template fail to integrate at the DSBs, but the NHEJ repair process might also insert or delete DNA fragments at random at the break sites.

The advent of CRISPR as the genome-editing tool

Though the identification of artificially engineered mega nucleases followed by ZFNs and TALENs successively improved the capability to edit the genome, the treatment of varied targets on the genome required redesign or re-engineering of a new set of proteins. The difficulties of cloning and engineering proteins somewhat limited these tools from common usage by the scientific community. In this sense, CRISPR has revolutionized the game since it is as effective as, if not more effective than, the existing tools when it comes to editing.

More importantly, it is much simpler and more adaptable to use. The CRISPR gene-editing tool, as we know it today, is an endonuclease protein whose DNA-targeting specificity and activity to cleave can be engineered by a short guide RNA. Even though the term CRISPR was created much later, these repeat elements were first observed in Escherichia coli by Dr. Nakata’s group. Curiously, the CRISPR repeat clusters were unlike the ordinary tandem repeats of the genome as they were demarcated by non-repetitive DNA spacers.

The time taken by researchers to discover the nature and source of such spacer sequences took over a decade. In the human genome project, genomes from several other organisms like several different phages were sequenced as well. The computational analysis of genomic sequences resulted in the observation of major characteristics of CRISPR repeat and spacer elements by researchers.

Evolution of second-generation CRISPR gene-modifying tools

The evolution of genome editing technology has been among the key developments in the CRISPR technology field. Unlike WT Cas9, which produces DSBs and indels at random in target sites, these so-called second-generation genome-modifying technologies can replace one base with another without creating DNA DSBs. The Nickase Case represents the base platform for the base editor tools that make it possible to convert C to T or A to G directly at the target site without causing DSBs.

Recently, Komor et al. demonstrated a fusion complex of nickase Cas9 fused with an APOBEC1 deaminase enzyme and Uracyl Glycosylase inhibitor protein effectively converts Cytosine to Thymine at the target site in the absence of inducing double-strand DNA breaks88. Interestingly, a transfer RNA adenosine deaminase has also been engineered and paired with nickase Cas9 to construct another novel base editor that executes direct A–G conversion at the target locations.

These new base-editing technologies significantly extend the scope of the application of genome targeting. The tools are also being enhanced for other applications by researchers. We and others have recently harnessed the power of this CRISPR base editor to modify the genetic code and introduce early STOP codons within genes. The CRISPR-STOP approach is a highly effective and less harmful alternative to WT-Cas9-mediated gene knockout studies. In addition to the APOBEC adenosine deaminase enzyme, the activation-induced adenosine deaminase enzyme has also been conjugated with the dCas9 enzyme.

CRISPR-mediated epigenome editing

The word epigenetics is genome extremely controversial. Here, we use the term epigenetic to imply the molecular mechanism of heritable variation in gene expression that is not a result of variation in DNA sequence information. While epigenetics is the process, the epigenome is all post-translational modifications and other chromatin features that are associated with regulatory elements of the genome. To this end, locus-specific epigenome map tools and technologies are expected to play an important role in allowing researchers to decipher the functional functions of chromatin modifications. They will allow the investigation of some of the long-standing questions of chromatin biology, such as the causal relationship between the existence of an epigenetic mark and gene expression.

CRISPR-mediated misdirections of chromatin topology

Yet another promising CRISPR genome front in the field of chromatin biology is the targeted chromatin loop architecture engineering. Targeted engineering of artificial loops between regulatory genomic regions provides a means to manipulate endogenous chromatin architectures to define the function and contribution of the latter to gene expression. Such a procedure can enable the creation of new enhancer-promoter contacts to overcome certain genome deficiencies. In addition, the abnormally active enhancer-promoter interaction may be inhibited.

Thus, these methods are of great therapeutic relevance as well. Toward this end, a demonstration that gene expression could be induced from a developmentally repressed endogenous locus by forced looping of the chromatin was an important step toward demonstrating the potential of this system. Researchers are now using dCas9-based platforms to achieve targeted and efficacious manipulation of chromatin architecture and DNA loop formation.

In a stunning recent work, Morgan et al. used two dimerizable protein domains from the plant hormone abscisic acid signaling pathway. Attachment of these protein-dimerization systems to two distinct dCas9 orthologues enabled forced chromatin loop formation between remote enhancer and promoter regions. Notably, this inducible chromatin loop resulted in increased gene expression at the β-globin locus in the proper K562 hematopoietic cells but not HEK293T cells.

Conclusion

Future breakthroughs in genome editing miniaturizing the size of existing Cas proteins or discovering smaller Cas9 proteins are highly desirable. As CRISPR technologies broaden in application and functionality, social and ethical issues surrounding their use are also growing, and the applications of these powerful tools require more scrutiny. One of these CRISPR applications with a long-term consequence is the so-called gene drive with the potential to attack an entire population or a species.

In this remarkable CRISPR application, researchers have demonstrated that a gene allele for the parasite-resistant phenotype in mosquitos can spread quickly across the population in a non-Mendelian fashion. Such applications can significantly fortify us in the fight against malaria-type diseases. But as such applications have worldwide implications, safety redundancies must be carefully designed, and other regulatory processes must be considered and implemented in advance.

The latest CRISPR technologies will undoubtedly continue to revolutionize basic as well as clinical and biotechnological research. But the path ahead is not smooth sailing. One of them is the potential for immunogenicity to CRISPR-Cas9 proteins. The most widely used Cas9 proteins are S. aureus and S. pyogenes. Notably, since these bacteria cause infectious disease in humans at high frequencies, recently, a publication reported that more than half of humans may already have pre-existing humoral and cell mediated adaptive immune responses to Cas9 proteins.