Different guide RNAs have different targeting efficacies.When performing a genome modification, several factors contribute to the success of the edit. Related: Sanger Sequencing Solutions for SARS-CoV-2 Research Importance of Evaluating Gene Editing Efficacy in Genome Modification Abbott and colleagues recently published a proof-of-concept study using this strategy to digest RNA genome from SARS-CoV-2 and Influenza A virus to inhibit viral replication in human lung epithelial cells. One of the powerful applications of these nucleases is to direct targeting and modification of RNA virus genomes, including the genomes of several respiratory pathogens. CRISPR for Genome Modification in Research of SARS-CoV-2 and Influenza A have been discovered and optimized to expand the selection of targets as well as features such as small size and protospacer adjacent motif (PAM) sequences. Several new nucleases, including RNA-guided nucleases such as Cas12a, Cas12b etc. The CRISPR/Cas9 system continues to be improved and adapted to new problems. Recent studies in macaques showed that Cas9 was effective in eliminating the proviral DNA of simian immunodeficiency virus (SIV), a virus closely related to HIV, from infected blood cells as well as tissue reservoirs of the virus and preventing its replication. A major bottleneck for achieving HIV cure is that the virus integrates into host genome and therefore infected patients must be on lifelong antiviral treatment to suppress virus replication. In addition to genetic diseases, CRISPR has also shown promise as a potential treatment strategy for HIV infection. Sensitivity and specificity are measures of a diagnostic test’s ability to correctly classify an animal as having a disease or not having a disease. Gene-edited mice exhibited a robust expression of dystrophin in the muscles and amelioration of the disease-associated phenotype.Ī diagnostic test with high sensitivity and specificity is the key to solving this problem. For instance, Bengtsson and colleagues showed the application of the system in correcting a Duchenne muscular dystrophy (dmd) gene mutation in an animal model of the disease through muscle specific expression of CRISPR/Cas9. Several applications of CRISPR have been demonstrated for the research of debilitating and life-threatening diseases. Applications of CRISPR in Disease Research Genetic and Infectious Diseases Muscular Dystrophy Unlike TALENs, which require a unique transcription activator-like (TAL) effector protein for each target DNA sequence, CRISPR only requires switching guide RNA sequences, making the latter much easier for gene editing applications. The cut made by the nuclease is then the site that gets modified, either by non-homologous end joining or by template directed repair. This gRNA then directs the CRISPR/Cas9 nuclease complex to the specific sequence. To target a gene, a guide RNA (gRNA) is designed that bears complementarity to the region of interest. This mechanism was subsequently adapted for eukaryotic genomes and was first shown to modify human and mice cells in 2013. The Spacer sequences, when transcribed and processed into small RNAs, form a functional complex with a nuclease (Cas9) which recognizes and digests the incoming foreign DNA. Briefly, it involves integration of short nucleic acid sequences of the foreign DNA (Spacer) into the host genome adjacent to an array of repetitive elements (Repeats). In its native role, CRISPR is an adaptive defense mechanism used by bacteria and other prokaryotes to protect against viruses and plasmid challenges. In 2020, Emmanuelle Charpentier and Jennifer Doudna, won the Nobel Prize in Chemistry for biochemically characterizing the Cas9-mediated DNA modification system. Related: Solutions for Infectious Disease Research The Advent of Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR)Īlthough other genome editing methods, such as TALENs (Transcription activator-like effector nucleases) and ZFNs (zinc finger nucleases), have been in use, CRISPR is newer and simpler gene editing technology. Given the implications of DNA modification on cell function, it is important to validate and understand the nature of such genetic edits using accurate and reliable tools. Methods such as CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats) have opened a world of possibilities for researchers to not only understand the genetic basis of several human pathologies, but also develop therapeutic strategies against both inborn as well as infectious diseases such as HIV, SARS-CoV-2 and others. The last decade has witnessed the rapid development and advancement of gene editing technologies.
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