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Emerging Nanotechnology-Enabled Approaches to Mitigate COVID-19 Pandemic
Published in Devarajan Thangadurai, Saher Islam, Charles Oluwaseun Adetunji, Viral and Antiviral Nanomaterials, 2022
Maria Shoukat, Samiullah Khan, Arshad Islam, Maleeha Azam, Malik Badshah
The CRISPR–Cas12-based lateral flow assay technique is recently established for diagnosis of COVID-19 infection. It is a good substitution for real-time RT-PCR technique, and it is easy to implement in short time duration of 40 minutes (Broughton et al. 2020). The FET-based biosensors use monoclonal antibodies against the SARS-CoV-2 spike protein with coating of the graphene sheets of the FET. This FET biosensor device can detect SARS-CoV-2 spike protein 100 fg/mL concentration in the viral transport medium and 1 fg/mL concentration in phosphate-buffered saline (PBS) (Seo et al. 2020).
CRISPER Gene Therapy Recent Trends and Clinical Applications
Published in Yashwant Pathak, Gene Delivery, 2022
Prachi Pandey, Jayvadan Patel, Samarth Kumar
As CRISPR–Cas system is found in bacteria, it undertakes evolution rapidly and eventually may produce new Cas genes which will encode new proteins. Thus, these proteins may have the budding potential for genome editing or other similar applications within the near future. Of late, Cas12a has been known to have great purpose in genome editing. CRISPR technology has also been of great use to treat cancer and other diseases. CRISPR–Cas9 technology experiments are normally performed in vitro in model organisms and stem cells like human pluripotent stem cells (hPSCs). Gene editing by this technology is also conducted in human embryonic stem cells (ESCs) in vitro to correct mutations, but research in ESCs gives rise to many ethical issues. However, it is undeniable that the genome editing of ESCs has the potential to give rise to organisms possessing outstanding desirable qualities. Safety measures are essential while using this technology to prevent its misuse or minimize the risk of the negative impact of genome editing.
The Evolution of COVID-19 Diagnostics
Published in Debmalya Barh, Kenneth Lundstrom, COVID-19, 2022
Praveen Rai, Ballamoole Krishna Kumar, Deekshit Vijaya Kumar, Prashant Kumar, Anoop Kumar, Shashi Kumar Shetty, Biswajit Maiti
CRISPR is short palindromic repeat DNA sequences usually found in prokaryotes as a part of their defense mechanism. CRISPR requires a small RNA fragment called the guide RNA (gRNA), which binds to the complementary target sequence, and a nuclease enzyme cleaves at the precise site. In the case of viral nucleic acid detection, the gRNA in turn binds to the target segment of the viral gene. Several CRISPR associated Cas proteins (cas9, cas12 or cas13) have been shown to exert nonspecific endonuclease activity to cleave DNA or RNA. More recently, it was reported that the CRISPR-Cas12a protein could detect nucleic acids of exogenous viruses, like the human papillomavirus from cervical cancer patients in raw plasma, without the need for RNA extraction [33]. Recently, a CRISPR-based technology DETECTR was used to detect SARS-CoV-2 in RNA extracted clinical specimens [32]. Subsequently, a combination of CRISPR-Cas13a with a reader device based on a mobile phone was developed for detection of SARS-CoV-2 RNA extracted from nasal swabs [34]. Furthermore, SHERLOCK (Specific High sensitivity Enzymatic Reporter unlocking) a CRISPR-Cas12b-based test for detecting SARS-CoV-2, was developed [35]. In addition, Ding et al. developed the All-In-One Dual CRISPR-Cas12a (AIOD-CRISPR) assay for SHERLOCK Testing in One-Pot (STOP) and visual detection of SARS-CoV-2 [32]. However, the majority of these methods involve isolation of SARS-CoV-2 RNA from the specimens that might further increase the risk of cross-contamination and virus transmission. To avoid such complications, a CRISPR-Cas12a-based point of care SARS-CoV-2 test was developed recently, where CRISPR Cas-12a was combined with RT-RPA to directly analyze lysed samples without the need for any RNA extraction. The test showed a sensitivity of 0.1 copies /μl for the detection of SARS-CoV-2 from clinical specimens in 60 minutes [36].
An overview: CRISPR/Cas-based gene editing for viral vaccine development
Published in Expert Review of Vaccines, 2022
Santosh Bhujbal, Rushikesh Bhujbal, Prabhanjan Giram
CRISPR/Cas gene editing requires two basic components: a Cas enzyme and guide RNA. These components are associated to form a ribonucleoprotein (RNP) complex. CRISPR/Cas system involves different types of Cas enzymes, among them, Cas9 and Cas12a are the most widely used for gene editing. The Cas9 enzyme is a nonspecific type II CRISPR locus, derived spCas9 from Streptococcus pyogenes SF370 [14]. The Cas12a enzyme, also known as Cpf1, is derived from Acidaminococcus sp. (AsCas12a) and Lachnospiraceae bacterium (LbCas12a) [18]. The RNP Complex: Cas protein and gRNA together shown to have a bacterial defense system and also some antiviral effect [19]; this is followed by three steps: 1) Acquisition- in which newly infected viral DNA invades the leading CRISPR locus, 2) Expression- in which the Cas gene is expressed and the CRISPR system is transcribed into a precursor CRISPR-RNA (Pre-crRNA) [20], which subsequently matures into a short mature crRNA with spacers and repeats. 3) Interference- if the virus DNA infects the bacteria again, the CRISPR/Cas9 system interferes, allowing the bacteria to keep a record of the infection and the CRISPR locus to serve as a genetic vaccination card for those bacteria [21,22].
Molecular detections of coronavirus: current and emerging methodologies
Published in Expert Review of Anti-infective Therapy, 2022
Mingkun Diao, Lang Lang, Juan Feng, Rongsong Li
Different CRISPR/Cas formats, such as CRISPR/Cas9 [28], Cas 12a [29], Cas 13 [30] as well as Cas14 [31], have been developed to detect different pathogens. For coronavirus detection, a technique called specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) [27] that combines isothermal amplification and Cas13a cleavage have been invented. In a clinical study conducted in Thailand with 154 nasopharyngeal and throat swab samples, SHERLOCK was reported to have a detection limit of 42 RNA copies of SARS-CoV-2 per sample, showing 100% specificity and 100% sensitivity with a fluorescence readout, and 100% specificity and 97% sensitivity in a lateral-flow assay (LFA) format readout [32]. STOP (SHERLOCK Testing in One Pot), a derivative of SHERLOCK, was shown to detect 100 copies of viral genome in saliva or nasopharyngeal swabs per reaction with comparable sensitivity to RT-PCR for SARS-CoV-2 [33]. Taking use of Cas12a, Broughton et al. developed a DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) assay in a LFA format readout. This method can also achieve comparable performance of US CDC approved RT-PCR assay for SARS-CoV-2 detection [34].
Potential of CRISPR/Cas system in the diagnosis of COVID-19 infection
Published in Expert Review of Molecular Diagnostics, 2021
V. Edwin Hillary, Savarimuthu Ignacimuthu, S. Antony Ceasar
CRISPR/Cas12 is also known as CRISPR-associated endonuclease from Prevotella and Francisella 1 (cpf1); it belongs to the Class 2 type V associated nuclease [49,50]. CRISPR/Cas12 system is an efficient system that creates staggered cuts in both ssDNA and dsDNA. The Cas12 is derived from Acidaminococcus species Cas12a (AsCas12a) and Lachnospiraceae bacterium Cas12a (LbCas12a), which naturally evolves to fight against foreign genetic elements. The CRISPR/Cas12 system requires only the crRNA to create staggered cuts at the targeted sequence. The Cas12 nuclease contains two domains namely (1) nuclease lope (NUC) domain and (2) RuvC domain for cleavage activity [51,52]. Like the Cas9 system, the Cas12 system targets the region besides the PAM region. Once Cas12 starts to encounter, it initiates R-loop formation which forms base-pair hybrids between the crRNA and the target sequence [53]. During this step, it matches the <17 bp target sequence and establishes an R-loop formation. Once R-loop is formed, the Cas12 nuclease uses its active RuvC domain and creates staggered cuts at the targeted ssDNA or dsDNA sequence with the presence of PAM sequence [50,54].