Covid-19: Biomedical Perspectives by Charles S. Pavia;Volker Gurtler; & Volker Gurtler

Author:Charles S. Pavia;Volker Gurtler; & Volker Gurtler [Pavia, Charles S. & Gurtler, Volker]
Language: eng
Format: epub
ISBN: 9780323853408
Publisher: Elsevier
Published: 2022-06-02T00:00:00+00:00

Fig. 1 Schematics for SARS-Cov-2 detection using CRISPR/Cas platform. The RNA is extracted from the patient using conventional RNA extraction method. For Cas12 based detection the RNA is amplified into dsDNA whereas for Cas13 based detection the amplified DNA is transcribed into ssRNA. The collateral activity of both Cas12 and Cas13 is used for the detection of SARS-CoV-2.

Fig. 2 (A) CRISPR/Cas13d array showing HEPN domains. (B) Schematic of CRISPR/Cas13d mechanism as an antiviral strategy. Cas13d shown as a green cloud and guide RNA (DR and spacer) together forms a Cas13d:crRNA complex required for viral RNA degradation. DR: Direct repeat.

Consolidating the studies on antiviral capabilities of subtypes of CRISPR/Cas13 systems, Cox and co-workers detected that among the different orthologues of Cas13a, b and c; Cas13b (PspCas13b) was the most specific and efficient for RNA knockdown in mammalian cells (Cox et al., 2017). LwaCas13a had two major issues; first it must be stabilized with monomeric super-folded GFP and secondly, the average RNA knockdown efficiency was around 50% whereas PspCas13b provided better efficiency as compared to LwaCas13a with 62.9% knockdown. Although these systems can be reprogrammed to target ssRNA, it is difficult to pack them into adeno-associated vectors (AAV) for in-vivo delivery due to their large size. In comparison to Cas13a (1250 aa), Cas13b (1150 aa), and Cas13c (1120 aa), Cas13d effector has an average length of 930 aa (the smallest class2 CRISPR effector) (Cox et al., 2017; Shmakov et al., 2015; Smargon et al., 2017). Overall, the CRISPR/Cas13d system seems to be the most practical system for a therapeutic approach to target the SARS-CoV-2 genome. The small but robust CRISPR/Cas13d system seems to be the latest technology for the RNA engineering toolbox.

This claim may be further supported by the following statements.

i. The size of Cas13d (approx. 930aa) is 17–26% smaller than other class2 type VI- CRISPR/Cas subtypes (Cas13a-c); which makes it suitable for packaging in low- capacity vectors, like AAV; making it particularly suitable for delivery in primary cells and in-vivo applications.

ii. It lacks any sequence constraints for flanking sequences i.e., Cas13d does not require the presence of PFS. This property makes it possible to target theoretically any ssRNA sequence.

iii. Modular activity of WYL1 for CRSISPR/Cas13d system: CRISPR/Cas13d locus consists of an accessory protein with the WYL domain (named for three amino acids conserved in the original domain) (Yan et al., 2018). Co-occurrence of Cas13d with accessory proteins having a WYL-domain increases the targeted cleavage, implying such proteins act as regulators for Cas13d activity.

iv. Comparison between Cas13a/b effectors and Cas13d effector (CasRx) as RNA regulating systems, showed knockdown of approximately 70% by Cas13a/b enzymes whereas 96% by CasRx.

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