Methods for in vitro CRISPR/CasRx-Mediated RNA Editing

Chuang, Yu-Fan; Wang, Peng‐Yuan; Kumar, Satheesh; Lama, Suraj; Lin, Fan-Li; Liu, Guei‐Sheung

doi:10.3389/fcell.2021.667879

Cited by 15 publications

(15 citation statements)

References 26 publications

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“…128 However, modulation of these silencing therapeutics will be key to their success, aiming for optimal protein concentrations that can lead to photoreceptor survival, also importantly avoiding off target effects. 26 Regulatory agencies closely overseeing the safety of novel therapeutic approaches such as CRISPR-mediated DNA (Cas9) and RNA (CasRx) 129 editing will be necessary. Gene regulation of certain novel approaches, including the RNA editors, might also aid their safety profile.…”

Section: Discussionmentioning

confidence: 99%

Genetic treatment for autosomal dominant inherited retinal dystrophies: approaches, challenges and targeted genotypes

Varela

Georgiadis²,

Michaelides

2022

Br J Ophthalmol

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Inherited retinal diseases (IRDs) have been in the front line of gene therapy development for the last decade, providing a useful platform to test novel therapeutic approaches. More than 40 clinical trials have been completed or are ongoing, tackling autosomal recessive and X-linked conditions, mostly through adeno-associated viral vector delivery of a normal copy of the disease-causing gene. However, only recently has autosomal dominant (ad) disease been targeted, with the commencement of a trial for rhodopsin (RHO)-associated retinitis pigmentosa (RP), implementing antisense oligonucleotide (AON) therapy, with promising preliminary results (NCT04123626).Autosomal dominant RP represents 15%–25% of all RP, with RHO accounting for 20%–30% of these cases. Autosomal dominant macular and cone-rod dystrophies (MD/CORD) correspond to approximately 7.5% of all IRDs, and approximately 35% of all MD/CORD cases, with the main causative gene being BEST1. Autosomal dominant IRDs are not only less frequent than recessive, but also tend to be less severe and have later onset; for example, an individual with RHO-adRP would typically become severely visually impaired at an age 2–3 times older than in X-linked RPGR-RP.Gain-of-function and dominant negative aetiologies are frequently seen in the prevalent adRP genes RHO, RP1 and PRPF31 among others, which would not be effectively addressed by gene supplementation alone and need creative, novel approaches. Zinc fingers, RNA interference, AON, translational read-through therapy, and gene editing by clustered regularly interspaced short palindromic repeats/Cas are some of the strategies that are currently under investigation and will be discussed here.

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Section: Discussionmentioning

confidence: 99%

Genetic treatment for autosomal dominant inherited retinal dystrophies: approaches, challenges and targeted genotypes

Varela

Georgiadis²,

Michaelides

2022

Br J Ophthalmol

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“…Moreover, the majority of the type VI-D orthologues loci were found to harbor an additional accessory component called the WYL domain. This WYL domain founds to play a crucial role in enhancing and auxiliary modulating the ssRNA cleavage activity in a dose-dependent manner ( Yan et al, 2018 ; Zhang et al, 2018 ; Chuang et al, 2021 ).…”

Section: Cas13d and Its Structural Featuresmentioning

confidence: 99%

“…This ternary complex is also called as “cleavage competent” complex because the HEPN1 and HEPN2 domain with two R-X 4-6 -H motifs forms a catalytic endoRNase HEPN-domain dimer which resulted in the formation of the bipartite active site to arbitrate the hydrolysis of the target as well as collateral ssRNAs. On the contrary, such “collateral cleavage’ by Cas13d has not been detected in mammalian cells and even in plants ( Figure 7 ) (Min et al, 2019; Chuang et al, 2021 ). Furthermore, studies have shown some distinct requirement to get optimal Cas13d endoRNase activity such as i) HEPN nuclease activity of Cas13d is strongly depend on the base pairing of target ssRNA i.e., binding of > 21-nucleotide complementarity with crRNA spacer causes full conformational activation and optimal cleavage activity of Cas13d while 18–20 nucleotide complementarity causes half-maximal cleavage activity; ii) presence of any mismatches in the two separate crRNA spacer region (an internal region (spacer nucleotides 5–8) and 3′-end region (spacer nucleotides 13–22) with target ssRNA, founds to be intolerant and completely eradicate the ssRNA cleavage activity; iii) presence of Mg 2+ and additional accessory component WYL domain increases the endoRNase activity; iv) Cas13d tends to cleave only structurally accessible ssRNA sequences, any secondary structure present in the target RNA sequence it fails to recognize and the RNA cleavage activity is abolished; v) Cas13d has exhibited no preference for PFS imposed ssRNA cleavage but has shown considerable preference for uracil bases in target ssRNA structures.…”

Section: Mechanism Of Cas13d Rna Cleavage Activitymentioning

confidence: 99%

Cas13d: A New Molecular Scissor for Transcriptome Engineering

Gupta¹,

Ghosh²,

Chakravarti³

et al. 2022

Front. Cell Dev. Biol.

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The discovery of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and its associated Cas endonucleases in bacterial and archaeal species allowed scientists to modify, utilized, and revolutionize this tool for genetic alterations in any species. Especially the type II CRISPR-Cas9 system has been extensively studied and utilized for precise and efficient DNA manipulation in plant and mammalian systems over the past few decades. Further, the discovery of the type V CRISPR-Cas12 (Cpf1) system provides more flexibility and precision in DNA manipulation in prokaryotes, plants, and animals. However, much effort has been made to employ and utilize the above CRISPR tools for RNA manipulation but the ability of Cas9 and Cas12 to cut DNA involves the nuisance of off-target effects on genes and thus may not be employed in all RNA-targeting applications. Therefore, the search for new and diverse Cas effectors which can precisely detect and manipulate the targeted RNA begins and this led to the discovery of a novel RNA targeting class 2, type VI CRISPR-Cas13 system. The CRISPR-Cas13 system consists of single RNA-guided Cas13 effector nucleases that solely target single-stranded RNA (ssRNA) in a programmable way without altering the DNA. The Cas13 effectors family comprises four subtypes (a-d) and each subtype has distinctive primary sequence divergence except the two consensuses Higher eukaryotes and prokaryotes nucleotide-binding domain (HEPN) that includes RNase motifs i.e. R-X4-6-H. These two HEPN domains are solely responsible for executing targetable RNA cleavage activity with high efficiency. Further, recent studies have shown that Cas13d exhibits higher efficiency and specificity in cleaving targeted RNA in the mammalian system compared to other Cas13 endonucleases of the Cas13 enzyme family. In addition to that, Cas13d has shown additional advantages over other Cas13 variants, structurally as well as functionally which makes it a prominent and superlative tool for RNA engineering and editing. Therefore considering the advantages of Cas13d over previously characterized Cas13 subtypes, in this review, we encompass the structural and mechanistic properties of type VI CRISPR-Cas13d systems, an overview of the current reported various applications of Cas13d, and the prospects to improve Cas13d based tools for diagnostic and therapeutic purposes.

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“…The application of CRISPR has important implications for the treatment of human genetic diseases, the discovery of new drugs and rapid diagnosis of diseases [ 20 – 22 ]. In the context of emerging infectious diseases, the CRISPR-Cas system, as a fast, accurate and convenient genome editing tool, provides a fast, inexpensive and highly sensitive diagnosis when used for nucleic acid detection [ 23 , 24 ].…”

Section: Crispr-cas Biology and Principlementioning

confidence: 99%