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RNA-Targeting CRISPR Systems
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RNA-Targeting CRISPR Systems

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Biochemistry".

Deadline for manuscript submissions: closed (20 August 2023) | Viewed by 14174

Special Issue Editor

Dr. Giancarlo Russo

E-Mail Website
Guest Editor
EMBL Partner Institute for Gene Editing, Life Sciences Center, Vilnius University, 10257 Vilnius, Lithuania
Interests: CRISPR-Cas; microbiology; next-generation sequencing; bioinformatics data analysis
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) systems are mechanisms of acquired defense in prokaryotes. CRISPR arrays are DNA stretches in the prokaryote genomic that store short genomic sequences (spacers) of bacteriophages from past attacks. Upon a new phage challenge, these spacers are spliced and the resulting RNA acts as a guide to binding to the complementary sequence in the attacking phage; the associated Cas effector then cleaves the phage DNA and the viral threat is neutralized.

Since their discovery, CRISPR-Cas systems have been engineered into the most promising genome editing tool: a programmable effector deliverable anywhere in a genomic sequence to modify a nucleotide sequence.

Initially, it seemed that CRISPR-Cas systems were capable of targeting exclusively DNA. Later it has been discovered that they can also cleave RNA. The main advantage of RNA-targeting CRISPR-Cas is that they act on a transient entity, therefore allowing modifications that might otherwise be deleterious or lethal if applied to the genome.

This Special Issue will focus on the growing number of applications of RNA-targeting CRISPR-Cas-based editing, such as transcriptional repression, RNA editing, guided isoform splicing, nucleic acid detection, expression regulation, and viral resistance. Additional suggested topics include RNA-targeting CRISPR systems structures and mechanisms of action, off-target activity in CRISPR-mediated RNA cleavage, and in silico methods for the discovery of new systems.

Dr. Giancarlo Russo
Guest Editor

Manuscript Submission Information

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Keywords

  • CRISPR-Cas
  • Cas13
  • Cmr
  • Csm
  • RNA editing
  • RNA knockdown
  • CRISPRi
  • off-target effects

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Published Papers (5 papers)

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Research

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14 pages, 2645 KiB  
Article
A New RNA-Dependent Cas12g Nuclease
by Natalia Gunitseva, Martha Evteeva, Aleksei Korzhenkov and Maxim Patrushev
Int. J. Mol. Sci. 2023, 24(23), 17105; https://doi.org/10.3390/ijms242317105 - 4 Dec 2023
Viewed by 1526
Abstract
The development of RNA-targeting CRISPR-Cas systems represents a major step forward in the field of gene editing and regulation. RNA editing presents a viable alternative to genome editing in certain scenarios as it offers a reversible and manageable approach, reducing the likelihood of [...] Read more.
The development of RNA-targeting CRISPR-Cas systems represents a major step forward in the field of gene editing and regulation. RNA editing presents a viable alternative to genome editing in certain scenarios as it offers a reversible and manageable approach, reducing the likelihood of runaway mutant variants. One of the most promising applications is in the treatment of genetic disorders caused by mutations in RNA molecules. In this study, we investigate a previously undescribed Cas12g nuclease which was found in metagenomes from promising thermophilic microbial communities during the expedition to the Republic of North Ossetia—Alania in 2020. The method outlined in this study can be applied to other Cas orthologs and variants, leading to a better understanding of the CRISPR-Cas system and its enzymatic activities. The cis-cleavage activity of the new type V-G Cas effector was indicated by in vitro RNA cleavage experiments. While CRISPR-Cas systems are known for their high specificity, there is still a risk of unintended cleavage of nontargeted RNA molecules. Ultimately, the search for new genome editing tools and the study of their properties will remove barriers to research in this area. With continued research and development, we may be able to unlock their full potential. Full article
(This article belongs to the Special Issue RNA-Targeting CRISPR Systems)
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11 pages, 3260 KiB  
Article
Site-Specific RNA Editing of Stop Mutations in the CFTR mRNA of Human Bronchial Cultured Cells
by Roberta F. Chiavetta, Simona Titoli, Viviana Barra, Patrizia Cancemi, Raffaella Melfi and Aldo Di Leonardo
Int. J. Mol. Sci. 2023, 24(13), 10940; https://doi.org/10.3390/ijms241310940 - 30 Jun 2023
Cited by 2 | Viewed by 1616
Abstract
It is reported that about 10% of cystic fibrosis (CF) patients worldwide have nonsense (stop) mutations in the CFTR gene, which cause the premature termination of CFTR protein synthesis, leading to a truncated and non-functional protein. To address this issue, we investigated the [...] Read more.
It is reported that about 10% of cystic fibrosis (CF) patients worldwide have nonsense (stop) mutations in the CFTR gene, which cause the premature termination of CFTR protein synthesis, leading to a truncated and non-functional protein. To address this issue, we investigated the possibility of rescuing the CFTR nonsense mutation (UGA) by sequence-specific RNA editing in CFTR mutant CFF-16HBEge, W1282X, and G542X human bronchial cells. We used two different base editor tools that take advantage of ADAR enzymes (adenosine deaminase acting on RNA) to edit adenosine to inosine (A-to-I) within the mRNA: the REPAIRv2 (RNA Editing for Programmable A to I Replacement, version 2) and the minixABE (A to I Base Editor). Immunofluorescence experiments show that both approaches were able to recover the CFTR protein in the CFTR mutant cells. In addition, RT-qPCR confirmed the rescue of the CFTR full transcript. These findings suggest that site-specific RNA editing may efficiently correct the UGA premature stop codon in the CFTR transcript in CFF-16HBEge, W1282X, and G542X cells. Thus, this approach, which is safer than acting directly on the mutated DNA, opens up new therapeutic possibilities for CF patients with nonsense mutations. Full article
(This article belongs to the Special Issue RNA-Targeting CRISPR Systems)
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16 pages, 4136 KiB  
Article
Harnessing the LdCsm RNA Detection Platform for Efficient microRNA Detection
by Zhenxiao Yu, Jianan Xu and Qunxin She
Int. J. Mol. Sci. 2023, 24(3), 2857; https://doi.org/10.3390/ijms24032857 - 2 Feb 2023
Cited by 1 | Viewed by 2010
Abstract
In cancer diagnosis, diverse microRNAs (miRNAs) are used as biomarkers for carcinogenesis of distinctive human cancers. Thus, the detection of these miRNAs and their quantification are very important in prevention of cancer diseases in human beings. However, efficient RNA detection often requires RT-PCR, [...] Read more.
In cancer diagnosis, diverse microRNAs (miRNAs) are used as biomarkers for carcinogenesis of distinctive human cancers. Thus, the detection of these miRNAs and their quantification are very important in prevention of cancer diseases in human beings. However, efficient RNA detection often requires RT-PCR, which is very complex for miRNAs. Recently, the development of CRISPR-based nucleic acid detection tools has brought new promises to efficient miRNA detection. Three CRISPR systems can be explored for miRNA detection, including type III, V, and VI, among which type III (CRISPR-Cas10) systems have a unique property as they recognize RNA directly and cleave DNA collaterally. In particular, a unique type III-A Csm system encoded by Lactobacillus delbrueckii subsp. bulgaricus (LdCsm) exhibits robust target RNA-activated DNase activity, which makes it a promising candidate for developing efficient miRNA diagnostic tools. Herein, LdCsm was tested for RNA detection using fluorescence-quenched DNA reporters. We found that the system is capable of specific detection of miR-155, a microRNA implicated in the carcinogenesis of human breast cancer. The RNA detection system was then improved by various approaches including assay conditions and modification of the 5′-repeat tag of LdCsm crRNAs. Due to its robustness, the resulting LdCsm detection platform has the potential to be further developed as a better point-of-care miRNA diagnostics relative to other CRISPR-based RNA detection tools. Full article
(This article belongs to the Special Issue RNA-Targeting CRISPR Systems)
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Review

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12 pages, 1083 KiB  
Review
RNA-Dependent RNA Targeting by CRISPR-Cas Systems: Characterizations and Applications
by Natalia Gunitseva, Marta Evteeva, Anna Borisova, Maxim Patrushev and Fedor Subach
Int. J. Mol. Sci. 2023, 24(8), 6894; https://doi.org/10.3390/ijms24086894 - 7 Apr 2023
Cited by 8 | Viewed by 3164
Abstract
Genome editing technologies that are currently available and described have a fundamental impact on the development of molecular biology and medicine, industrial and agricultural biotechnology and other fields. However, genome editing based on detection and manipulation of the targeted RNA is a promising [...] Read more.
Genome editing technologies that are currently available and described have a fundamental impact on the development of molecular biology and medicine, industrial and agricultural biotechnology and other fields. However, genome editing based on detection and manipulation of the targeted RNA is a promising alternative to control the gene expression at the spatiotemporal transcriptomic level without complete elimination. The innovative CRISPR-Cas RNA-targeting systems changed the conception of biosensing systems and also allowed the RNA effectors to be used in various applications; for example, genomic editing, effective virus diagnostic tools, biomarkers, transcription regulations. In this review, we discussed the current state-of-the-art of specific CRISPR-Cas systems known to bind and cleave RNA substrates and summarized potential applications of the versatile RNA-targeting systems. Full article
(This article belongs to the Special Issue RNA-Targeting CRISPR Systems)
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Other

Jump to: Research, Review

15 pages, 994 KiB  
Perspective
Current Bioinformatics Tools to Optimize CRISPR/Cas9 Experiments to Reduce Off-Target Effects
by Muhammad Naeem and Omer S. Alkhnbashi
Int. J. Mol. Sci. 2023, 24(7), 6261; https://doi.org/10.3390/ijms24076261 - 27 Mar 2023
Cited by 17 | Viewed by 5191
Abstract
The CRISPR-Cas system has evolved into a cutting-edge technology that has transformed the field of biological sciences through precise genetic manipulation. CRISPR/Cas9 nuclease is evolving into a revolutionizing method to edit any gene of any species with desirable outcomes. The swift advancement of [...] Read more.
The CRISPR-Cas system has evolved into a cutting-edge technology that has transformed the field of biological sciences through precise genetic manipulation. CRISPR/Cas9 nuclease is evolving into a revolutionizing method to edit any gene of any species with desirable outcomes. The swift advancement of CRISPR-Cas technology is reflected in an ever-expanding ecosystem of bioinformatics tools designed to make CRISPR/Cas9 experiments easier. To assist researchers with efficient guide RNA designs with fewer off-target effects, nuclease target site selection, and experimental validation, bioinformaticians have built and developed a comprehensive set of tools. In this article, we will review the various computational tools available for the assessment of off-target effects, as well as the quantification of nuclease activity and specificity, including web-based search tools and experimental methods, and we will describe how these tools can be optimized for gene knock-out (KO) and gene knock-in (KI) for model organisms. We also discuss future directions in precision genome editing and its applications, as well as challenges in target selection, particularly in predicting off-target effects. Full article
(This article belongs to the Special Issue RNA-Targeting CRISPR Systems)
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