CRISPR-Cas-Led Revolution in Diagnosis and Management of Emerging Plant Viruses: New Avenues Toward Food and Nutritional Security

Plant viruses pose a serious threat to agricultural production systems worldwide. The world's population is expected to reach the 10-billion mark by 2057. Under the scenario of declining cultivable land and challenges posed by rapidly emerging and re-emerging plant pathogens, conventional strategies could not accomplish the target of keeping pace with increasing global food demand. Gene-editing techniques have recently come up as promising options to enable precise changes in genomes with greater efficiency to achieve the target of higher crop productivity. Of genome engineering tools, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) proteins have gained much popularity, owing to their simplicity, reproducibility, and applicability in a wide range of species. Also, the application of different Cas proteins, such as Cas12a, Cas13a, and Cas9 nucleases, has enabled the development of more robust strategies for the engineering of antiviral mechanisms in many plant species. Recent studies have revealed the use of various CRISPR-Cas systems to either directly target a viral gene or modify a host genome to develop viral resistance in plants. This review provides a comprehensive record of the use of the CRISPR-Cas system in the development of antiviral resistance in plants and discusses its applications in the overall enhancement of productivity and nutritional landscape of cultivated plant species. Furthermore, the utility of this technique for the detection of various plant viruses could enable affordable and precise in-field or on-site detection. The futuristic potential of CRISPR-Cas technologies and possible challenges with their use and application are highlighted. Finally, the future of CRISPR-Cas in sustainable management of viral diseases, and its practical utility and regulatory guidelines in different parts of the globe are discussed systematically.

The CRISPR-Cas system: its origin, functions and applications in biotechnology: A Review

The CRISPR-Cas system is a genome editing system seen in prokaryotic immune system. Bacteria and archaea protect itself against invading viruses and plasmid by targeting RNA or DNA of the invading element predominantly using this gene-editing tool. The CRISPR- Cas defense mechanism is carried out in three stages; adaptation stage where the spacers are inserted into the CRISPR locus, the expression stage where crRNA is formed by transcription of the CRISPR loci and the interference stage where the invading element is destroyed by the crRNA and cas proteins. The CRISPR-cas has been involved in many other functions apart from the immune defense they include; DNA repair, regulation of virulence, genome evolution, inhibit biofilm formation etc. The application of CRISPR-Cas system include genome engineering, agriculture to efficiently target and mutate plants, improve crop yield and crop resistance, in medicine to eradicate genetic diseases. However, ethical considerations are a major setback of CRISPR-Cas application especially in medicine. CRISPR-Cas has been used in variety of species including cultured human cell, rice, drosophila and mice.

CRISPR-Based Approaches for the High-Throughput Characterization of Long Non-Coding RNAs

Over the last decade, tens of thousands of new long non-coding RNAs (lncRNAs) have been identified in the human genome. Nevertheless, except for a handful of genes, the genetic characteristics and functions of most of these lncRNAs remain elusive; this is partially due to their relatively low expression, high tissue specificity, and low conservation across species. A major limitation for determining the function of lncRNAs was the lack of methodologies suitable for studying these genes. The recent development of CRISPR/Cas9 technology has opened unprecedented opportunities to uncover the genetic and functional characteristics of the non-coding genome via targeted and high-throughput approaches. Specific CRISPR/Cas9-based approaches were developed to target lncRNA loci. Some of these approaches involve modifying the sequence, but others were developed to study lncRNAs by inducing transcriptional and epigenetic changes. The discovery of other programable Cas proteins broaden our possibilities to target RNA molecules with greater precision and accuracy. These approaches allow for the knock-down and characterization of lncRNAs. Here, we review how various CRISPR-based strategies have been used to characterize lncRNAs with important functions in different biological contexts and how these approaches can be further utilized to improve our understanding of the non-coding genome.

Definition of CRISPR Cas12a Trans-Cleavage Units to Facilitate CRISPR Diagnostics

The CRISPR diagnostic (CRISPR-Dx) technology that employs the trans-cleavage activities has shown great potential in diagnostic sensitivity, specificity, convenience, and portability, and has been recognized as the next-generation diagnostic methods. However, due to the lack of standardized definition of Cas trans-cleavage enzymatic units, it is difficult to standardize the present CRISPR-Dx systems, which have undoubtedly impeded the development of the CRISPR-Dx industry. To solve the problem, we here first systematically optimized the reaction systems for Cas12a, and then defined its trans-cleavage units (transU), which we believe will be of great importance and interest to researchers in both molecular diagnostic industry and basic research. Moreover, a simple protocol was provided to facilitate a step-by-step measurement of the Cas12a transU, which can also act as a reference for the definition of the transU for other Cas proteins.

In Silico Analysis of Pathogenic CRB1 Single Nucleotide Variants and Their Amenability to Base Editing as a Potential Lead for Therapeutic Intervention

Mutations in the Crumbs homolog 1 (CRB1) gene cause both autosomal recessive retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA). Since three separate CRB1 isoforms are expressed at meaningful levels in the human retina, base editing shows promise as a therapeutic approach. This retrospective analysis aims to summarise the reported pathogenic CRB1 variants and investigate their amenability to treatment with currently available DNA base editors. Pathogenic single nucleotide variants (SNVs) were extracted from the Leiden open-source variation database (LOVD) and ClinVar database and coded by mutational consequence. They were then analyzed for their amenability to currently available DNA base editors and available PAM sites from a selection of different Cas proteins. Of a total of 1115 unique CRB1 variants, 69% were classified as pathogenic SNVs. Of these, 62% were amenable to currently available DNA BEs. Adenine base editors (ABEs) alone have the potential of targeting 34% of pathogenic SNVs; 19% were amenable to a CBE while GBEs could target an additional 9%. Of the pathogenic SNVs targetable with a DNA BE, 87% had a PAM site for a Cas protein. Of the 33 most frequently reported pathogenic SNVs, 70% were targetable with a base editor. The most common pathogenic variant was c.2843G>A, p.Cys948Arg, which is targetable with an ABE. Since 62% of pathogenic CRB1 SNVs are amenable to correction with a base editor and 87% of these mutations had a suitable PAM site, gene editing represents a promising therapeutic avenue for CRB1-associated retinal degenerations.

Potential Use of CRISPR/Cas13 Machinery in Understanding Virus–Host Interaction

Prokaryotes have evolutionarily acquired an immune system to fend off invading mobile genetic elements, including viral phages and plasmids. Through recognizing specific sequences of the invading nucleic acid, prokaryotes mediate a subsequent degradation process collectively referred to as the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)–CRISPR-associated (Cas) (CRISPR–Cas) system. The CRISPR–Cas systems are divided into two main classes depending on the structure of the effector Cas proteins. Class I systems have effector modules consisting of multiple proteins, while class II systems have a single multidomain effector. Additionally, the CRISPR–Cas systems can also be categorized into types depending on the spacer acquisition components and their evolutionary features, namely, types I–VI. Among CRISPR/Cas systems, Cas9 is one of the most common multidomain nucleases that identify, degrade, and modulate DNA. Importantly, variants of Cas proteins have recently been found to target RNA, especially the single-effector Cas13 nucleases. The Cas13 has revolutionized our ability to study and perturb RNAs in endogenous microenvironments. The Cas13 effectors offer an excellent candidate for developing novel research tools in virological and biotechnological fields. Herein, in this review, we aim to provide a comprehensive summary of the recent advances of Cas13s for targeting viral RNA for either RNA-mediated degradation or CRISPR–Cas13-based diagnostics. Additionally, we aim to provide an overview of the proposed applications that could revolutionize our understanding of viral–host interactions using Cas13-mediated approaches.

Bioinformatic Identification of CRISPR Leader Motifs

Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas) proteins serve as a sophisticated adaptive immune system to defend bacteria and archaea from viral infection. CRISPR mediated immunity occurs in three stages which allow the bacteria to adapt and respond to new as well as previously encountered viruses. The initial step of CRISPR adaptation requires the help of the Integration Host Factor (IHF) and a stretch of 200 base pairs known as the CRISPR leader to ensure encounters with new viruses are properly recorded in the host organism’s immunological memory. A bioinformatic analysis of over 15,000 CRISPR leaders reveals that IHF is a prevalent and widespread feature of CRISPR adaptation across several different CRISPR subtypes and host organisms.

Bioinformatic Identification of CRISPR Leader Motifs

Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas) proteins serve as a sophisticated adaptive immune system to defend bacteria and archaea from viral infection. CRISPR mediated immunity occurs in three stages which allow the bacteria to adapt and respond to new as well as previously encountered viruses. The initial step of CRISPR adaptation requires the help of the Integration Host Factor (IHF) and a stretch of 200 base pairs known as the CRISPR leader to ensure encounters with new viruses are properly recorded in the host organism’s immunological memory. A bioinformatic analysis of over 15,000 CRISPR leaders reveals that IHF is a prevalent and widespread feature of CRISPR adaptation across several different CRISPR subtypes and host organisms.

Application of Cas12j for Streptomyces editing and cluster activation

In recent years, CRISPR-Cas toolboxes for Streptomyces editing have rapidly accelerated natural product discovery and engineering. However, Cas efficiencies are also oftentimes strain dependent, subsequently a variety of Cas proteins would allow for flexibility and enable genetic manipulation within a wider range of Streptomyces strains. In this work, we have further expanded the Cas toolbox by presenting the first example of Cas12j mediated editing in Streptomyces sp. A34053. In our study, we have also observed significantly improved editing efficiencies with Acidaminococcus sp. Cas12j compared to Cas12a, Francisella tularensis subsp. novicida U112's type V-A Cas (FnCpf1).