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Review
. 2021 Jan 12:11:609376.
doi: 10.3389/fmicb.2020.609376. eCollection 2020.

Next-Generation Sequencing and the CRISPR-Cas Nexus: A Molecular Plant Virology Perspective

Muhammad Shafiq Shahid  1 , Muhammad Naeem Sattar  2 , Zafar Iqbal  2 , Amir Raza  1 , Abdullah M Al-Sadi  1
Affiliations
Review

Next-Generation Sequencing and the CRISPR-Cas Nexus: A Molecular Plant Virology Perspective

Muhammad Shafiq Shahid et al. Front Microbiol. .

Abstract

In recent years, next-generation sequencing (NGS) and contemporary Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) technologies have revolutionized the life sciences and the field of plant virology. Both these technologies offer an unparalleled platform for sequencing and deciphering viral metagenomes promptly. Over the past two decades, NGS technologies have improved enormously and have impacted plant virology. NGS has enabled the detection of plant viruses that were previously undetectable by conventional approaches, such as quarantine and archeological plant samples, and has helped to track the evolutionary footprints of viral pathogens. The CRISPR-Cas-based genome editing (GE) and detection techniques have enabled the development of effective approaches to virus resistance. Different versions of CRISPR-Cas have been employed to successfully confer resistance against diverse plant viruses by directly targeting the virus genome or indirectly editing certain host susceptibility factors. Applications of CRISPR-Cas systems include targeted insertion and/or deletion, site-directed mutagenesis, induction/expression/repression of the gene(s), epigenome re-modeling, and SNPs detection. The CRISPR-Cas toolbox has been equipped with precision GE tools to engineer the target genome with and without double-stranded (ds) breaks or donor templates. This technique has also enabled the generation of transgene-free genetically engineered plants, DNA repair, base substitution, prime editing, detection of small molecules, and biosensing in plant virology. This review discusses the utilities, advantages, applications, bottlenecks of NGS, and CRISPR-Cas in plant virology.

Keywords: CRISPR; CRISPR associated (Cas) proteins; genome editing; next generation sequencing (NGS); plant viruses.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
A generalized CRISPR-Cas-based resistance induction model. The identification, recruitment, and cleavage of the invading pathogen’s nucleic acid (fungi, bacteria, viruses, and/or phytoplasma) are achieved in three fundamental steps. 1. Acquisition: the invaded DNA (bar in red color) is integrated into the CRISPR-array (white rectangles) as new spacers; 2. Expression: the pre-CRISPR and crRNAs machinery is triggered and expressed in the invaded cell; 3. Interference: the mature crRNA hybridized to the invading pathogen genome and subsequently recognized by the Cas proteins. The coordination of Cas helicase and nuclease with CRISPR RNA machinery results in the invader’s nucleic acid cleavage.
FIGURE 2
FIGURE 2
Overview of the host–virus interaction, the role of HTS, CRISPR-Cas system, and the discovery of unknown viruses.
FIGURE 3
FIGURE 3
A schematic approach to engineer CRISPR-Cas-based antiviral resistance in crop plants. (A) Designing of sgRNA to target the virus-encoded genes or the host susceptibility factors. (B) Construction of a cassette to express multiple sgRNAs and Cas protein under suitable promoters. The Cas protein can be expressed under CaMV-35S promoter and NOS terminator sequences, while sgRNAs can be expressed under the RNA polymerase-III promoter. Direct repeat (DR) sequences can follow the cloning of individual sgRNAss. (C) A suitable recombinant binary vector carrying the multiplexed sgRNAs cassette can be employed for the stable in vitro genetic transformations in plants. (D) The successful GE plants are tested for resistance against single or multiple plant viruses through mechanical, Agrobacterium- or vector-mediated plant inoculation techniques. The plants expressing effective CRISPR-Cas machinery would show resistance against the invading viruses. (E) In planta CRISPR-Cas-based genetic resistance model against potato virus Y (PVY): 1. CRISPR-Cas components are transcribed in the successfully transformed plants. 2. The Cas protein and respective sgRNAs are assembled to make a complex. 3. Mechanical or vector-mediated transmission of PVY in the primary plant cell. 4. Uncoating and subsequent host-mediated translation of the viral RNA genome. Multiple viral proteins help in viral replication at the periphery of the endoplasmic reticulum. 5. The Cas:sgRNA complex recruits and subsequently binds to the targeted PVY genome. 6. The activation of the Cas:sgRNA complex disrupts the genes involved either in viral translation, virion assembly, or long-distance movement through plasmodesmata. (F) In planta CRISPR-Cas-based genetic resistance model against an ssDNA plant virus, cotton leaf curl Kokhran virus (CLCuKoV), and it associated DNA-satellites. 1. CRISPR-Cas components are transcribed in the successfully transformed plants. 2. The Cas protein and respective sgRNAs are assembled to form a complex. 3. Agrobacterium- or vector-mediated transmission of CLCuKoV and associated DNA-satellites in the primary plant cell. 4. The ssDNA viral genome replication in the cellular nuclei following rolling circle replication (RCR) via dsRNA intermediates. The Replication associated protein (Rep) initiates RCR and generates free 3’-hydroxyl end to prime ssDNA synthesis by nicking dsDNA molecules. 5. The Cas:sgRNAs complex recruits and subsequently binds to the targeted genes encoded by CLCuKoV and/or DNA-satellites genomes, respectively. 6. The activation of the Cas:sgRNA complex cleaves the viral genes involved either in viral translation, virion assembly, or long-distance movement through plasmodesmata. (G) A recessive resistance model mediated by host factors against plant viruses (RNA or DNA). During the infection cycle, plant viruses interact with several host factors to endure their successful infection. Many host factors are known as promising candidates in antiviral breeding, which do not perturb the plant development or growth if mutated. These host factors aid virus replication (1–3), transcription (4–5), translation (6–8), or intercellular long-distance movement (9–10). These host factors can be targeted through CRISPR-Cas based GE to break their interaction with viral proteins.
FIGURE 4
FIGURE 4
Different Cas proteins that have been opted widely to engineer antiviral resistance in plants. (A) The conventional CRISPR-Cas9-based genome editing is mediated through a single effector protein Cas9, the crRNA, and a tracrRNA. The crRNA and tracrRNA associated with Cas9 endonuclease are hybridized and subsequently bind to the target region with the help of ∼20 nucleotide (nt) guide RNA (gRNA) sequence upstream of the PAM sequence, respectively. The recognition lobe (REC) is responsible for recognizing this crRNA:tracrRNA:target DNA complex. The PAM interacting domain (PI) recognizes the PAM sequence. The HNH and RuvC domains in the NUC lobe cleave the target strand and the non-target strand through a blunt-ended double-stranded break (DSB) at the upstream of the PAM sequence, respectively. (B) The conventional Cas9 has been modified to reduce off-target mutations. SpCas9 is generated by introducing point mutations in one of the two nuclease domains, RuvC, which produces single-stranded breaks (SSB) rather than DSB. (C) The specificity of Cas9 protein is also enhanced by fusing catalytically inactive “dead-Cas9” (dCas9) to an RNA-guided Fok1 nuclease. (D) Unlike CRISPR-Cas9, the CRISPR-Cas12a-based GE is mediated through a single effector protein (Cas12a) associated with a single crRNA. The REC lobe recognizes the Cas12a:crRNA complex, which subsequently binds to the target strand specifically at the target region downstream of the PAM sequence. The PI domain recognizes the PAM sequence of ∼23–25 nt long gRNA, thus helping specific DNA-binding. A DSB is introduced at the target region, with the Nuc domain responsible for cleaving the target strand 18 nt downstream of the PAM, while the non-target strand is cleaved by RuvC domain 23 nt downstream of the PAM, respectively. (E) The CRISPR-Cas13-based GE is mediated via a single effector protein (Cas13) associated with single crRNA. The REC lobe is responsible for recognizing the Cas13 and crRNA complex, which binds to the recognition site based on sequence complementarity with the ssRNA substrate directed by the gRNA sequence of crRNA. The sequence-specific cleavage of the ssRNA substrate is mediated through HEPN1 and HEPN2 domains. Instead of PAM sequences, Cas13a protein is directed toward the ssRNA target via a single protospacer flanking sequence (PFS). (F) The CRISPR-Cas13b is distinct from Cas13a due to the presence of a suppressor and enhancer Cas genes directed by two PFS. (G) The CRISPR-Cas14a-based ssDNA GE is mediated via single-effector Cas14a in association with crRNA and a 130-bp-long tracrRNA. The crRNA:tracrRNA complex does not require the presence of the PAM sequence in the ssDNA substrates. Nevertheless, ssDNA cleavage by the RuvC domain requires sequence-specific complementarity of 20 nt in the crRNA guide sequence to the ssDNA substrate.
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