approach is RNA interference (RNAi) where host RNAi machinery is used to target
viral RNA (Zaidi et al. 2016) but it has
many off target effects (Romay 2017). Also, viruses evolve rapidly by recombination
and mutation thus counter these strategies. With the advent of new techniques,
the most efficient strategy for controlling viruses is genome engineering where
precise alteration in genome is made and among them, CRISPR Cas9 is the most
widely used strategy providing broad spectrum and durable viral resistance in
crops (Khatodia et al. 2017).
CRISPR/Cas9 system is an RNA-guided, site-specific genome-editing technology
that scales from single-gene loci to whole genomes. In this application,
accuracy and uniformity of oligo synthesis is critical to ensure representation
of guides and specificity of targeting. In this article, we summarize the
recent progress in CRISPR/Cas9 system, mechanisms underlying and illustrate a
strategy to develop crop plants resistance to multiple viral infections.
Genome engineering (GE)
Genome engineering (GE)
refers to techniques in which genome of an organism is altered at specific site
thus enabling introduction of trait of interest in plants (Sovova et al. 2016; Zaidi et al. 2017).
In order to avoid public and political concerns regarding use of transgenes in
crops, the transgene can be eliminate from improved variety (Woo et al. 2015; Kanchiswamy 2016;
Zhang et al. 2016). GE is carried out by
nucleases (SSNs) which creates double
stranded break in DNA at specific site (Fig. 1). The double strand break can
then be repaired by either non-homologous end-joining (NHEJ) or
homology-directed repair (HDR) (Aouida et al. 2014,
Ali et al. 2015b; Piatek and Mahfouz 2016). NHEJ pathway disrupts translational reading
frame of coding sequence by introduction of small insertion or deletion at site
of various lengths and is error prone (Sander and Joung 2014). HDR uses
donor DNA as template and thus used to introduce desired sequences through
recombination (Ding et al. 2016).
1. Mechanism for repairing of double strand break caused by sequence-specific
nucleases (SSNs) by non-homologous
end-joining (NHEJ) and homology-directed repair (HDR) methods.
Precise and efficient
genome-targeting technologies are needed to enable systematic reverse
engineering of causal genetic variations by allowing selective perturbation of
individual genetic elements. Genome editing involves three programmable
nucleases: zinc ?nger nucleases (ZFNs) (Pabo et al.
2001), transcription activator–like e?ector nucleases (TALENs)
(Boch et al. 2009; Moscou and Bogdanove 2009), and clustered regularly interspaced short
palindromic repeat (CRISPR) and CRISPR associated proteins (Cas) system (van
der Oost 2013) (Fig. 2). ZFNs and TALENs are
artificial chimeric dimer proteins having engineered DNA binding domain joined
to a nonspecific DNA cleavage domain from the FokI restriction enzyme (Kim et al. 1996). The ZFNs recognize three nucleotides while
TALENs recognize single nucleotide. The ef?ciency of ZFN for targeted gene
mutagenesis is lower (1.7–19.6 %) than that of TALEN (30–48 %) (Townsend et al. 2009; Lloyd
et al. 2005; Shukla et al. 2009). The major drawback of
ZFNs and TALENs is that designing of DNA binding protein to target sequence of
interest is expensive as well as laborious (Ceasar et al. 2016).