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Abstract
Genome-editing, a recent technological advancement in the field of life sciences, is one of the great examples of techniques used to explore the understanding of the biological phenomenon. Besides having different site-directed nucleases for genome editing over a decade ago, the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) based genome editing approach has become a choice of technique due to its simplicity, ease of access, cost, and flexibility. In the present review, several CRISPR/Cas based approaches have been discussed, considering recent advances and challenges to implicate those in the crop improvement programs. Successful examples where CRISPR/Cas approach has been used to improve the biotic and abiotic stress tolerance, and traits related to yield and plant architecture have been discussed. The review highlights the challenges to implement the genome editing in polyploid crop plants like wheat, canola, and sugarcane. Challenges for plants difficult to transform and germline-specific gene expression have been discussed. We have also discussed the notable progress with multi-target editing approaches based on polycistronic tRNA processing, Csy4 endoribonuclease, intron processing, and Drosha ribonuclease. Potential to edit multiple targets simultaneously makes it possible to take up more challenging tasks required to engineer desired crop plants. Similarly, advances like precision gene editing, promoter bashing, and methylome-editing will also be discussed. The present review also provides a catalog of available computational tools and servers facilitating designing of guide-RNA targets, construct designs, and data analysis. The information provided here will be useful for the efficient exploration of technological advances in genome editing field for the crop improvement programs.
Highlights
Cells have several inherent mechanisms for the repair of double-strand DNA breaks (DSBs) [1,2].These DNA repair mechanisms have been acknowledged as important approaches for targeted gene modification or editing, by introducing precise breaks in the genome at specific sites
Earlier approaches to modify the genomic DNA and RNA included self-splicing introns, cross-linking agents like psoralen or bleomycin or other chemical reagents coupled with chemical recognition of DNA sequences using polyamides or peptide nucleic acids (PNAs), and homing endonucleases encoded by introns [3,4,5,6,7]
CRISPR/CRISPR associated protein 9 (Cas9) is still in preliminary stages of development, the most frequently targeted genes used for genome editing includes the genes which have been proved to be an scorable marker for plants (Table 1)
Summary
Cells have several inherent mechanisms for the repair of double-strand DNA breaks (DSBs) [1,2] These DNA repair mechanisms have been acknowledged as important approaches for targeted gene modification or editing, by introducing precise breaks in the genome at specific sites. Cas and sgRNAin sgRNA and Cas are sgRNA, expressed in RuvC-like the plant which leads to break (DSB), resulting coding sequences are cloned into a vector (blue), together or individually, which is transformed intoin activation of DNA repair machinery leading to the modification of DNA sequence and subsequently the plant cells. Homology directed repair; Indel: insertion or deletion mutations; NHEJ: non-homologous end joining; Cas: CRISPR associated protein. Double-stranded break; dsREPAIR: sgRNA: single guide RNA; tracrRNA: transactivating double-strand repair; HDR: homology directed repair; Indel: insertion or deletion mutations; NHEJ: non-homologous end joining; sgRNA: single guide RNA; tracrRNA: transactivating CRISPR RNA.
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