Genome Editing in Plants: Exploration of Technological Advancements and Challenges

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.

Keywords: CRISPR/Cas; biotic and abiotic stress tolerance; methylome-editing; multi-target editing; plant transformation; promoter bashing.

Figure 1

An overview of the different aspects covered in the present review related to the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) based genome editing in plants.

Figure 2

Generalized process for CRISPR/Cas mediated genome editing in plants. sgRNA is composed of a spacer (black), and crRNA and tracrRNA (both shown here in red), and Cas9 is composed of two domains: HNH and RuvC-like domain. HNH domain cleaves the DNA strand complementary to the sgRNA, and RuvC-like domain cleaves the other DNA strand. Cas and sgRNA coding sequences are cloned into a vector (blue), together or individually, which is transformed into the plant cells. The sgRNA and Cas9 are expressed in the plant which then leads to double-strand break (DSB), resulting in activation of DNA repair machinery leading to the modification of DNA sequence and subsequently in the protein coded by sequences and conclusively in the phenotype. The final step is the screening of mutations, which is usually done by PCR and sequencing. Abbreviations: Cas9: CRISPR associated protein 9; crRNA: CRISPR RNA; DSB: double-stranded break; dsREPAIR: 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.

Figure 3

Multigene targeting via CRISPR/Cas9 using PTG/Cas9 method. (A) A eukaryotic pre-tRNA with a depiction of post-transcriptional processing by RNaseP and RNaseZ (depicted as blue and red arrows respectively), splicing out 5′leader and 3′ trailer respectively. (B) Here, each gRNA with target-specific sequence (labelled here as circles of different colors) and conserved gRNA sequence (blank rectangle) is fused to a tRNA coding sequence (rectangles with boxes), which is cleaved after transcription by RNaseP and RNaseZ to release mature tRNAs and gRNAs (with lines of same colors as the circles). These processed gRNAs direct Cas9 to the target site, which then causes a double-strand break (DSB), which is repaired by NHEJ or Homologous recombination (HR).

Figure 4

Schematic representation of multiplex genome editing by utilizing intron polycistronic transfer RNA-guide RNAs (inPTGs). Here, figure (A) depicts the regular small nuclear ribonucleoprotein (snRNP) mediated splicing mechanism. (B) Introns are engineered to code for fused polycistronic tRNA-gRNAs (PTGs). (C) PTGs are further processed to release individual gRNAs (shown here in different colors) via the tRNA processing machinery. Individual gRNAs can then go on to target their complementary loci in the genome.

Figure 5

Multiplex gene editing using CRISPR system Yersinia (Csy4) endonuclease, shown here as blue circles. Csy4 restriction sites are cloned between each sgRNA, and Csy4 endonuclease gene is also cloned in the same vector. Expression of Csy4 endonuclease results in the separation of individual sgRNAs, which can then go on to target their respective sites.

Figure 6

Drosha based approach for multiplex gene editing. In this system, gRNAs and miRNAs are cloned in a tandem array. Dicer cleaves the miRNA ends and thereby separating gRNAs also. The pathway on the left explains the general scheme for miRNA mediated mRNA targeting, and the one on right side explains miRNA-based gRNA multiplexing system. Abbreviations. miRNA: micro RNA; gRNA: guide RNA; RISC: RNA induced gene silencing complex; Cas9: CRISPR associated protein 9; RNase: ribonuclease; sgRNA: single guide RNA. Here, sgRNA and gRNA imply the same entity.

Figure 7

(A) Precision base editing by utilizing cytidine deaminase fused with dCas9. With the help of guide RNA (gRNA), Cas9 make complex at a specific target site and then the cytidine deaminase act on cysteine present on the opposite strand. The deamination process converts cysteine (C) to uracil (U) which later gets converted into adenine-thymine base-pair during DNA replication by the inbuilt mismatch repair mechanism. (B,C) CRISPR mediated methylome editing. Dead Cas (dCas) is fused to a DNA methyltransferase (DNMT3A in case of animals) or a demethylase, such as ten-eleven translocation dioxygenase (tet) in animals, which can be used to edit the epigenome.