Gene Editing and Crop Improvement Using CRISPR-Cas9 System

Abstract

Advancements in Genome editing technologies have revolutionized the fields of functional genomics and crop improvement. CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat)-Cas9 is a multipurpose technology for genetic engineering that relies on the complementarity of the guideRNA (gRNA) to a specific sequence and the Cas9 endonuclease activity. It has broadened the agricultural research area, bringing in new opportunities to develop novel plant varieties with deletion of detrimental traits or addition of significant characters. This RNA guided genome editing technology is turning out to be a groundbreaking innovation in distinct branches of plant biology. CRISPR technology is constantly advancing including options for various genetic manipulations like generating knockouts; making precise modifications, multiplex genome engineering, and activation and repression of target genes. The review highlights the progression throughout the CRISPR legacy. We have studied the rapid evolution of CRISPR/Cas9 tools with myriad functionalities, capabilities, and specialized applications. Among varied diligences, plant nutritional improvement, enhancement of plant disease resistance and production of drought tolerant plants are reviewed. The review also includes some information on traditional delivery methods of Cas9-gRNA complexes into plant cells and incorporates the advent of CRISPR ribonucleoproteins (RNPs) that came up as a solution to various limitations that prevailed with plasmid-based CRISPR system.

Keywords: CRISPR ribonucleoproteins; CRISPR/Cas system; disease resistance; gene expression regulation; genome editing; metabolic engineering; nutrition improvement.

FIGURE 1

Various genome-editing tools. (A) Zinc-finger nucleases (ZFNs) act as dimer. Each monomer consists of a DNA binding domain and a nuclease domain. Each DNA binding domain consists of an array of 3–6 zinc finger repeats which recognizes 9–18 nucleotides. Nuclease domain consists of type II restriction endonuclease Fok1. (B) Transcription activator-like nucleases (TALENs): these are dimeric enzymes similar to ZFNs. Each subunit consists of DNA binding domain (highly conserved 33–34 amino acid sequence specific for each nucleotide) and Fok1 nuclease domain. (C) CRISPR/Cas9: Cas9 endonuclease is guided by sgRNA (single guide RNA: crRNA and tracrRNA) for target specific cleavage. 20 nucleotide recognition site is present upstream of protospacer adjacent motif (PAM).

FIGURE 2

Genome editing with site-specific nucleases (SSNs). The double stranded breaks (DSBs) introduced by CRISPR/Cas9 complex can be repaired by non-homologous end joining (NHEJ) and homologous recombination (HR). (A) NHEJ repair can produce heterozygous mutations, biallelic mutations (two different mutations at each chromosome) and homozygous mutations (two independent identical mutations) leading to gene insertion or gene deletion. (B) In the presence of donor DNA digested with the same endonuclease leaving behind similar overhangs, HR can be achieved leading to gene modification and insertion.

FIGURE 3

Key discoveries and advances in CRISPR/Cas9 technology.

FIGURE 4

Mechanism of CRISPR/Cas9 action: in the acquisition phase foreign DNA gets incorporated into the CRISPR loci of bacterial genome. CRISPR loci is then transcribed into primary transcript and processed into crRNA with the help of tracrRNA during crRNA biogenesis. During interference, Cas9 endonuclease complexed with a crRNA and cleaves foreign DNA near PAM region.

FIGURE 5

Simplified flow chart representing CRISPR/Cas9 mediated plant genome editing. After the selection of the target site, sgRNAs are designed using various bioinformatic softwares and packed into specific vectors along with codon optimized Cas9. After delivery into plant cells, putative transformants can be screened by multiple assays and used for further analysis.

FIGURE 6

Various applications of CRISPR/Cas9 system many of which are yet to be tested in plants.

FIGURE 7

Schematic representation of Cas9 nuclease activity and its modifications. SpCas9 endonucleases create DSBs in target DNA through the activity of RuvC and HNH nuclease domains. SpCas9 nucleases can be converted into DNA nickase by substitution of its key amino acids D10A and H840A that produces single stranded breaks. Site directed mutagenesis in D10A produces Cas9n D10A and mutation in HNH domain produces Cas9n (H840A). Mutations in both catalytic residues modify Cas9 to an inactive dead Cas9 (dCas9).

FIGURE 8

Proposed workflow for DNA free genome editing. Cas9 is expressed purified from E. coli. In vitro transcription of single guide RNA (sgRNA) and transcribed in vitro and RNP complex formation. RNPs and DNA precipitation onto 0.6 μm gold particles followed by Particle bombardment in targeted cells. Plants regeneration without any selective agent from bombarded cells and screened for mutations via PCR/restriction enzyme assay and deep sequencing.