A CRISPR way for accelerating cereal crop improvement: Progress and challenges

Abstract

Humans rely heavily on cereal grains as a key source of nutrients, hence regular improvement of cereal crops is essential for ensuring food security. The current food crisis at the global level is due to the rising population and harsh climatic conditions which prompts scientists to develop smart resilient cereal crops to attain food security. Cereal crop improvement in the past generally depended on imprecise methods like random mutagenesis and conventional genetic recombination which results in high off targeting risks. In this context, we have witnessed the application of targeted mutagenesis using versatile CRISPR-Cas systems for cereal crop improvement in sustainable agriculture. Accelerated crop improvement using molecular breeding methods based on CRISPR-Cas genome editing (GE) is an unprecedented tool for plant biotechnology and agriculture. The last decade has shown the fidelity, accuracy, low levels of off-target effects, and the high efficacy of CRISPR technology to induce targeted mutagenesis for the improvement of cereal crops such as wheat, rice, maize, barley, and millets. Since the genomic databases of these cereal crops are available, several modifications using GE technologies have been performed to attain desirable results. This review provides a brief overview of GE technologies and includes an elaborate account of the mechanisms and applications of CRISPR-Cas editing systems to induce targeted mutagenesis in cereal crops for improving the desired traits. Further, we describe recent developments in CRISPR-Cas-based targeted mutagenesis through base editing and prime editing to develop resilient cereal crop plants, possibly providing new dimensions in the field of cereal crop genome editing.

Keywords: CRISPR/Cas; TALENs; base editing; cereals; crop improvement; food security; genome editing; prime editing.

FIGURE 1

Steps involved in CRISPR/Cas9-based gene editing of cereals. Selection of target gene and gRNA design; cloning of Cas9 and gRNA in a suitable vector; vector delivery into the plants via Agrobacterium tumefaciens or particle bombardment, and screening of mutant/edited cereal crops using Sanger sequencing/NGS/RE/PCR.

FIGURE 2

Successful application of CRISPR/Cas genome editing in cereals.

FIGURE 3

Cytosine and adenine base editors. (A) Cytosine base editors (CBEs), composed of a nickase Cas9 (nCas9) fused to a deaminase and UGI; conversion of C-G into T-A base pairs. (B) Adenine base editors (ABEs) are composed of a dead (d) or nickase (n) Cas9 (d/nCas9) fused to two TadA, with one evolved to edit adenine in DNA (TadA*) and one wild type (TadA). ABEs convert A-T into G-C base pairs.

FIGURE 4

Illustration of fused nCas9, AID, and UNG enzyme complexes to perform a series of functions, including specific DNA binding, cleaving of the amine group from C, and creating AP sites followed by cellular repair to achieve specific base editing. (A) Glycosylase base editor–mediated C-A transition. (B) Glycosylase base editor–mediated C-G transition.

FIGURE 5

(A) Prime editors (PEs) are precise genome editing tools that directly write new genetic information into a specified DNA target site using a Cas9 nickase (nCas9; H840A) fused to an engineered reverse transcriptase (RT). (B) The RT is programmed with a prime editing gRNA (pegRNA) that specifies the target site and encodes the desired edit. PegRNA is a modified sgRNA with 3′ extension of the RT template and primer-binding site (PBS) sequences. (C) The nCas9 (catalytically impaired Cas9 harbouring a H840A mutation) is used to nick the editing strand of the double-stranded DNA target. (D) and (E) Next, the nicked strand is used for priming the reverse transcription of an edit-encoding extension (RT template) on the pegRNA directly into the target site. (F) This results in a branched intermediate consisting of two competing single-stranded DNA flaps. (G) The 3′ flap contains the edited sequence, whereas the 5′ flap contains the unedited sequence. (H) The 5′ flap is preferentially cleaved by structure-specific endonucleases such as FEN1 (Flap endonuclease 1: a central component of DNA metabolism) or 5′ exonucleases such as Exo1 (Human exonuclease 1) in mammalian cells. Ligation of the 3′ flap incorporates the edited DNA strand into the heteroduplex DNA containing one edited strand and one unedited strand. (I) Finally, to resolve the heteroduplex, DNA repair machinery permanently installs the desired edit by copying the information from the edited strand to the complementary strand.

FIGURE 6

Prime editing-mediated genetic modifications and their potential use in cereals. (A) Various kinds genetic/sequence manipulations/modifications that are potentially possible through prime editing in plants. (B) Different applications of prime editing in various cereal crops. The rectangles specify mutation and different colors within them indicate different types of mutations. The yellow colored ovals denote the DNA segment inserted or replaced using prime editing. Cas9n, Cas9 nickase; SNP, single-nucleotide polymorphism; RT, reverse transcriptase.