Genome editing for plant research and crop improvement

Abstract

The advent of clustered regularly interspaced short palindromic repeat (CRISPR) has had a profound impact on plant biology, and crop improvement. In this review, we summarize the state-of-the-art development of CRISPR technologies and their applications in plants, from the initial introduction of random small indel (insertion or deletion) mutations at target genomic loci to precision editing such as base editing, prime editing and gene targeting. We describe advances in the use of class 2, types II, V, and VI systems for gene disruption as well as for precise sequence alterations, gene transcription, and epigenome control.

Present and future prospects for wheat improvement through genome editing and advanced technologies

“…As a simple, versatile, and robust system, CRISPR/Cas has been extensively applied in various organisms, including plants ( Zhan et al., 2021 ). Despite its importance and widespread use, large-scale mutation screening remains time-consuming, labor-intensive, and costly, especially in polyploid wheat plants.…”

“…Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (CRISPR-Cas), a versatile, simple, and inexpensive system for precise, sequence-specific modifications of DNA sequences, including targeted mutagenesis for gene knockout, single-base substitution, and gene/allele replacement in vivo , has dominated the genome-editing field over the past few years. The applications of CRISPR/Cas in both plant biological sciences and crop improvement are particularly important in the context of global climate change and in the face of current agricultural, environmental, and ecological challenges ( Li and Xia, 2020 ; Ma et al., 2015 ; Zhan et al., 2021 ). Once double-strand DNA breaks (DSBs) are generated by CRISPR/Cas, the DSBs are repaired by the error-prone non-homologous end joining (NHEJ) pathway, accurate homology-directed repair (HDR), or both NHEJ and HDR ( Cong et al., 2013 ; Danner et al., 2017 ; Jinek et al., 2012 ; Puchta, 1998 ; Zetsche et al., 2015 ) ( Figure 1 A).…”

“…Another strategy is prime editing (PE), which enables all 12 types of base substitutions and small indels, substantially expanding the scope and capabilities of precise genome editing ( Anzalone et al., 2019 ). To date, various CRISPR/Cas toolboxes have been developed and allow for targeted mutagenesis at specific genome loci, transcriptome regulation and epigenome editing, base editing, and precise targeted gene/allele replacement or tagging in plants ( Zhan et al., 2021 ). In particular, precise replacement of an existing allele with an elite allele in a commercial variety through HDR is the holy grail of genome editing for crop improvement, as it has been very difficult, laborious, and time-consuming to introgress these elite alleles into commercial varieties without any linkage drag from parental lines within a few generations in crop-breeding practice ( Li and Xia, 2020 ).…”

Strong and tunable anti-CRISPR/Cas9 activity of AcrIIA4 in plants

“…Although different CRISPR-based architectures in plants have been shown to induce both e cient mutagenesis and powerful transcriptional activation when expressed constitutively 5,6 , the spatialtemporal control on such activities would considerably expand the range of their potential applications.…”

“…In addition to gene editing through site-speci c DNA cleavage and subsequent repair by the host machinery, CRISPR/Cas-based platforms provide new ways of engineering other catalytic and non-catalytic genome-associated functions, such as DNA base modi ers, epigenetic effectors or programmable transcription factors (PTFs); the latter enabling new control strategies for gene expression known as CRISPR activation (CRISPRa) or repression (CRISPRi) 2,3 . This is achieved by coupling nuclease-deactivated versions of Cas (dCas) to transcriptional activator or repressor domains, which then act as functionally-customized RNA-guided DNA-binding complexes 2,4 .Although different CRISPR-based architectures in plants have been shown to induce both e cient mutagenesis and powerful transcriptional activation when expressed constitutively 5,6 , the spatialtemporal control on such activities would considerably expand the range of their potential applications.To this date, the control of CRISPR/Cas activity in plants has been attempted mainly at the transcriptional level using inducible promoters controlling expression of Cas transcripts [7][8][9] . However, in practical terms, the control of effector function at the transcriptional level alone is only partially effective due to inherent leakiness and noise.…”