Perspectives of CRISPR/Cas-mediated cis-engineering in horticulture: unlocking the neglected potential for crop improvement

Abstract

Directed breeding of horticultural crops is essential for increasing yield, nutritional content, and consumer-valued characteristics such as shape and color of the produce. However, limited genetic diversity restricts the amount of crop improvement that can be achieved through conventional breeding approaches. Natural genetic changes in cisregulatory regions of genes play important roles in shaping phenotypic diversity by altering their expression. Utilization of CRISPR/Cas editing in crop species can accelerate crop improvement through the introduction of genetic variation in a targeted manner. The advent of CRISPR/Cas-mediated cis-regulatory region engineering (cis-engineering) provides a more refined method for modulating gene expression and creating phenotypic diversity to benefit crop improvement. Here, we focus on the current applications of CRISPR/Cas-mediated cis-engineering in horticultural crops. We describe strategies and limitations for its use in crop improvement, including de novo cis-regulatory element (CRE) discovery, precise genome editing, and transgene-free genome editing. In addition, we discuss the challenges and prospects regarding current technologies and achievements. CRISPR/Cas-mediated cis-engineering is a critical tool for generating horticultural crops that are better able to adapt to climate change and providing food for an increasing world population.

Abiotic Stress Responses in Plants: Current Knowledge and Future Prospects

“…Recently, the use of targeted genome editing using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein9 nuclease (Cas9) (CRISPR/Cas) has generated a lot of interest in various fields of plant biology including abiotic stress management [109]. CRISPR/Cas has been adopted in the field of plant developmental biology for characterizing genes as well as to underpin the molecular mechanisms behind various plant traits [110]. It has been used in the model plants such as Arabidopsis and tobacco earlier and likewise, now it is being utilized effectively for crop plants like sorghum, rice, wheat, maize, soybean as well as woody plants.…”

Challenges and Prospects of New Plant Breeding Techniques for GABA Improvement in Crops: Tomato as an Example

“…However, like most plant species, almost no knowledge has been generated regarding the structure of promoters and other regulatory sequences in tomato, and no prior information is currently available for CREs and their interactions in the promoter of SlGABA-T1. Even though prior knowledge would facilitate and speed the process, cis-engineering could be implemented if a multiplexed CRISPR/Cas9 system was developed (Li et al, 2020). For this, we assembled a vector containing four gRNAs that was designed within a region 2 kbp upstream of the SlGABA-T1 start codon and transformed in Micro-Tom (Figure 2A).…”

“…Many examples have reported that mutations in cis -regulatory elements (CREs) produce significant phenotypic and morphological changes that have been selected during domestication ( Meyer and Purugganan, 2013 ; Swinnen et al., 2016 ). In tomato, changes in CREs and promoter regions were translated in elongated ( SlSUN ) and larger fruits ( FW2.2 , FW3.2 , SlWUS ) or those with improved β-carotene contents ( Slcys-B ) ( van der Knaap et al., 2014 ; Li et al., 2020 ) as just a few relevant examples. However, despite the great potential of this approach, currently, the vast majority of CRISPR/Cas9 studies focus on targeting coding sequences for null allele editing or controlling transcription by activation (CRISPRa) or inhibition (CRISPRi), and only 15 studies so far have reported successful applications of cis -engineering ( Li et al., 2020 ).…”

“…In tomato, changes in CREs and promoter regions were translated in elongated ( SlSUN ) and larger fruits ( FW2.2 , FW3.2 , SlWUS ) or those with improved β-carotene contents ( Slcys-B ) ( van der Knaap et al., 2014 ; Li et al., 2020 ) as just a few relevant examples. However, despite the great potential of this approach, currently, the vast majority of CRISPR/Cas9 studies focus on targeting coding sequences for null allele editing or controlling transcription by activation (CRISPRa) or inhibition (CRISPRi), and only 15 studies so far have reported successful applications of cis -engineering ( Li et al., 2020 ). One of them was successfully conducted in tomato by cis -engineering the promoters of genes controlling fruit size, inflorescence branching and plant architecture, achieving multiple cis -regulatory alleles with a continuum of quantitative variation ( Rodríguez-Leal et al., 2017 ).…”

<strong>SHERLOCK and DETECTR: CRISPR-Cas Systems as Potential Rapid Diagnostic Tools for Emerging Infectious Diseases and Cancer-Associated Mutations</strong>

“…Rapid nucleic acid detection is an important part of many applications in human health and biotechnology, including the identifying of infectious diseases, agricultural pathogens, or circulating DNA or RNA associated with disease. [17][18][19] Standard methods to amplify nucleic acids for detection (such as PCR) are effective but require instrumentation that is not portable, precluding their deployment in the field [20,21]. CRISPR-Cas-based approaches are being tested to treat hereditary, infectious, and many other diseases.…”