CRISPR/Cas genome editing in tomato improvement: Advances and applications

Abstract

The narrow genetic base of tomato poses serious challenges in breeding. Hence, with the advent of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein9 (CRISPR/Cas9) genome editing, fast and efficient breeding has become possible in tomato breeding. Many traits have been edited and functionally characterized using CRISPR/Cas9 in tomato such as plant architecture and flower characters (e.g. leaf, stem, flower, male sterility, fruit, parthenocarpy), fruit ripening, quality and nutrition (e.g., lycopene, carotenoid, GABA, TSS, anthocyanin, shelf-life), disease resistance (e.g. TYLCV, powdery mildew, late blight), abiotic stress tolerance (e.g. heat, drought, salinity), C-N metabolism, and herbicide resistance. CRISPR/Cas9 has been proven in introgression of de novo domestication of elite traits from wild relatives to the cultivated tomato and vice versa. Innovations in CRISPR/Cas allow the use of online tools for single guide RNA design and multiplexing, cloning (e.g. Golden Gate cloning, GoldenBraid, and BioBrick technology), robust CRISPR/Cas constructs, efficient transformation protocols such as Agrobacterium, and DNA-free protoplast method for Cas9-gRNAs ribonucleoproteins (RNPs) complex, Cas9 variants like PAM-free Cas12a, and Cas9-NG/XNG-Cas9, homologous recombination (HR)-based gene knock-in (HKI) by geminivirus replicon, and base/prime editing (Target-AID technology). This mini-review highlights the current research advances in CRISPR/Cas for fast and efficient breeding of tomato.

RNA virus-mediated gene editing for tomato trait breeding

“…Owing to their specificity and versatility, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) systems are widely used for efficient genome editing in numerous plant species, including tomato 2 . Current methods in tomato genome editing mainly use the Streptococcus pyogenes Cas9 nuclease and single-guide RNA (sgRNA) molecules that are delivered into plant cells through stable Agrobacterium -mediated transformation of constructs for the simultaneous Cas9 and sgRNA expression 2 . Stable transformation of CRISPR/Cas9 constructs for multiplexed gene editing allows rapid breeding of tomato traits.…”

“…We reasoned that PVX-mediated sgRNA expression in MT-Cas9 would result in editing events of the tomato genome. For proof-of-concept VIGE assays, we assembled pLX-PVX::sgPDS for expression of a sgRNA targeting PHYTOENE DESATURASE ( PDS ; Solyc03g123760) (Figure S1A; Table S1), whose loss-of-function mutants have a reported photobleaching phenotype 2 . Cotyledons of MT-Cas9 seedlings were agroinoculated with pLX-PVX::sgPDS, and samples were collected from upper uninoculated leaves after 6 weeks.…”

Generation of parthenocarpic tomato plants in multiple elite cultivars using the CRISPR/Cas9 system

“…The breeding, including conventional breeding and modern biotechnology, have been involved in producing elite tomato cultivars which possess superior traits (Abewoy Fentik 2017;Sharma et al 2019;Xia et al 2021). New plant breeding technologies such as transgenesis and targeted mutagenesis have been adopted widely in recent years to quickly develop desired traits in tomatoes (Bai 2017;Vu et al 2020;Tiwari et al 2023). Gene editing systems, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9), base editors (BEs), and prime editors (PEs), have been applied in crops to target specific genes with high accuracy and produce transgene-free plants (Zhu & Zhu 2022).…”

“…Among these tools, CRISPR/Cas9 has shown significant potential in editing beneficial genes, promoting elite tomato production (Li et al 2018;Pan et al 2016;Reem & van Eck 2019;Santillán Martínez et al 2020;Yang et al 2022). This technology has been used to enhance biotic and abiotic stress resistance, improve fruit quality, and accelerate the domestication of tomatoes (Wang et al 2019;Tiwari et al 2023). However, the efficiency of gene editing tools depends on several factors, including the expression level of sgRNA and Cas proteins, the delivery efficiency of CRISPR/Cas reagents, the specificity of the gRNAs, and the transformation and regeneration systems (Ma et al 2023).…”

Laser-Induced Breakdown Spectroscopy and Microscopy Study of Human Dental Tissues

“…Thus, spectroscopic characterization of the plasma light will reveal the elements present in the target. The identification of many elemental lines, including both the wavelength and the intensity within the emission spectrum, will form a unique spectral fingerprint of the target, such as calcium [13][14][15][16].…”