Improvement of the LbCas12a-crRNA system for efficient gene targeting in plants

Abstract

Plant gene targeting (GT) can be utilized to precisely replace up to several kilobases of a plant genome. Recent studies using the powerful clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) nucleases significantly improved plant GT efficiency. However, GT for loci without associated selection markers is still inefficient. We previously utilized Lachnospiraceae bacterium Cas12a (LbCas12a) in combination with a replicon for tomato GT and obtained high GT efficiency with some selection markers. In this study, we optimize and advance our GT system by using a temperature-tolerant LbCas12a (ttLbCas12a) in combination with various crRNA forms and chemical treatments to suppress the canonical non-homologous end-joining pathway in tomato. Our work demonstrates the significance of the selection of gene scissors, the appropriate design and number of LbCas12a crRNAs, the use of chemical treatments, and the establishment of favorable experimental conditions for further enhancement of plant HDR to enable efficient GT in tomato.

CRISPR-Cas-based Microhomology-Mediated End Joining for Precise Gene Replacement in Plant

“…Since the MMEJ-mediated precise editing efficiency was still low to be practically applicable for crop improvement, significant improvement of the editing system is needed. NU7441, a small chemical that was shown to inhibit DNA-dependent protein kinase (DNA-PKcs), an important cNHEJ component, enhancing HR-mediated repair of DSBs (Robert et al, 2015; Vu et al, 2021a; Zhao et al, 2006), also significantly elevated the MMEJ-mediated DSB repair products in mammalian cells (Dutta et al, 2017). We next tested if NU7441 can facilitate MMEJ repair using various concentrations of the chemical and the MJ.HPAT3-1donors.…”

“…Construction of plasmid for Agrobacterium-mediated transformation in tomato. For stable transformation and assessment of the cNHEJ and MMEJ-mediated precision gene replacement in tomatoes, we designed and cloned the gRNA expression cassettes (Supplementary file 1) using the Golden-gate cloning system as described previously 9,15 . Two gRNA expression cassettes (gR1.HPAT3 and gR2.HPAT3 for SlHPAT3; gR1.HKT1;2 and gR2.HKT1;2 for SlHKT1;2) were used to generate two DSBs at each targeted site.…”

“…With HGT, we can precisely edit genes from a single base to kilobase scales, while narrower-range precise gene editing could be achieved with the PE editing tools. However, HGT still requires improvement for practical applications to a wide range of Arabidopsis loci (Zhang et al, 2022) and crop plant loci due to low editing efficiency and inaccessibility of the genomic contexts of the loci (Vu et al, 2021a; Wang et al, 2017; Zhang et al, 2022). In the case of prime editors, while substantial improvements have been made to achieve efficient editing in monocots (Lin et al, 2021; Zong et al, 2022), it has not been so successful in dicot plants (Lu et al, 2020b; Vu et al, 2021b).…”