Insights into the molecular mechanisms of CRISPR/Cas9-mediated gene targeting at multiple loci in Arabidopsis

Abstract

Homologous recombination-mediated gene targeting (GT) enables precise sequence knockin or sequence replacement, and thus is a powerful tool for heritable precision genome engineering. We recently established a clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9)-mediated approach for heritable GT in Arabidopsis (Arabidopsis thaliana), but its broad utility was not tested, and the underlying molecular mechanism was unclear. Here, we achieved precise GT at 14 out of 27 tested endogenous target loci using the sequential transformation approach and obtained vector-free GT plants by backcrossing. Thus, the sequential transformation GT method provides a broadly applicable technology for precise genome manipulation. We show that our approach generates heritable GT in the egg cell or early embryo of T1 Arabidopsis plants. Analysis of imprecise GT events suggested that single-stranded transfer DNA (T-DNA)/VirD2 complexes produced during the Agrobacterium (Agrobacterium tumefaciens) transformation process may serve as the donor templates for homologous recombination-mediated repair in the GT process. This study provides new insights into the molecular mechanisms of CRISPR/Cas9-mediated GT in Arabidopsis.

Precise and heritable gene targeting in rice using a sequential transformation strategy

“… 40 Furthermore, DNA methylation was not altered, and de novo DNA methylation was not established at the GT loci in Arabidopsis . 23 , 27 , 35 These reports suggest that DNA methylation status is generally unaffected by GT events in Arabidopsis . In this study, we examined whether DNA methylation at homology arm regions was altered by CRISPR-Cas9-mediated GT in rice.…”

“…According to our work in Arabidopsis , the GT-specific primer set detects some false-positive signals, which likely come from nonheritable GT events in minor somatic tissues that were not detected by the external primer set. 27 , 35 , 36 OsFTL1-GFP GT-positive T0 rice plants were obtained from the parental lines #167 and #180, which yielded seven and two plants, respectively ( Figure 2 B and Table 3 ). All GT-positive T0 rice plants could be confirmed by Southern blot analysis using SpeI and HpaI restriction enzymes ( Figures 2 B and S1 A).…”

“… 27 Only DD45 pro:Cas9, which stably expresses the Cas9 nuclease in egg cells and the early embryo in parental lines, yielded heritable GT, indicating that high DSB efficiency in Agrobacterium -infected cells may be the most important factor for GT establishment. 35 Agrobacterium releases a large amount of single-stranded transfer DNA (ssT-DNA) into the infected cells, which are germline cells, as in the case of the flower dipping method for Arabidopsis . 42 , 43 We proposed that the released ssT-DNA could be the most likely candidate of the donor molecule for HDR-mediated GT.…”

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

“…However, efficient precision gene editing remains challenging in plants (Schindele et al, 2020). With the advent of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) technology (Jinek et al, 2012), plant precision editing has been revolutionized with homology-directed gene targeting (HGT) (Cermak et al, 2015; Vu et al, 2020; Wang et al, 2017; Wolter and Puchta, 2019; Zhang et al, 2022) and prime editing (PE) (Anzalone et al, 2020; Lin et al, 2021). 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.…”

“…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).…”