Abstract
CRISPR/Cas systems have revolutionized genome engineering in plants (Hassan et al. 2021) via specific control of genetic modifications and transcriptional activities in plants (Liu et al. 2018; Wolter et al. 2018; Pan et al. 2022). However, there are increasing public concerns about the biosafety and security surrounding the emergence of CRISPR tools, e.g., chimeras caused by continuously CRISPR/Cas activity in dividing cells, chromosomal instability and genotoxicity induced by off-target DNA cleavage of CRISPR/Cas systems and CRISPR/Cas-based gene drives to non-target populations, to name a few (Abraham et al. 2020; Yuan et al. 2021a; Calvache et al. 2022; Shin et al. 2022). Hence, there is an urgent need for developing more controllable and precise CRISPR/Cas-based genome engineering tools in plants. Anti-CRISPR (Acr) proteins, as natural inhibitors for CRISPR-Cas systems, have apparent utility in biodesign strategies aimed at regulating Cas activities (Marino et al. 2020). Acr proteins inhibit CRISPR/Cas activities by either blocking 1) DNA binding activity or 2) DNA cleavage activity of Cas proteins (Marino et al. 2020). Multiple Acr proteins have been tested in mammalian cells and yeast (Saccharomyces cerevisiae), including AcrIIA4 (inhibiting SpCas9 activity), AcrVA1 (inhibiting Cas12a activity), and AcrIIA5 (potentially inhibiting all Cas9 orthologs) (Marino et al. 2020; Zhang and Marchisio 2022). With these demonstrations, Acr proteins are now recognized as promising mechanisms to “safeguard” CRISPR/Cas systems as a means of mitigating risk associated with unwanted genome editing. To this end, AcrIIA4 and AcrIIA5 have been implemented in biodesign for reducing off-target effects, limiting CRISPR/Cas activity to particular environment (e.g., blue-light), and/or restricting gene editing activity to a specific cell type (Zhang and Marchisio 2021). In plants, only AcrIIA4 and AcrVA1 have been evaluated and so far only in a single plant species (Nicotiana benthamiana) based on transient expression through leaf infiltration and viral delivery (Calvache et al. 2022). AcrIIA5 activity remains to be evaluated in plants and performance differences between transient or stable expression of Acr proteins remains unanswered. Therefore, we evaluated the performance of AcrIIA4 and AcrIIA5 activity in different types of plants, including herbaceous and woody plant species, using both transient expression and stable transformation approaches. More specifically, we tested the effects of AcrIIA4 and AcrIIA5 activity on the SpCas9-based adenine base editor (ABE7) in the herbaceous plant Arabidopsis thaliana and N. benthamiana, and the woody plant hybrid poplar ‘717’ (Populus tremula × P. alba hybrid clone INRA 717‐1B4), using both leaf-infiltration and protoplast-based transient expression. The activity of AcrIIA4 to ABE7 was then further investigated in Arabidopsis via Agrobacterium-mediated stable genetic transformation.
