Strong and tunable anti‐CRISPR/Cas activities in plants

Abstract

CRISPR/Cas has revolutionized genome engineering in plants. However, the use of anti-CRISPR proteins as tools to prevent CRISPR/Cas-mediated gene editing and gene activation in plants has not been explored yet. This study describes the characterization of two anti-CRISPR proteins, AcrIIA4 and AcrVA1, in Nicotiana benthamiana. Our results demonstrate that AcrIIA4 prevents site-directed mutagenesis in leaves when transiently co-expressed with CRISPR/Cas9. In a similar way, AcrVA1 is able to prevent CRISPR/Cas12a-mediated gene editing. Moreover, using a N. benthamiana line constitutively expressing Cas9, we show that the viral delivery of AcrIIA4 using Tobacco etch virus is able to completely abolish the high editing levels obtained when the guide RNA is delivered with a virus, in this case Potato virus X. We also show that AcrIIA4 and AcrVA1 repress CRISPR/dCas-based transcriptional activation of reporter genes. In the case of AcrIIA4, this repression occurs in a highly efficient, dose-dependent manner. Furthermore, the fusion of an auxin degron to AcrIIA4 results in auxin-regulated activation of a downstream reporter gene. The strong anti-Cas activity of AcrIIA4 and AcrVA1 reported here opens new possibilities for customized control of gene editing and gene expression in plants.

An Intein-Mediated Split–nCas9 System for Base Editing in Plants

“…Recently, it was reported that a split prime editor system could mediate endogenous base transversion and insertion in human cells . And it has been shown that Tobacco etch virus (TEV)-based system and Potato virus X (PVX)-based vector can infect N. benthamiana simultaneously . Therefore, the identification of multiple valid split sites provides more choice for split SpCas9 and can be potentially used for the delivery of CRISPR/Cas9-based tools such as base editor, prime editor, and CRISPR interference/activation through viruses/nanoparticles-mediated transformation.…”

Small Molecules for Enhancing the Precision and Safety of Genome Editing

“…On the other hand, small molecules can be used to deactivate Acr proteins and induce genome editing. Calvache et al fused AcrIIA4 to minimal auxin-inducible degron (mAID), such that the fusion protein can be degraded by auxin treatment through the ubiquitin-proteasome pathway ( Figure 6 C) [ 100 ]. Indeed, treatment with auxin (indole-3-acetic acid) degraded the Acr fusion proteins and induced dCas9-based gene activation in plant cells.…”

Comprehending the evolution of gene editing platforms for crop trait improvement

“…NHEJ is the active DNA repair mechanism in nature wherein Cas9-induced DSBs are repaired ( Moore and Haber 1996 ). NHEJ can, therefore, lead to small insertions or deletions that could yield a host of mutations ( Calvache et al, 2022 ; Wada et al, 2022 ). Such mutations are beneficial while knocking out a targeted gene using CRISPR/Cas9 systems.…”

“…To this effect, HDR is a more obvious choice of DSB repair mechanism for incorporation of desired sequences following cleavage by Cas9. In plants, GE HDR relies on a DNA template along with the gDNA and Cas9 for a successful DSB repair ( Calvache et al, 2022 ; Wada et al, 2022 ). In plants, through genetic engineering, many outstanding repairs have been achieved via HDR, leading to gene replacement, DNA correction, and targeted knockouts.…”