Materials and methods

Plant-based biosensors for detecting CRISPR-mediated genome engineering

Construction of vector

To build the BS1, the mutant gfp1a and gfp1b were created by inserting a gblcoks containing frame shift mutation sequences at the 5’ end of GFP coding sequence of pGFPGUSPlus (Plasmid #64401 in addgene) using the NEBuilder HiFi DNA Assembly Cloning Kit (New England BioLabs, Catalog #E5520S). Then, a gblocks harboring a U6 promoter, sgRNA and terminator was assembled into the mutant gfp1a and gfp1b through NEBuilder HiFi DNA Assembly. To build the BS2 and BS3, the mutant gfp2a and gfp2b were created by replacing the GFP coding sequence of pGFPGUSPlus with a gblcoks containing PTC mutation. BS2 was then assembled by inserting a gblocks harboring a U6 promoter, sgRNA and terminator into the mutant gfp2a and gfp2b through NEBuilder HiFi DNA Assembly. BS3 was assembled by inserting a gblocks harboring a U6 promoter, pegRNA and terminator into the mutant gfp2b. To build the BS4, a gblocks harboring a U3 promoter, sgRNA and terminator was inserted into the vector ProOsTPR-like:mCherry 11 using NEBuilder HiFi DNA Assembly. All gblocks were synthesized from Integrated DNA Technologies. All plasmids were confirmed by Sanger sequencing.

Protoplast transformation

The isolation and transformation of Arabidopsis and poplar protoplast were performed as described previously. 12

Arabidopsis transformation

The floral dip method of transformation was performed by immersion of Arabidopsis wild type ‘Col-0’ inflorescences in a suspension of Agrobacterium ‘GV3101’ with modification as described by Yuan et al. 13

Tobacco leaf infiltration

N. benthamiana leaves were infiltrated using a syringe without a needle as described by Li. 14

Article TitlePlant-based biosensors for detecting CRISPR-mediated genome engineering

Abstract

CRISPR/Cas has recently emerged as the most reliable system for genome engineering in various species. However, concerns about risks associated with CRISPR/Cas9 technology are increasing on potential unintended DNA changes that might accidentally arise from CRISPR gene editing. Developing a system that can detect and report the presence of active CRIPSR/Cas tools in biological systems is therefore very necessary. Here, we developed the real-time detection systems that can spontaneously indicate CRISPR-Cas tools for genome editing and gene regulation including CRISPR/Cas9 nuclease, base editing, prime editing and CRISPRa in plants. Using the fluorescence-based molecular biosensors, we demonstrated that the activities of CRISPR/Cas9 nuclease, base editing, prime editing and CRIPSRa can be effectively detected in transient expression via protoplast transformation and leaf infiltration (in Arabidopsis, poplar, and tobacco) and stable transformation in Arabidopsis.

Competing Interest Statement

The authors have declared no competing interest.


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