DNA constructs used in this work were assembled using GoldenBraid25. AcrIIA4 protein sequence, as originally reported by Rauch et al.26 fused to SV40 NLS and mAID47 sequence, as reported by Brosh et al.21 (modified as N-tag or C-tag), were codon optimized for N. benthamiana using IDT Codon Optimization Tool (https://eu.idtdna.com/CodonOpt) and subsequently domesticated at https://gbcloning.upv.es/do/synthesis/. Additionally, a N. benthamiana codon optimized AcrIIA4 protein version without SV40-NLS, B3-B4 version, was domesticated using GoldenBraid GB Domesticator tool (https://gbcloning.upv.es/do/domestication/). Level 1 assemblies of transcriptional units from individual Level 0 parts were performed through Golden Gate-like multipartite BsaI reactions. All Level 0 and Level 1 assemblies were performed as previously reported 19. Level 0 assemblies were confirmed by restriction analysis and Sanger sequencing and Level 1 assemblies were verified by restriction analysis. An exhaustive list of all plasmids used in this work are listed in Supplementary Table 2.
N. benthamiana transient expression
For transient expression assays, plasmids were transferred to Agrobacterium tumefaciens strain C58. Five to six weeks old N. benthamiana plants grown at 24°C and 16h (light)/ 20°C and 8h (darkness) conditions were used. Agroinfiltration was carried out as previously reported27. Briefly, overnight A. tumefaciens cultures were pelleted and resuspended in agroinfiltration solution (10 mM MES, pH 5.6, 10 mM MgCl2 and 200 μM acetosyringone) to an optical density of 0.1 at 600nm (OD600). Bacterial suspensions were incubated for 2h at room temperature on a horizontal rolling mixer, and then mixed for co-expression experiments, in which more than one GB element was used. Finally, agroinfiltrations were carried out through the abaxial surface of the three youngest fully expanded leaves of each plant with a 1ml needle-free syringe. For some experiments, agroinfiltrations were carried out with small modifications from the general procedure described above. For dose-response assays, SV40-AcrIIA4 TU (GB3344) was resuspended in agroinfiltration solution to an OD600 of 0.1 and subsequently diluted to 0.05, 0.01, 0.005, and 0.001 using a culture carrying an empty vector to maintain the final OD600 at 0.1. For the time-course assay, the strain carrying the SV40-AcrIIA4 TU (GB3344) was agroinfiltrated at OD600 of 0.05 at 0, 24, 48 and 72 h after infiltration of the dCasEV2.1 with the DFR gRNA (GB2513) and the pSlDFR reporter construct (GB1160). Detailed information of the experimental design can be found in the Supplementary Materials and Methods section.
Luciferase activity and determination of relative transcriptional activity
Leaf samples were collected at 4 days post infiltration (dpi). For the determination of the FLuc/RLuc activity, one 0.8 cm diameter disc per agroinfiltrated leaf was excised. Leaf discs were frozen in liquid nitrogen and subsequently homogenized with 180 μl of Passive Lysis buffer, followed by 15 min of centrifugation at 14000 x g at 4°C. 10 μl of crude extract were mixed with 40 μl of LARII and Firefly luciferase (FLuc) activity was determined using a GloMax 96 microplate luminometer (Promega) with a 2-s delay and a 10-s measurement time. After the measurements, 40 μl of Stop&Glo Reagent were added per sample and Renilla luciferase (RLuc) activity was determined using the same protocol. Sample FLuc/RLuc ratios were calculated as the mean value of the three independent agroinfiltrated leaves. Relative transcriptional activities (RTAs) were calculated as the Fluc/Rluc ratios of the pSlDFR reporter in each sample normalized with the Fluc/Rluc ratios produced by a pNos reporter (GB1116) assayed in parallel and expressed in relative promoter units (rpu)19.
Determination of Cas9-mediated editing activity
Genomic DNA was extracted from leaf samples 5 dpi following the CTAB protocol28. The DNA was used as template for PCR amplification of the targeted sites with primers listed on Supplementary Table 3 and using MyTaq™ DNA Polymerase (Bioline). Subsequently, PCR products were analyzed in 1% agarose gel electrophoresis, purified with the ExoSAP-IT™ PCR Product Cleanup Reagent (Applied Biosystems™) following manufacturer instructions and Sanger-sequenced. Finally, sequencing results were analyzed using Synthego CRISPR Performance Analysis (https://ice.synthego.com/#/) to determine the ICE score.
RNA isolation and reverse transcription-quantitative PCR (RT-qPCR)
Leaf samples from infiltrated plants were harvested at 4 dpi and 100 mg of tissue were used for total RNA isolation using the Thermo Scientific™ GeneJET RNA Purification Kit. Total RNA was treated with Recombinant DNase I (RNase-free) (Takara) following manufacturer’s instructions. Aliquots of 1 μg of the treated RNA were used for cDNA synthesis with oligo dT using PrimeScript™ RT-PCR kit (Takara). cDNAs (0.4 μl) were used to determine the expression levels for each gene in triplicated 25 μl reactions with the SYBR® Premix Ex Taq (Takara) using the Applied biosystem 7500 Fast Real Time PCR system. N. benthamiana F-BOX gene was used as internal reference29. Calculations of each sample were carried out according the comparative ΔΔCT method30. Primers used for qRT-PCR reactions are listed in Supplementary Table 3.
This study describes the strong anti-CRISPR activity of the bacterial AcrIIA4 protein in Nicotiana benthamiana, a model plant used as molecular farming platform. The results demonstrate that AcrIIA4 abolishes site-directed mutagenesis in leaves when transiently co-expressed with CRISPR/Cas9. We also show that AcrIIA4 represses CRISPR/dCas9-based transcriptional activation (CRISPRa) of both reporter and endogenous genes 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-Cas9 activity of AcrIIA4 reported here opens new possibilities for customized control of gene editing and gene expression in plants.