Strong and tunable anti-CRISPR/Cas9 activity of AcrIIA4 in plants

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.

evidence of their functionality in plants exists. Here we study the ability of an Acr protein from a Listeria monocytogenes prophage (AcrIIA4) to inhibit the activity of Streptococcus pyogenes Cas9 (SpCas9) in Nicotiana benthamiana.
First, we analyzed the ability of AcrIIA4 to prevent Cas9 site-speci c mutagenesis in plant tissues using three previously well characterized targets: the xylosyltransferase gene (XT) and two different genes of the Squamosa-promoter binding protein-like (SPL) family (Supplementary Table 1). As shown in Fig. 1A and 1B, transient expression of speci c gRNAs along with the Cas9 resulted in average editing e ciencies of 6%, 8% and 10% for each target respectively, whereas co-in ltrations of the same editing constructs with a transcriptional unit (TU) expressing a nuclear-localized AcrIIA4 under the constitutive CaMV 35S promoter reduced the editing e ciencies to undetectable levels in all three targets. These results con rmed the ability of AcrIIA4 to prevent Cas9-mediated editing.
dCas9-based programmable transcriptional activators (PTAs) are becoming widely used in plants for customized regulation of gene expression. We previously developed a collection of dCas9-based PTAs for plant gene control, being the strongest one the so-called dCasEV2.1 activator complex 16 . dCasEV2.1 comprises three elements, each one encoded in a different TU: (i) a modi ed scaffold RNA, which includes anchoring sites for the phage MS2 coat protein (MCP) 17 ; (ii) a dCas9 fused to the EDLL plant activator domain, and (iii) the MCP fused to the synthetic VP64-p65-Rta (VPR) activator domain. We rst validated the ability of AcrIIA4 to prevent dCasEV2.1-based gene activation in plants using a transient assay based on a luciferase reporter. The reporter system comprised a Fire y luciferase CDS (Fluc) driven by the Solanum lycopersicum DFR promoter (pSlDFR) and a dCasEV2.1 complex targeted to position − 147 of the pSlDFR. Luminescence assays showed that the relative transcription activity (RTA) conferred by pSlDFR changed from 0.25 ± 0.28 in its basal state (with dCasEV2.1 loaded with an unrelated gRNA) to 4.96 ± 1.59 in the activated stage (with dCasEV2.1 loaded with a promoter-speci c gRNA Fig. 1B).
It has been suggested that AcrIIA4 inhibits Cas9 activity by a binding mimicking mechanism, occupying the PAM DNA-binding site with higher a nity than the DNA substrate 18 . Accordingly, its anti-Cas activity is expected to be dependent on AcrIIA4 concentration. To test this hypothesis, we performed a doseresponse experiment, in ltrating decreasing amounts of the agrobacterium culture that drives expression of AcrIIA4. As expected, AcrIIA4 anti-Cas9 activity showed a strong dose-dependent effect ( Fig. 2A). The two highest optical densities (OD 600 ) assayed, namely 0.1 and 0.05, resulted in the complete inhibition of the dCasEV2.1-mediated activation, with RTA values of ~ 0.09. Based on previous estimations of the multiplicity of transformation (MOT) as a function of the agrobacterium concentration in N. benthamiana leaf agroin ltration 19 , we estimated that a complete dCasEV2.1 inhibition was reached with between ve and seven T-DNA copies per cell. The lowest OD 600 that resulted in a signi cant reduction of the dCasEV2.1-mediated activation was 0.005 (equivalent to an estimated three T-DNA copies per cell on average). This is a bacterial concentration 20 times lower than that used for transferring the dCasEV2.1 construct, which leads to approximately seven T-DNA copies per cell on average. Considering that all transcriptional units in the assay are under the control of the CaMV35S promoter, these results suggest a strong anti-Cas9 activity of AcrIIA4 in plants, a very e cient expression of AcrIIA4, or both.
A key factor to consider when regulating different components in a genetic circuit is time course dependency. Therefore, we decided to test the ability of the AcrIIA4 to suppress an ongoing dCasEV2.1 activation. For this analysis, AcrIIA4 construct was in ltrated simultaneously, or with a delay of 24,48 and 72 h after the in ltration of the reporter/dCasEV2.1/gRNA mix, and all tissue samples were subsequently collected 96 h after the initial in ltration. As shown in Fig. 2B, anti-Cas9 treatment is effective up to 24 h post initial in ltration, but this e ciency is substantially lost at longer timepoints.
These results indicate that displacement of dCas9-DNA activation complexes by AcrIIA4 is a relatively slow or ine cient process and that PTA inhibition by AcrIIA4 is more successfully achieved by early formation of inhibitory complexes rather than by binding competition.
As a rst attempt to regulate anti-CRISPR activity post-translationally, we created and assayed an Nterminal fusion of the AcrIIA4 with the temperature-dependent K2 degron, which was earlier described to induce temperature-dependent degradation when translationally fused to other proteins-of-interest 20 (Fig. 2C). We assayed two K2 con gurations with or without the nuclear localization signal SV40. As shown in Fig. 2D, K2-AcrIIA4 fusions showed considerably reduced ability to repress dCasEV2.1dependent luciferase activation in permissive temperature (14ºC), however the remaining AcrIIA4 repression activity was reduced even further when incubated at restricted temperature (28ºC), resulting in higher luminescence values indicative of partial degron functionality. To explore alternative regulatory options with wider dynamic range, we next analyzed N-and C-terminal fusions of the AcrIIA4 to the minimal auxin inducible degron (AID) mAID47 21 , again with or without the SV40 signal, as schematically depicted in Fig. 3A. AID was previously used for reprogramming Arabidopsis thaliana development when coupled to dCas9-based repressors 22 , as well as for inducing protein depletion in other eukaryotic systems 21,23,24 . Results in Fig. 3B show that most of the mAID fusions assayed partially retained the anti-Cas9 activity of native AcrIIA4, with some con gurations showing almost full activity as it was the case for the mAID N-terminal fusion without nuclear localization signal (mAID-AcrIIA4). Upon auxin treatment, all protein fusions showed a clear de-repression trend indicative of auxin-mediated degradation, however the increase in reporter activity was only statistically signi cant for the above-mentioned mAID-AcrIIA4 fusion (four-fold activation). This is still an incomplete de-repression when compared with the luminescence levels obtained when AcrIIA4 is not present, thus indicating that this degron fusion needs further optimization to reach its full potential. To further con rm the auxin-dependency of the observed de-repression, we conducted a dose-response analysis. As shown in Fig. 3C, a near lineal dependency with the concentration of hormone in the treatment medium was clearly recorded. Auxin treatment was most effective when applied simultaneously with agroin ltration, progressively losing its effect thereafter (Fig. 3D), suggesting that effective post-translation regulation occurs preferentially before the formation of the AcrIIA4-dCas9 complex.
As a nal application test, we analyzed the ability of AcrIIA4 to prevent dCasEV2.1-mediated gene activation of two endogenous N. benthamiana genes, Niben101Scf00305g05035 (NbDFR), encoding a dihydro avonol reductase, and Niben101Scf00156g02004 (NbAN2), encoding a MYB factor involved in phenylpropanoid biosynthesis. Both genes were earlier shown to be e ciently activated with the dCasEV2.1 system 16 . N. benthamiana leaves were agroin ltrated with the dCasEV2.1 system targeting NbDFR or NbAN2 promoters with or without AcrIIA4. At 4 dpi leaf samples were collected and qRT-PCR assays were performed for each gene. To estimate gene expression levels, agroin ltrated NbDFR samples were used as negative control for NbAN2 samples and vice versa. As shown in Fig. 4, the in ltration of dCasEV2.1 targeting NbDFR or NbAN2 promoter regions conferred strong activation reaching up to 400 and 30 fold respectively, although the absolute levels were highly dependent on the age of the leaf. Remarkably, co-in ltration of AcrIIA4 strongly repressed CRISPR-mediated activation in both genes, resulting in a signi cant reduction of the relative gene expression values in all samples assayed.
In view of the results shown here, we conclude that AcrIIA4 is a potent tool for the spatial-temporal regulation of CRISPR/Cas9 activity in plants, Furthermore, in combination with programable transcriptional activation factors such as dCasEV2.1, AcrIIA4 will facilitate the customization of endogenous gene expression in plants.

Materials And Methods
GoldenBraid Cloning DNA constructs used in this work were assembled using GoldenBraid 25 . 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 (modi ed 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 con rmed by restriction analysis and Sanger sequencing and Level 1 assemblies were veri ed by restriction analysis. An exhaustive list of all plasmids used in this work are listed in Supplementary conditions were used. Agroin ltration was carried out as previously reported 27 . Brie y, overnight A. tumefaciens cultures were pelleted and resuspended in agroin ltration solution (10 mM MES, pH 5.6, 10 mM MgCl 2 and 200 µM acetosyringone) to an optical density of 0.1 at 600 nm (OD 600 ). Bacterial suspensions were incubated for 2 h 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, agroin ltrations were carried out through the abaxial surface of the three youngest fully expanded leaves of each plant with a 1 ml needle-free syringe. For some experiments, agroin ltrations were carried out with small modi cations from the general procedure described above. For dose-response assays, SV40-AcrIIA4 TU (GB3344) was resuspended in agroin ltration solution to an OD 600 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 nal OD 600 at 0.1.
For the time-course assay, the strain carrying the SV40-AcrIIA4 TU (GB3344) was agroin ltrated at OD 600 of 0.05 at 0, 24, 48 and 72 h after in ltration 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 in ltration (dpi). For the determination of the FLuc/RLuc activity, one 0.8 cm diameter disc per agroin ltrated 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 Fire y luciferase (FLuc) activity was determined using a GloMax 96 microplate luminometer (Promega) with a 2s 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 agroin ltrated 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 protocol 28 . The DNA was used as template for PCR ampli cation 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, puri ed 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 in ltrated plants were harvested at 4 dpi and 100 mg of tissue were used for total RNA isolation using the Thermo Scienti c™ GeneJET RNA Puri cation 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 reference 29 . Calculations of each sample were carried out according the comparative ΔΔCT method 30 . Primers used for qRT-PCR reactions are listed in Supplementary  Figure 1 AcrIIA4 prevents Cas9 editing in N. benthamiana. A) Schematic representation of AcrIIA4 activity. B) Editing e ciency of NbXT2 and NbSPL in the absence and in the presence of SV40-AcrIIA4. N. benthamiana leaves were agroin ltrated with a Cas9 TU, a P19 TU and the corresponding gRNA, with or without the SV40-AcrIIA4 TU. Editing e ciencies were determined with Synthego. C) CRISPRa repression of SV40-AcrIIA4. Bars show normalized Fluc/Rluc ratios of N. benthamiana leaves expressing dCasEV2.1 and the corresponding speci c or unspeci c gRNAs, with or without AcrIIA4. Error bars indicate SD (n=3). Statistical analyses were performed using unpaired t-Test (P Value <0.05). Variables carrying not signi cant differences are coupled within the same statistical groups marked with the same letters.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. AntiCasSupplementaryInformation.docx