Materials and methods

RNA silencing by CRISPR in plants does not require Cas13

Designing CRISPR-Cas13a machinery for in planta expression

To develop prokaryotic CRISPR-Cas13a machinery as a platform for in planta transcript-silencing, sequences of LbuCas13a and LbaCas13a effectors were N. benthamiana codon optimized along with 3x-FLAG tag or 3x-HA tag at the N-terminus, and custom synthesized (Genscript, Piscataway, NJ) (Supplementary Table S1). These fragments were assembled using HiFi DNA assembly (New England Biolabs, Ipswich, MA). The integrity of the constructs was confirmed by Sanger sequencing (Genewiz, South Plainfield, NJ).

Turnip mosaic virus engineered to express GFP (TuMV-GFP)20 and the endogenous phytoene desaturase (PDS) gene were selected as targets for CRISPR-Cas13a interference. For crRNA designs, Lba- or LbuCas13a specific direct repeats with 28 nucleotide spacer sequences complementary to the target were expressed by the Arabidopsis thaliana U6 promoter (Supplementary Table S2). For TuMV targeting, three single crRNAs targeting different regions of TuMV namely 5’untranslated region (5’ UTR), Helper component Proteinase (HcPro), viral genome linked protein (Vpg), and a poly crRNA containing aforementioned individual crRNAs in an array were designed and constructed (Fig. 1 and Supplementary Table S3). Similar to TuMV, the PDS transcript was targeted using three single crRNAs namely, s-guide 1, s-guide 2, and s-guide 3 and a multi-guide crRNA containing the three single guides (Supplementary Tables S3 and S4). To create mismatch guides corresponding to PDS multi-guide crRNA, the nucleotide sequence was altered at positions 5-6 bp, 10-11bp, and 21-22 bp from the 5’ end of each crRNA (Supplementary Table S4). A non-targeting crRNA was designed as a negative control. To create the sgRNA2 construct, we assembled the single-guide 2 target sequence with the transactivating crRNA (tracrRNA). The same strategy was used to construct sgRNA2 50%mm in which single-guide 2 crRNA had mismatches at every-other nucleotide. The NT-sgRNA negative control contained the Cas9 tracrRNA sequence and a non-plant target sequence (Supplementary Table S4).

Cloning of CRISPR-Cas13a machinery

A backbone harboring AtU6 promoter sequence with one Lbu or Lba specific direct repeat sequence and Bsa_I Golden Gate site was custom synthesized (IDT, Coralville, IA) for expressing crRNAs. This backbone was cloned into entry vector _pENTR (Thermo Scientific, Waltham MA) using Topo cloning. Spacer sequences were ordered as oligos and cloned using Bsa_I Golden Gate site. Gateway assembly (Invitrogen) was used to clone the promoter and crRNA cassette into the destination vector _pGWB413 containing or lacking Cas13a effector (Supplementary Table S1).

Cloning crRNA for TRV systemic delivery

For systemic expression of crRNA using TRV, pea early browning virus (PEBV) promoter sequence with LbuCas13a specific direct repeat and Bsa_I Golden gate site were custom synthesized (IDT, Coralville, IA) and cloned into Gateway entry vector _PCR8 (Supplementary Table S1). Three single guide and multi-guide crRNA sequences targeting NbPDS, and a multi-guide crRNA targeting SlPDS were ordered as oligos and cloned using Golden gate assembly (Supplementary Table S5). The cassette harboring PEBV promoter and TuMV, NbPDS, or SlPDS targeting crRNAs was PCR amplified with primers having EcoR_I and _Mlu_I restriction sites and cloned into _EcoR_I and _Mlu_I digested _pTRV2 vector (Supplementary Table 6).

Cloning of intron hairpin RNAi (hpRNAi) cassette

For cloning of PDS hpRNAi construct, a 197 bp sequence of PDS gene was custom synthesized as sense and antisense arm along with PDK intron sequence with 25 bp overhang complementarity to pGWB413 vector (Supplementary Table S1). All the fragments were assembled using HiFi DNA assembly (New England Biolabs, Ipswich, MA) expressed by the 35S promoter.

Agro-infiltration of N. benthamiana and Solanum lycopersicum

N. benthamiana plants were grown and maintained in growth chamber at 23°C with 16-hour day and 8 hour light cycle and 70% humidity. Four-week-old plants were used for leaf spot agroinfiltration to test Cas13a interference against TuMV-GFP. Binary constructs harboring Cas13a homologs with or without crRNA (targeting TuMV or PDS transcript), TuMV-GFP infectious clone (a gift from Dr. James Carrington) were individually transformed into chemically competent Agrobacterium tumefaciens strain GV3101. Single colonies for each construct were inoculated into LB medium with antibiotics and grown overnight at 28 °C. Next day, the cultures were centrifuged and suspended in agroinfiltration buffer (10mM MgCl2, 10mM MES buffer pH 5.7 and 100μM acetosyringone), and incubated at ambient temperature for 2-3 hours. For TuMV interference assay, Agrobacterium cells harboring Cas13a with crRNA targeting TuMV were infiltrated at an OD600 of 1.0 into adaxial side of four-week-old N. benthamiana leaves using a 1.0 ml needleless syringe. Two days later, Agrobacterium cells harboring TuMV-GFP were infiltrated into same areas at an OD600 of 0.3. After five days, interference activity of Cas13a against the TuMV-GFP was assayed by visualizing GFP in infiltrated leaves under UV light using a hand-held UV lamp (Fisher Scientific, Waltham, MA) and a Nikon camera.

For PDS silencing, leaves of four-week-old N. benthamiana plants were infiltrated with Agrobacterium cultures harboring LbuCas13a with crRNAs targeting PDS and leaf samples were collected at 5 days post inoculation. For TRV mediated crRNA delivery, assays used three-week-old N. benthamiana plants. A single colony of Agrobacterium harboring crRNAs targeting PDS were inoculated into LB medium with antibiotics and grown overnight at 28 °C. Next day, the cultures were centrifuged and resuspended into infiltration buffer at an OD600 of 0.6. The cultures were incubated at ambient temperature for 2-3 hours and infiltrated into N. benthamiana. Two upper leaves were collected two-weeks after TRV infiltration. Control plant infiltrated with TRV expressing an RNAi antisense fragment were used to help track systemic TRV movement. Infiltration of tomato plants was performed similarly to N. benthamiana except that Agrobacterium cells were resuspended into infiltration buffer at an OD600 0f 2.0. The cultures were incubated at ambient temperature for 2-3 hours and infiltrated into three-week-old tomato plants. Data was collected two-weeks after TRV infiltration in the lower leaves.

RNA isolation, cDNA synthesis, qRT-PCR and northern blotting

Total RNA was isolated from Agro-infiltrated leaf samples and upper leaf tissue following systemic TRV movement using Trizol (Ambion) 54. For first strand cDNA synthesis, DNase treated 1 μg total RNA was reverse transcribed using either random hexamers or oligo(dT20) and SuperScript II reverse transcriptase (Thermo Fisher Scientific) according to the manufacturer’s instructions. Quantitative PCR was performed using SYBR Select Master Mix (Applied Biosystem) and gene specific primers (Supplementary table) for PDS and TuMV. EF1α gene was used as internal house-keeping reference for PDS and TuMV qRT-PCR 55 The experiments were repeated three times with three biological and two technical replicates. Relative expression values were plotted using ggplot2 in R 56,57. For detection of PDS transcript, 20 μg of total RNA was separated on a denaturing 1.2% agarose gel and blotted on a Hybond-N+ (Roche) membrane. RNA was crosslinked using UV light and hybridized with a DIG labelled probe (PCR DIG probe synthesis kit, Sigma). For detection of LbuCas13a the membrane was stripped and probed with DIG labelled Cas13a specific probe and signal detected on a Licor Odyssey imaging system (LI-COR Bioscience, Lincoln, NE).

Real time quantification of PDS and TuMV transcripts using Nanocounting technology

For direct RNA quantification of PDS and TuMV transcripts using NanoString technology, we collected sequence data for different N. benthamiana genes including PDS, three house-keeping genes for normalization (PP2aa2, EF1α, RPL23a), LbuCas13a, HCPro and coat protein (Supplementary Table 7). The sequence information was utilized to design two probes for each target gene. Total RNA samples (300 ng total RNA) and probe master mix were supplied to the Huntsman Cancer Institute, University of Utah for Nanostring quantification following manufacturer specifications. The nano-counting data was analyzed using the nSolver software.

Western blotting

For western blotting, total protein was isolated from Agrobacterium infiltrated leaves using extraction buffer (50mM Tris-Cl, 1% β-Mercaptoethanol and protease inhibitor cocktail (Roche, Basel, Switzerland)). Total proteins were boiled with loading buffer (100mM Tris-Cl, 20% Glycerol, 4% SDS, 10% β-Mercaptoethanol and 0.2mg/ml bromophenol blue) and resolved on 12% SDS-PAGE gel. The proteins were transferred from SDS-PAGE gel to PVDF membrane (GE healthcare, Chicago, IL). Membrane blocking and antibody incubations were performed using iBind western device (Thermo Fisher Scientific, Waltham, MA) according to the instrument manual. Finally, the membrane was treated with ECL Select western blotting detection reagent (GE healthcare, Chicago, IL) and signal was detected with Licor Odyssey imaging system (LI-COR Bioscience, Lincoln, NE).

Small RNA sequencing and analysis

Two separate small RNA sequencing experiments were conducted. For results shown in (Fig. 3a-e), Cas13 and crRNA guides and controls were expressed in N. benthamiana leaves using agrobacterium spot infiltration as described. Total RNA was extracted from infiltrated leaves using Trizol following manufactures guidelines. For results shown in (Fig. 3f-l), crRNA guides and controls were expressed from TRV using agrobacterium infiltration as described. Total RNA was extracted from upper leaves following systemic TRV movement using Trizol. Total RNA samples were sent to the Beijing Genomics Institute (BGI Group, Hong Kong). Twenty-four small RNA libraries were constructed following the DNBseq small RNA library protocol. Briefly, small RNA were isolated from PAGE gel corresponding to size 18-30 nt. Adapters were ligated and first strand synthesis performed according to DNBseq small RNA library protocol. Libraries were PCR amplified and size selected and sequenced on the DNBseq platform (BGI Tech Solutions, Hong Kong, China).

Small RNA reads for both experiments were trimmed 58,59, and aligned using STAR (v2.7.3a) 60 to a modified version of the N. benthamiana genome (v1.0.1)61. The modifications included removing all contigs with less than 70K nt, adding the coding sequence of LbuCas13a as a contig, and masking one of the two paralogs coding for PDS. The coding sequence for PDS on contig Niben101Scf14708, position 12885-21779 (gene23) was masked in order to ensure unique mapping to a single PDS locus on contig Niben101Scf01283, position 197129-205076 (gene 2002). Uniquely mapped read counts for the exons were extracted per base-pair using samtools (v1.3)62 and bedtools ‘coverage’ (v2.29.2) 63. To compare between sequenced samples, mapped reads were normalized to library size (i.e. total uniquely mapped reads per library) using the equation (number of reads mapped at a nucleotide position (1 / number of uniquely mapped reads in library) 1M), referred to as counts per million (CPM). The size distribution of uniquely mapped reads were analyzed for 21, 22, and 24 nt sRNA. The average number of uniquely mapped sRNA to the PDS transcript was calculated for the duplicate samples for each size class. The proportion of each size class was determined by the equation, ((average number of reads per size class / sum of average number of reads per size class)*100). Analyses were carried out using Python3 (v3.8.2) libraries NumPy (v1.18.1), Pandas (1.0.3) and plotted with Matplotlib (v3.2.1) 6467. Processed files, additional information and the reference genome used for mapping are provided through the GEO53 Series accession number GSE171980. (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE171980).

Generating stable transgenic Arabidopsis plants

TTG1-targeting three single guides (guide-1, −2, −3) and a multi-guide crRNA (Supplementary Table 8), and non-targeting (NT) oligos were annealed and ligated into pENTR backbone containing Bsa_I Golden gate site. Gateway assembly was used to transfer guide crRNA to _pGWB413 destination vector with or without 3xHA-LbuCas13a. Stable transgenic Arabidopsis plants expressing TTG1 guides with or without LbuCas13a were generated using Agrobacterium-mediated floral dip 68 Similarly, stable Arabidopsis controls with a NT crRNA, a 197 bp hairpin construct against TTG1 (a gift from Dr. Steven Strauss), and no guide transformation control (only 3xFLAG-LbuCas13a) were generated. One month after floral dip, T1 seeds were collected and stored at 4°C.

Arabidopsis phenotyping

Transformed T1 Arabidopsis seedlings were identified using rapid selection protocol 69. Selection was conducted on ½ MS media with a Kanamycin concentration of 100 μg/ml. Positive transformants (n = 36) for each TTG1 crRNA with or without LbuCas13a and TTG1 hairpin controls were transferred to soil and grown under optimal conditions. Control Arabidopsis Col-0 plants were germinated on ½ MS media without Kanamycin and transferred to soil. Seventh leaf from ten individual plants for each construct was imaged under a dissecting microscope equipped with a Nikon camera and trichomes were counted using multi-point feature in ImageJ software 70. For each construct, RNA was extracted from 10th leaf of five individual plants with varying leaf trichomes to quantify TTG1 expression using qRT-PCR. AtEF1α was used as internal house-keeping control for normalizing TTG1 expression (Supplementary Table 6). Selected individual plants for each construct were self-pollinated to collect T2 seed. Five technical replicates of each selected plant/line were used for analyzing total flavonoids, in 5 mg seed, using modified aluminum chloride (AlCl3) colorimetric method 71. Total flavonoids content was estimated using the following formula: flavonoids (mg/g) = concentration obtained through quercetin calibration curve × (volume of extract/seed weight).

To determine the inheritance of GIGS and Cas13-mediated gene silencing, 10 T2 plants from selected T1 lines were transferred to soil after Kanamycin selection. Seventh leaf from 10 individual T2 plants was imaged for counting leaf trichomes. Statistical comparisons between the transformation control (no guide) and each selected line was performed. TTG1 expression in the top rosette leaf from three individual T2 plants was analyzed using qRT-PCR. Five individual T2 plants for each line were self-pollinated to collect T3 seed. Total flavonoid content was analyzed in T3 seeds from five independent seed lots (five biological replicates). Similarly, proanthocyanidins content was measured using DMACA-HCl method from three seed lots 72. Proanthocyanidins were measured at 640 nm and reported as per gram of seed weight. Total flavonoid and proanthocyanidin analyses were repeated twice, the averaged values for each seed lot were used for statistical comparisons. Absorbance of flavonoids and anthocyanin was measured using Thermo Spectronic 3 UV-Visible Spectrophotometer. While absorbance of proanthocyanidins was measured through Synergy H1 Hybrid Multi-Mode Microplate Reader (Agilent Technologies, Winooski, Vermont).

For leaf anthocyanin quantification, one-week-old T3 seedlings after Kanamycin selection were transferred into ½ MS media + 3% sucrose and subjected to light stress (500 μmol m−2 s−1) for one week. 200 mg of leaf tissue was used for quantifying anthocyanin 73. Anthocyanin analysis was repeated twice with 5 replicates in each batch. Anthocyanin content was calculated by using following formula (absorbance/35,000× dilution factor×647 × 1,000 per mg of sample extracted (in mg g-1 fresh weight). Representative plantlets following sucrose treatment showing anthocyanin pigmentation were imaged with a dissecting microscope equipped with a Nikon camera. To test TTG1 expression in T3 generation, seventh leaf from three individual plants was analyzed using qRT-PCR. To determine the expression of LbuCas13a, RT-PCR was conducted on cDNA synthesized for qRT-PCR. Western blot analysis with HA-tag antibody was conducted on one-week-old T3 seedlings post Kanamycin selection.

Article TitleRNA silencing by CRISPR in plants does not require Cas13

Abstract

RNA-targeting CRISPR-Cas can provide potential advantages over DNA editing, such as avoiding pleiotropic effects of genome editing, providing precise spatiotemporal regulation and expanded function including anti-viral immunity. Here, we report the use of CRISPR-Cas13 in plants to reduce both viral and endogenous RNA. Unexpectedly, we discovered that crRNA designed to guide Cas13 could, in the absence of the Cas13 protein, cause substantial reduction in RNA levels as well. We demonstrate Cas13-independent guide-induced gene silencing (GIGS) in three plant species, including stable transgenic Arabidopsis. We determined that GIGS utilizes endogenous RNAi machinery despite the fact that crRNA are unlike canonical triggers of RNAi such as miRNA, hairpins or long double-stranded RNA. These results suggest that GIGS offers a novel and flexible approach to RNA reduction with potential benefits over existing technologies for crop improvement. Our results demonstrate that GIGS is active across a range of plant species, evidence similar to recent findings in an insect system, which suggests that GIGS is potentially active across many eukaryotes.


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