key: cord-0744201-hl1r0qww authors: Singsuksawat, Ekapot; Onnome, Suppachoke; Posiri, Pratsaneeyaporn; Suphatrakul, Amporn; Srisuk, Nittaya; Nantachokchawapan, Rapirat; Praneechit, Hansa; Sae-kow, Chutimon; Chidpratum, Pala; Hongeng, Suradej; Avirutnan, Panisadee; Duangjinda, Thaneeya; Siridechadilok, Bunpote title: Potent programmable antiviral against dengue virus in primary human cells by Cas13b RNP with short spacer and delivery by virus-like particle. date: 2021-05-01 journal: Mol Ther Methods Clin Dev DOI: 10.1016/j.omtm.2021.04.014 sha: 35daef4b481c489de4a0eb3801fd08ccdd030749 doc_id: 744201 cord_uid: hl1r0qww With sequencing as a standard frontline protocol to identify emerging viruses such Zika virus and SARS-CoV-2, direct utilization of sequence data to program antivirals against the viruses could accelerate drug development to treat their infections. CRISPR-Cas effectors are promising candidates that could be programmed to inactivate viral genetic material based on sequence data but several challenges such as delivery and design of effective crRNA need to be addressed to realize practical use. Here, we showed that virus-like particle (VLP) could deliver PspCas13b-crRNA ribonucleoprotein (RNP) in nanomolar range to efficiently suppress dengue virus infection in primary human target cells. Shortening spacer length could significantly enhance RNA-targeting efficiency of PspCas13b in mammalian cells compared to the natural length of 30 nucleotides without compromising multiplex targeting by a crRNA array. Our results demonstrate the potentials of applying PspCas13b RNP to suppress RNA virus infection, with implications in targeting host RNA as well. . Though the site of flaviviral RNA replication is associated with ER membrane 19 , 102 localizing PspCas13b to ER with tail-anchor sequences such as SQS or VAMP2 20 failed to 103 suppress DENV2-mCherry infection (Supplementary Figure 1b) . The suppression of 104 DENV2-mCherry by mCh3 crRNA was specific as other reporter DENV2 could not be 105 suppressed by the crRNA in both single-virus infection and co-infection settings 106 ( Supplementary Figures 1c-d) . Using BHK-21-Cas13b, we individually tested 51 crRNAs 107 against the targets on DENV2 reporter viruses, DENV2-16681 (NS5 gene), and ZIKV-108 SV0010 (NS2A gene) (Supplementary Table 1 (Figure 1b) . However, the delivery at 24 hpi 147 failed to reduce infection (Figure 1b) . The same experiment in BHK-21 also showed similar 148 effect of delivery time on the reduction of infectious titer (Figure 1c) . Together, these results infection but the efficacy depends on the delivery time. 151 Shortening spacer length could improve knock down activity of PspCas13b. 153 Our results in Supplementary Figure 1e also suggested that the knock-down efficiency 155 could be improved for several targets. Using BHK-21-Cas13b, we tested whether spacer 156 length could affect viral suppression efficiency for a subset of these crRNAs. Strikingly, 157 shortening spacer length to 26-18 nucleotides of mCh3 crRNA could drastically enhance the 158 suppression of DENV2-mCherry with maximum knock-down activity achieved between 20 159 and 22 nucleotides and without cross suppressing nontarget DENV2-mAmetrine (Figure 2a) . 160 We also found that 22-nucleotide spacer length could improve targeting efficiency against a 161 ZIKV NS2A region compared to crRNAs with 30-nucleotide spacer (30-nt crRNA) tiled 162 against it (Figure 2b) . Shortening spacer enhanced the knock-down activity of several 163 crRNAs with varying efficiency (Supplementary Figures 3a-b) . Nevertheless, the knock-164 down activities of two crRNAs (mAmet1 and 8681) were aggravated by 22-nt spacer 165 ( Supplementary Figures 3a-b) . We selected three pairs of 30-nt vs. 22-nt crRNAs for 166 testing by VLP delivery in BHK-21 cells. The knock-down enhancement was recapitulated 167 with Cas13b RNP delivery by VLP for 22-nt vs. 30-nt mCh3 crRNAs (Figure 2c) . In contrast 168 to the results in BHK-21-Cas13b, the 22-nt mAmet-1 crRNA could knock down DENV2-169 mAmetrine with much less PspCas13b than its 30-nt counterpart (Figure 2c) . The dose-170 response curves indicated that the 22-nt mCh3 and mAmet1 crRNAs needed 15-20 folds less 171 of PspCas13b than 30-nt crRNAs to achieve the same level of virus suppression (Figure 2c) . We tested whether the enhancement effect of short spacer could be observed in different 178 settings. VLP delivery of Cas13b with 22-nt mCh3 crRNA to 293T, hDC, and iMHC showed 179 superior knock-down activity over its 30-nt counterpart with Cas13b dose at 2,575 180 ng/100,000 cells (Figure 2d) , indicating that the effect was not cell-specific. In BHK-21-181 Cas13b was required to achieve the same level of suppression compared to Cas13b-single 201 crRNA by 3-10 folds (Figure 3d vs. Figure 2c) . The knock-down activities by multiplex 202 VLP against reporter DENV2 generally conformed to what had been observed with singlet 203 VLPs, though the enhancement effect of 22-nt spacer for mAmeterine was drastically 204 reduced in the multiplex targeting (Figure 3d vs. Figure 2c) . (Figures 1 and 2d) . We found that shorter Overall, our study addressed two key aspects of the implementation of antiviral CRISPR-287 Cas13 against RNA viruses. We showed that virus-like particle was an effective delivery 288 vehicle to deliver PspCas13b RNP into a variety of primary cells. We showed that crRNA 289 The isolated CD14 + monocytes were cultured in R10 supplemented with 5% HI-human AB 342 serum to allow differentiation into macrophages. 39 Half volume of medium was replaced with 343 fresh medium every 3 days. The macrophages were harvested for experiments on day 13. The 344 macrophages were then verified by flow cytometry analysis with CD163-FITC mAb For these cells, the VLP-treated cells were then maintained for 24 hours at 37°C before VLP into hDC, 50,000 cells of hDC were centrifuged at 450 x g, 5 min, 4°C and the cell 498 pellet was resuspended with 300 µl R10 + 8 µg/ml polybrene. 100 µl of VLP delivery media 499 (R10) was added to hDC. The cells were then transferred to one well of 24-well plate and 500 cultured for 48 hours at 37°C before harvest. VLP delivery of YFP was performed with the 501 same protocol as VLP delivery of Cas13b. The preformed RNP was then added to 10 µl in vitro cleavage reaction that contained 20 ng 562 of mCherry RNA, 1 µl of 10x Cut smart buffer (NEB), 4 U of RNase IN and incubated at 563 37°C for 2 hours. The reaction was stop with 1% SDS and then extracted by 564 phenol/chloroform for ethanol precipitation. The RNA pellet was resuspended with 1x RNA 565 loading buffer (4 mM EDTA, pH 8.0, 2.7% formaldehyde, 20% glycerol, 7.7 M formamide, 566 80 mM MOPS, 20 mM sodium acetate, 0.025% (v/v) bromophenol blue) and heated at 95°C 567 for 10 min and then put on ice 2 min before loading on 8% TBE-polyacrylamide gel with 8 568 M urea. 569 different conditions. 45 The analysis and drawing of Cumming plot (Figure 1b) were 572 performed on https://www.estimationstats.com/. Significance tests for multiple two groups 573 and shared-control groups were performed by one-way ANOVA. Nonparametric, two-sided 574 permutation t-test was used to calculate p-values. 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Each 95% confidence 749 interval is indicated by the ends of the vertical error bars. The VLPs were delivered with 750 equal Cas13b dose of 1,720 ng/100,000 cells. The percent inhibition was calculated by the 751 frequency of The results represent the data from three donors and duplicate experiments. c) The 753 reduction of infectious titers by PspCas13b + 8681-nt crRNA against BHK-21 cells. The results are from triplicate experiments. The infections were performed as in four replicates. Error bar = standard deviation Figure 3: Short spacer did not inhibit crRNA processing and multiplex targeting of The sequences 30-nt and 22-nt crRNA arrays used for 775 testing in vitro crRNA processing and multiplex targeting. DR = direct repeat. b) In vitro 776 crRNA processing analyzed by 8M urea PAGE. 3x = crRNA array with three crRNAs, 2x = 777 crRNA array with two crRNAs, 1x = singlet crRNAs. c) Targeting of multiple reporter 778 DENV2 with the crRNA array in single-virus The measurements for crRNA arrays were 780 done in four replicates. For three-virus infections (right bar plot), p-values were calculated 781 from side-by-side comparison between no crRNA and HAK with the same reporter DENV2 * p-value < 0.05, ns = p-value > 0.05. d) Dose-response curves of multiplex VLP-Cas13b RNP generated with a crRNA array. The measurements were done in duplicate. Error bar = 784 standard deviation