Funding support (grant number BT/PR10906/MED/29/860/2014) for this study was provided by the Department of Biotechnology, Government of India.

NOTE: The JFH1 strain (WT) used here was a kind gift from Takaji Wakita, National Institute of Infectious Diseases. The human hepatoma cell line, Huh7.5, was a kind gift from Charles Rice, The Rockefeller University. A schematic of the method is shown in Figure 1.
1. Genome-wide substitution mutagenesis of JFH1 using error-prone PCR
<ol>
	<li>To perform ep-PCR, prepare the master mix for four sets of experiments with primers J-For 5&#39;-GTTTTCCCAGTCAGCACGTTGTAAAACGACGGC-3&#39; and J-Rev 5&#39;-CATGATCTGCAGAGAGACCAGTTACGGCACTCTC -3&#39; (Figure 1A), along with the reaction components described in Table 1 without the pJFH1 template (plasmid isolated from JFH1 strain).</li>
	<li>Aliquot the master mix into four tubes, add 100 ng, 50 ng, 25 ng, and 10 ng of template into the individual tubes, and adjust the total volume of the reaction to 50 &#181;L. Apply the cycling conditions described in Table 2 to amplify a 9736 base pair (bp) fragment comprising the T7 promoter and full-length HCV genome (Figure 2). 
	NOTE: The ep-PCR product must contain the T7 promoter sequence to facilitate in vitro transcription of the viral genome. Therefore, the forward primer must be located upstream of the T7 promoter of the viral cDNA clone, and the reverse primer sequence should end at the 3&#39;-end of the virus genome to facilitate transcription run-off. Optimize each component of the ep-PCR reaction within the range recommended by the manufacturer of Taq DNA polymerase to achieve high-yield amplification. In addition to varied template amounts, unbalanced dNTPs (especially low dATP and/or dGTP concentration) can also be used to increase diversity in the length of viral genomes from 6-8 kb long3.</li>
	<li>Estimate the ep-PCR product amplified by loading equal volumes of the PCR products (5 &#181;L) and comparing to known amounts of 1 kilobase (kb) DNA ladder by running a 0.8% Tris-acetate-EDTA (TAE)-based agarose gel electrophoresis11. Purify the product using a column purification kit as directed by the manufacturer.</li>
	<li>Estimate the concentration of the purified product by measuring the absorbance at 260 nm using a microvolume spectrophotometer12. Vacuum concentrate the ep-PCR product if needed to obtain a product concentration &#8805;100 ng/&#181;L. 
	&#8203;NOTE: More accurate estimation can be made by loading two-fold serial dilutions of ep-PCR product and comparing their intensities to the known amounts of 1 kb DNA ladder.</li>
</ol>
2. Viral RNA synthesis
<ol>
	<li>Set up a 40 &#181;L reaction to digest 5 &#181;g of clonal-pJFH1 with 4 U of XbaI enzyme at 37 &#176;C for 2 h, followed by column purification and measuring the absorbance at 260 nm in a microvolume spectrophotometer12. Set up a 20 &#181;L in vitro transcription reaction of the column-purified ep-PCR product or clonal-pJFH1 (WT) using a commercially available large-scale RNA production system according to the manufacturer&#39;s instructions.</li>
	<li>In a 200 &#181;L microcentrifuge tube, mix approximately 1 &#181;g of the purified ep-PCR product (mutant libraries) or XbaI digested clonal-pJFH1, 10 &#181;L of 2x buffer containing ribonucleotides, and 2 &#181;L of T7 enzyme mix. Mix all the components and briefly spin down the components. Incubate the reaction mixture at 37 &#176;C for 45 min, followed by the addition of 1 U RNase-free DNase enzyme and incubation at 37 &#176;C for 30 min (Figure 1C).</li>
	<li>Purify in vitro synthesized RNA using an RNA cleanup kit as per the manufacturer&#39;s instructions, elute in 40 &#181;L of RNase- and DNase-free water, and estimate the concentration of the purified RNA by measuring the absorbance at 260 nm using a microvolume spectrophotometer12. Make aliquots as needed (5 &#181;g or 2 &#181;g) to avoid freeze-thaw and store them at &#8722;80 &#176;C. Verify the integrity and size of the viral transcripts by using 2 &#181;g of RNA for a MOPS formaldehyde 0.8% agarose gel electrophoresis13 (Figure 3).</li>
</ol>
3. Estimation of the proportion of mutations in ep-PCR products (mutant libraries)
NOTE: In this step, the proportion of nucleotides mutated by subcloning the product obtained in step 2. was estimated to demonstrate the advantage of employing ep-PCR to create genetic heterogeneity using two full genome mutant libraries (ML50 and ML25) and clonal pJFH1-derived viral RNAs. The proportion of mutations was estimated in the HCV NS5A gene, which was also the phenotypic readout gene (drug resistance) in this study.
<ol>
	<li>Set up a 20 &#181;L cDNA synthesis reaction by adding approximately 1 &#181;g of the viral RNA synthesized in step 2., 5 &#181;M reverse primer 5&#39;-GTGTACCTAGTGTGTGCCGCTCTA-3&#39;, and 200 U reverse transcriptase as per the manufacturer&#39;s recommendations.</li>
	<li>Using the cDNA, amplify a 2571 bp fragment that comprises the complete NS5A gene. To do this, prepare a reaction mixture containing 0.5 &#181;M for 5272F and 7848R primers (final concentration; Table 3), 1.5 mM MgCl2,&#160;1x PCR buffer, and 1 U high-fidelity Taq polymerase in a total volume of 50 &#181;L and run an amplification cycle using the conditions described in Table 4.</li>
	<li>Run the product on a 0.8% TAE-based agarose gel to confirm the product size of 2571 bp and then purify the product using a column purification kit as directed by the manufacturer. Elute the column-purified product in 40 &#181;L of sterile water.</li>
	<li>To add a 3&#39; A overhang to the product, add 0.5 &#181;M dATP and 1 U of low-fidelity Taq DNA polymerase and incubate the entire PCR product at 70 &#176;C for 30 min along with 1x PCR buffer and 1.5 mM MgCl2 (final concentration). Purify the mixture using a column purification kit as directed by the manufacturer 
	NOTE: Alternatively, excise the ~9.7 kb long product from the gel from step 1.4. and perform cloning using a commercially available kit for cloning of long-PCR product according to the manufacturer&#39;s instructions or perform NGS of the ep-PCR product using an Illumina platform2.</li>
	<li>Set up a ligation reaction by adding 5 &#181;L of 2x ligation buffer, 50 ng of the T-vector DNA, approximately 3-fold molar excess of the insert DNA synthesized in step 3.4., 3 Weiss units of T4 DNA ligase, and nuclease-free water to a total volume of 10 &#181;L. Incubate this reaction at room temperature for 3 h and then add 100 &#181;L of Escherichia coli DH5&#945; with the ligated DNA; heat shock the cells at 42 &#176;C for 35 s (Figure 1B).</li>
	<li>Add 1 mL of Luria-Bertani (LB) medium (without antibiotic) and incubate with gentle shaking for 1 h at 37 &#176;C for recovery. Centrifuge the cell suspension at 13,800 x g, discard the supernatant, and resuspend in 200 &#181;L of fresh LB medium.</li>
	<li>Plate 100 &#181;L of the transformed E. coli DH5&#945;&#160;cells on an LB plate containing 50 &#181;g/mL ampicillin and incubate at 37 &#176;C for 16 h. Set up minipreps of 25-30 colonies in 5 mL of LB medium + 50 &#181;g/mL ampicillin and grow overnight at 37 &#176;C.</li>
	<li>The next day, extract the plasmids with a plasmid purification kit according to the manufacturer&#39;s instructions and perform restriction enzyme digestion in 10 &#181;L volume with 200 ng of the isolated plasmids, 2 U of EcoR1, and 1x restriction buffer for all the colonies.</li>
	<li>After incubating the digestion mixtures at 37 &#176;C for 3 h, resolve the products on 0.8% TAE-based agarose gel to confirm plasmid DNA insertion.</li>
	<li>Perform Sanger sequencing of the plasmids of 25 positive clones using M13 Forward, M13 reverse, 6208-SPF, and 6748-SPF primers (Table 3) to determine the proportion of mutations (expressed as the proportion of nucleotides mutated) in the viral RNAs derived from the libraries and clonal pJFH1 (Figure 1B and Figure 4).</li>
</ol>
4. Viral RNA transfection of the Huh7.5 cell line
NOTE: Use RNase/DNase-free tissue culture materials and work in a sterile class II biosafety cabinet. Work in the recommended containment facility as per the biosafety guidelines of the organization.
<ol>
	<li>Maintain Huh7.5 cells by incubating in a 5% CO2 incubator at 37 &#176;C in a T75 cm2 tissue culture flask containing 15 mL of complete Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) supplemented with 10% (vol/vol) fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 &#181;g/mL; Figure 1D).</li>
	<li>Split the cells in a 1:3 ratio when confluency reaches 90%. Wash the cells with 1x prewarmed phosphate-buffered saline (PBS; pH 7.4). Add 5 mL of 1x trypsin-EDTA solution to completely cover the cell layer, gently swirl the flask, and then incubate the flask at 37 &#176;C for 5 min. 
	NOTE: Incubation time may vary; incubate the cells until the cell layer is completely detached from the cell culture flask surface.</li>
	<li>Add 10 mL of 1x prewarmed complete DMEM, disperse the cells by repeat pipetting, and collect the cell suspension in a 15 mL or 50 mL sterile centrifuge tube. Centrifuge the cell suspension at 252 x g&#160;for 5 min at room temperature. Discard the supernatant, resuspend the cell pellet in 6 mL of complete DMEM, and incubate the cells at 37 &#176;C in a humidified incubator with 5% CO2.</li>
	<li>For continuous maintenance of na&#239;ve, transfected, or virus-infected Huh7.5 cells, repeat step 4.2. and step 4.3.</li>
	<li>1 day prior to transfection, split the cells as described in steps 4.1.-4.3., count the number of viable cells using a hemocytometer, seed the Huh7.5 cells at a density of 0.6 x 106 in 35 mm dishes in 2 mL of complete DMEM, and incubate the cells at 37 &#176;C in a humidified incubator with 5% CO2.</li>
	<li>On the following day, prepare the transcript-lipid complex. To do this, dilute 10 &#181;L of transfection reagent in 50 &#181;L of minimal essential medium, and separately dilute 5 &#181;g of viral transcripts in 50 &#181;L of minimal essential medium.</li>
	<li>Incubate both mixtures at room temperature for 10 min, and then mix them in a single sterile microcentrifuge tube and further incubate the mixture at room temperature for 30 min to form the transcript-lipid complex.</li>
	<li>After 16 h of cell incubation, remove the culture medium from the culture dish, wash the cells 2x with prewarmed 1x PBS, and add 1.5 mL of minimal essential medium. Slowly add the transcript-lipid complex to the dish and gently swirl with hands to ensure uniform distribution of the complexes.</li>
	<li>Incubate the cells in a 5% CO2 incubator at 37 &#176;C for 10 h. Then, remove the medium, wash the transfected cells 2x with 1 mL of prewarmed 1x PBS, and add 2 mL of complete DMEM.</li>
</ol>
5. Virus production
<ol>
	<li>Split the transfected Huh7.5 cells every 2 days or 3 days when they reach 90% confluency. Collect virus supernatants at every split and store them at &#8722;80 &#176;C for further analysis.</li>
	<li>At every split, monitor the spread of the virus using a focus forming assay (FFA) as described in step 6.2. Harvest the virus until the virus spread reaches &#62;80% of transfected cells (Figure 1D). 
	NOTE: Split the transfected cells at 1:1 ratio for the first few passages to rescue the maximum number of viral variants. Virus spread slows and viability decreases with increasing proportions of mutations in the full-genome MLs2.</li>
</ol>
6. Quantification of virus titers
<ol>
	<li>Quantification of viral RNA
	<ol>
		<li>Isolate viral RNA from 140 &#181;L of culture supernatants using a viral RNA isolation kit according to the manufacturer&#39;s instructions.</li>
		<li>Set up a 10 &#181;L qRT-PCR reaction using a commercial qRT-PCR kit according to the manufacturer&#39;s instructions. Use the forward primer R6-130-S17, reverse primer R6-290-R19 (final concentration 0.2 &#181;M each), and probe R9-148-S21FT (final concentration 0.3 &#181;M) for HCV RNA quantification (Table 3). Set the run conditions as 48 &#176;C for 20 min, 95 &#176;C for 10 min, and then 45 cycles of 95 &#176;C for 15 s and 60 &#176;C for 1 min. 
		NOTE: The probe should contain the fluorescent reporter dye 6-carboxyfluorescein (FAM) and the quencher dye 6-carboxy-tetramethyl-rhodamine (TAMRA) at the 5&#39; and 3&#39; ends, respectively.</li>
		<li>Run reactions in a real-time PCR machine. Setup negative controls, i.e., RNA extracted from the supernatants of mock-infected cells and nuclease-free water simultaneously to ensure the absence of cross-contamination.</li>
		<li>In parallel, generate a standard curve using a 10-fold serially diluted known copy number of HCV transcripts (1 x 108 to 0) for the quantification of viral RNA. Perform the quantification in triplicate (Figure 1D).</li>
	</ol>
	</li>
	<li>Infectious virus titer quantification using 50% tissue culture infectious dose (TCID50)
	<ol>
		<li>Approximately 16 h prior to adding the virus, plate 6.5 x 103 Huh7.5 cells/well in a 96-well plate and incubate the cells at 37 &#176;C in a 5% CO2 incubator.</li>
		<li>In a biosafety class II cabinet, perform 10-fold serial dilutions (1 mL each) covering 1 x 10&#8722;1 to 1 x 10&#8722;6 dilutions (eight dilutions) of the harvested virus (obtained when the virus spread reaches &#62;80% of cells as described in step 5.). Add 100 &#181;L/well (with eight replicates) of the diluted virus to infect the Huh7.5 cells and keep the plate in an incubator at 37 &#176;C with 5% CO2&#160;for 3 days.</li>
		<li>After 3 days, wash the infected cells 3x with 0.1 mL of PBS each time, and fix and permeabilize the cells with 0.1 mL of ice-cold methanol at &#8722;20 &#176;C for 20 min. Wash the wells with 1x PBS 3x and then 1x with PBS-T (1x PBS/0.1% [v/v] Tween-20).</li>
		<li>After removing the PBS-T, block the cells for 30 min at room temperature with 0.1 mL of 1% bovine serum albumin (BSA) containing 0.2% skimmed milk in PBST. Remove the blocking solution and treat the cells for 5 min with 0.1 mL of 3% H2O2 prepared in 1x PBS.</li>
		<li>Again, wash the cells 2x with 1x PBS and 1x with PBS-T, then add 50 &#181;L/well of anti-NS5A 9E10 mAb (1:10000, stock 1 mg/mL; a kind gift from Charles Rice, The Rockefeller University), and incubate at room temperature for 1 h. Wash the wells again 3x with 1x PBS and 1x with PBS-T.</li>
		<li>Add 50 &#181;L/well of HRP-conjugated goat anti-mouse secondary IgG (1:4000), incubate for 30 min at room temperature, and then remove the unbound antibody by washing the wells with 0.1 mL of 1x PBS.</li>
		<li>Add 30 &#181;L of DAB (diaminobenzidine tetrahydrochloride) color substrate and incubate the plate with gentle rocking for 10 min at room temperature. The substrate develops a brown precipitate on the well surface that indicates HCV NS5A antigen.Remove the DAB solution and wash the wells 2x with 1x PBS and 1x with distilled water. Add 100 &#181;L of PBS containing 0.03% sodium azide.</li>
		<li>Examine each well under an inverted light microscope using a 10x objective. Count the number of positive wells. If the well contains one or more NS5A-positive cells, then it is positive; if the well shows an absence of NS5A-positive cells, then it is negative. Use a Reed and Muench calculator to estimate the endpoint dilution that infects 50% of the wells (TCID50)14,15. A reciprocal of the dilution required to yield the TCID50 is the virus infectivity titer (foci forming units) per unit volume. 
		&#8203;NOTE: Virus infectivity titers vary for different mutant libraries.</li>
	</ol>
	</li>
</ol>
7. Drug-resistant viral variant selection
<ol>
	<li>Dissolve pibrentasvir (PIB), an NS5A inhibitor, in 100% DMSO to a concentration of 1 mM and further dilute it in complete DMEM to a concentration of 10 nM.</li>
	<li>To determine the 50% effective concentration of PIB, seed Huh7.5 cells at a density of 0.6 x 106/well in a 6-well plate and incubate the cells at 37 &#176;C in a 5% CO2 incubator. After 16 h of cell incubation, add a virus dose of either ML50 or clonal JFH1 virus to infect 50% of cells, and then, at 12 h post-infection (h p.i.), treat the cells with a two-fold dilution series of PIB ranging from 0.5-100 pM. Measure the FFA and quantify FFU as in step 6.2. after 3 days of incubation.</li>
	<li>Plot the dose-response curves using FFU-reduction assay values in statistical analysis software. From the sigmoidal curve, obtain the 50% effective concentration (EC50; Figure 5). 
	NOTE: The effective concentration range may vary depending on the class, between HCV antivirals of the same class, and depending on the mutant library.</li>
	<li>For the experiment, infect na&#239;ve Huh7.5 cells at 70% confluence with an ML50 virus (variants derived from ML50 RNA) dose to infect 50% of the cells for 12 h, and then transfer the infected cells onto 6-well plates 24 h post-infection.</li>
	<li>Add 1x EC50 PIB (47.3 pM) to the infected cells after 16 h of cell split. Do this after every cell split for six consecutive passages (18 days), followed by three drug-free passage cycles, and monitor virus spread using FFAs as described in step 6.2. Scale-up FFA reagents as per the growth area. Harvest viral supernatants at each passage and store them at &#8722;80 &#176;C until use. 
	NOTE: Viral spread to greater than 50% of cells during treatment follow-up may be defined as a breakthrough, and viral spread during the drug withdrawal period may be defined as viral relapse16.</li>
	<li>Extract viral RNA from the supernatant on day 18, as described in step 6.1.1., and then synthesize cDNA, as shown in step 3.1. Amplify the NS5A gene using 5 &#181;L of 1:5 diluted cDNA and the PCR components described in step 3.2. Then, follow steps 3.3.-3.5. to determine NS5A drug resistance mutations in 6-8 positive bacterial plasmids using the NS5A sequencing primers 6186F, 6862F, and 6460R (Table 3 and Table 4). 
	NOTE: Here in this study, we reported substitutions in NS5A of variants selected during PIB treatment at 1x EC50. This will rule out the contribution of substitutions other than NS5A in reduced susceptibility to PIB.</li>
</ol>

<ol>
	<li>Desselberger, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+genetics+of+rotavirus.">Reverse genetics of rotavirus.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (9), 2106-2108 (2017).</li><li>Singh, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+mutagenesis+of+hepatitis+C+virus+reveals+ability+of+genome+to+overcome+detrimental+mutations.">Genome-wide mutagenesis of hepatitis C virus reveals ability of genome to overcome detrimental mutations.</a> Journal of Virology. 94 (3), 01327 (2020).</li><li>Agarwal, S., Baccam, P., Aggarwal, R., Veerapu, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+synthesis+and+phenotypic+analysis+of+mutant+clouds+for+hepatitis+E+virus+genotype+1.">Novel synthesis and phenotypic analysis of mutant clouds for hepatitis E virus genotype 1.</a> Journal of Virology. 92 (4), 01932 (2018).</li><li>Edmonds, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+bacterium-free+method+for+generation+of+flavivirus+infectious+DNA+by+circular+polymerase+extension+reaction+allows+accurate+recapitulation+of+viral+heterogeneity.">A novel bacterium-free method for generation of flavivirus infectious DNA by circular polymerase extension reaction allows accurate recapitulation of viral heterogeneity.</a> Journal of Virology. 87 (4), 2367-2372 (2013).</li><li>Arumugaswami, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+functional+profiling+of+hepatitis+C+virus+genome.">High-resolution functional profiling of hepatitis C virus genome.</a> PLoS Pathogens. 4 (10), 1000182 (2008).</li><li>Heaton, N. S., Sachs, D., Chen, C. -. J., Hai, R., Palese, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+mutagenesis+of+influenza+virus+reveals+unique+plasticity+of+the+hemagglutinin+and+NS1+proteins.">Genome-wide mutagenesis of influenza virus reveals unique plasticity of the hemagglutinin and NS1 proteins.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (50), 20248-20253 (2013).</li><li>Eyre, N. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+mutagenesis+of+dengue+virus+reveals+plasticity+of+the+NS1+protein+and+enables+generation+of+infectious+tagged+reporter+viruses.">Genome-wide mutagenesis of dengue virus reveals plasticity of the NS1 protein and enables generation of infectious tagged reporter viruses.</a> Journal of Virology. 91 (23), 01455 (2017).</li><li>Cobb, R. E., Chao, R., Zhao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+evolution:+Past,+present,+and+future.">Directed evolution: Past, present, and future.</a> AIChE Journal. American Institute of Chemical Engineers. 59 (5), 1432-1440 (2013).</li><li>Sen, S., Venkata Dasu, V., Mandal, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developments+in+directed+evolution+for+improving+enzyme+functions.">Developments in directed evolution for improving enzyme functions.</a> Applied Biochemistry and Biotechnology. 143 (3), 212-223 (2007).</li><li>Yuan, L., Kurek, I., English, J., Keenan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory-directed+protein+evolution.">Laboratory-directed protein evolution.</a> Microbiology and Molecular Biology Reviews. 69 (3), 373-392 (2005).</li><li>Green, M. R., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+DNA+by+agarose+gel+electrophoresis.">Analysis of DNA by agarose gel electrophoresis.</a> Cold Spring Harbor Protocols. 2019 (1), (2019).</li><li>Desjardins, P., Conklin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NanoDrop+microvolume+quantitation+of+nucleic+acids.">NanoDrop microvolume quantitation of nucleic acids.</a> Journal of Visualized Experiments. (45), e2565 (2010).</li><li>Rio, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Denaturation+and+electrophoresis+of+RNA+with+formaldehyde.">Denaturation and electrophoresis of RNA with formaldehyde.</a> Cold Spring Harbor Protocols. 2015 (2), 219-222 (2015).</li><li>Lindenbach, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+HCV+infectivity+produced+in+cell+culture+and+in+vivo.">Measuring HCV infectivity produced in cell culture and in vivo.</a> Methods in Molecular Biology. 510, 329-336 (2009).</li><li>Lei, C., Yang, J., Hu, J., Sun, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+calculation+of+TCID50+for+quantitation+of+virus+infectivity.">On the calculation of TCID50 for quantitation of virus infectivity.</a> Virologica Sinica. 36 (1), 141-144 (2021).</li><li>Soni, S., Singh, D., Aggarwal, R., Veerapu, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+fitness+of+hepatitis+C+virus+increases+resistance+to+direct-acting+antivirals.">Enhanced fitness of hepatitis C virus increases resistance to direct-acting antivirals.</a> The Journal of General Virology. 103 (2), (2022).</li><li>Khan, S., Soni, S., Veerapu, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HCV+replicon+systems:+Workhorses+of+drug+discovery+and+resistance.">HCV replicon systems: Workhorses of drug discovery and resistance.</a> Frontiers in Cellular Infection Microbiology. 10, 325 (2020).</li><li>Cadwell, R. C., Joyce, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Randomization+of+genes+by+PCR+mutagenesis.">Randomization of genes by PCR mutagenesis.</a> PCR Methods and Applications. 2 (1), 28-33 (1992).</li></ol>

The authors have nothing to disclose.

In this study, we have detailed a simple and rapid FL-MRS procedure that integrates ep-PCR18 and reverse genetics for synthesizing HCV full-genome libraries, which can then be used in a cell culture system to generate replication-competent variants for the screening of drug-resistant phenotypes. The use of low-fidelity Taq DNA polymerase is a prerequisite of ep-PCR that allows the incorporation of substitutions during PCR amplification of a full-length viral genome. We tested several low-fidelity ...

Forward genetic screening begins with a viral phenotype of interest and then, through sequencing its genome and comparing it to that of the original strain, attempts to identify the mutation(s) causing that phenotype. In contrast, in reverse genetic screens, random mutations are introduced in a target gene, followed by an examination of the resultant phenotype(s)1. For the reverse genetics approach, in vitro mutagenesis is the most widely used technique to create a pool of variants that are subsequently screened for phenotypes of interest. Various genetic tools have been reported for achieving genome-wide random mutagenesis of RNA viruses, including error-prone PCR (ep-PCR)2,3, circular polymerase extension4, and Mu-transposon insertion mutagenesis 5,6,7. The latter two methods yield libraries harboring limited sequence diversity and are prone to the introduction of large insertions and deletions, which are highly lethal for viruses and severely limit the recovery of infectious viral variants.
ep-PCR is a well-known powerful mutagenesis technique widely used in protein engineering to generate mutant enzymes for the selection of phenotypes with desired properties, such as enhanced thermal stability, substrate specificity, and catalytic activity8,9,10. This technique is easy to perform because it requires simple equipment, does not involve tedious manipulations, uses commercially available reagents, and is quick; moreover, it generates high-quality libraries.
Here, we developed a novel method for full-length mutant RNA synthesis (FL-MRS) to generate complete genomes of hepatitis C virus (HCV) by integrating ep-PCR, which induces random genome-wide substitution mutagenesis and reverse genetics. Even a single nucleotide insertion or deletion is highly deleterious for positive-sense RNA viruses ([+]ssRNA); hence, PCR-based substitution mutagenesis is the preferred method for the iterative generation of large, diverse libraries of full (+)ss RNA virus genomes with good viability.
FL-MRS is a straightforward approach that can be applied to any positive-sense RNA virus with a ~10 kb genome length through the meticulous design of a primer set that binds to the viral cDNA clone. pJFH1 is an infectious cDNA clone that encodes the HCV genotype 2a and can recapitulate all steps of the virus life cycle. By using the FL-MRS approach, we demonstrated the synthesis of randomly mutagenized full-genome libraries (mutant libraries [MLs]) to produce replication-competent JFH1 variants for which there was no prior knowledge of the properties associated with mutations. Upon exposure to an antiviral, some of the viral variants quickly overcame the drug pressure with the desired phenotypic change. Using the protocol described here, a plethora of viral variants can be generated, creating opportunities to study the evolution of (+)ssRNA viruses.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 kb plus DNA ladder&#160;</td>
			<td>Thermo Fisher</td>
			<td>10787018</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml centrifuge tube</td>
			<td>Tarsons</td>
			<td>500010</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml centrifuge tube</td>
			<td>Tarsons</td>
			<td>546021</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm cell culture dish&#160;</td>
			<td>Tarsons</td>
			<td>960010</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml centrifuge tube</td>
			<td>Tarsons</td>
			<td>546041</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Merck</td>
			<td>A6283</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose&#160;</td>
			<td>HiMedia</td>
			<td>MB080</td>
			<td></td>
		</tr>
		<tr>
			<td>Agrose gel electrophoresis unit</td>
			<td>BioRad</td>
			<td>1704406</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety Cabinet, ClassII</td>
			<td>ESCO</td>
			<td>AC2 4S</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>HiMedia</td>
			<td>MB083</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge&#160;</td>
			<td>Eppendorf</td>
			<td>5424-R</td>
			<td></td>
		</tr>
		<tr>
			<td>CFX Connect Real-Time PCR Detection System</td>
			<td>BioRad</td>
			<td>1855201</td>
			<td></td>
		</tr>
		<tr>
			<td>Cloning plates 90 mm&#160;</td>
			<td>Tarson</td>
			<td>460091</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator&#160;</td>
			<td>New Brunswick</td>
			<td>Galaxy 170R</td>
			<td></td>
		</tr>
		<tr>
			<td>Colibri Microvolume Spectrometer</td>
			<td>Titertek-Berthold</td>
			<td>11050140</td>
			<td></td>
		</tr>
		<tr>
			<td>DAB&#160;Substrate&#160;Kit&#160;</td>
			<td>Abcam</td>
			<td>ab94665</td>
			<td></td>
		</tr>
		<tr>
			<td>dATP Solution</td>
			<td>NEB</td>
			<td>N0440S</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxynucleotide (dNTP) Solution Set</td>
			<td>NEB</td>
			<td>N0446</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethyl Pyrocarbonate (DEPC)&#160;</td>
			<td>SRL chemical</td>
			<td>46791</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulphoxide (DMSO)</td>
			<td>HiMedia</td>
			<td>MB058</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM high glucose</td>
			<td>Lonza</td>
			<td>BE12-604F</td>
			<td></td>
		</tr>
		<tr>
			<td>EcoR1-HF</td>
			<td>NEB</td>
			<td>R3101</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA tetrasodium salt dihydrate</td>
			<td>HiMedia</td>
			<td>GRM4918</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium Bromide</td>
			<td>Amresco</td>
			<td>X328</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum&#160;</td>
			<td>Gibco</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde&#160;</td>
			<td>Fishser Scientific</td>
			<td>12755</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Documentation System</td>
			<td>ALPHA IMAGER</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP</td>
			<td>Thermo Fisher</td>
			<td>A16066</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide 30%</td>
			<td>Merck</td>
			<td>107209</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nickon</td>
			<td>&#160;ECLIPSE Ts2</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth&#160;</td>
			<td>HiMedia</td>
			<td>M1245</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000&#160;</td>
			<td>Thermo Fisher</td>
			<td>116680270</td>
			<td>transfection reagent</td>
		</tr>
		<tr>
			<td>Mechanical Pipette Set</td>
			<td>Eppendorf&#160;&#160;</td>
			<td>3120000909</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Merck</td>
			<td>106009</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Tips 0.2-10 &#181;l&#160;</td>
			<td>Tarsons</td>
			<td>521000</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Tips 10 - 100 &#181;l&#160;</td>
			<td>Tarsons</td>
			<td>521010</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Tips 200-1000 &#181;l&#160;</td>
			<td>Tarsons</td>
			<td>521020</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS buffer&#160;</td>
			<td>GeNei</td>
			<td>3601805001730</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonessential aminoacids (NEAA)</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>One Shot TOP10 Chemically Competent&#160;E. coli</td>
			<td>Invitrogen</td>
			<td>C404010</td>
			<td>E.coli DH5&#945;</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Gibco</td>
			<td>1105-021</td>
			<td>minimal essential medium</td>
		</tr>
		<tr>
			<td>PCR tubes 0.2 ml</td>
			<td>Tarsons</td>
			<td>510051</td>
			<td></td>
		</tr>
		<tr>
			<td>Pencillin/streptomycin&#160;</td>
			<td>Gibco</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>pGEM-T Easy Vector System</td>
			<td>Promega</td>
			<td>A1360</td>
			<td>T-vector DNA</td>
		</tr>
		<tr>
			<td>Phosphate buffer saline (PBS)</td>
			<td>HiMedia</td>
			<td>TI1099</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion&#160;High-Fidelity DNA Polymerase</td>
			<td>NEB</td>
			<td>M0530S</td>
			<td></td>
		</tr>
		<tr>
			<td>Pibrentasvir</td>
			<td>Cayman Chemical</td>
			<td>27546</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette controller&#160;</td>
			<td>Gilson&#160;</td>
			<td>F110120</td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum Taq DNA Polymerase&#160;</td>
			<td>Thermo</td>
			<td>10966034</td>
			<td></td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>GraphPad</td>
			<td></td>
			<td>statistical analysis software</td>
		</tr>
		<tr>
			<td>QIAamp Viral RNA Mini kit&#160;</td>
			<td>Qiagen</td>
			<td>52904</td>
			<td>viral isolation kit</td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit</td>
			<td>QIAGEN</td>
			<td>28104</td>
			<td>cokum purification kit</td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit&#160;</td>
			<td>QIAGEN</td>
			<td>74104</td>
			<td>RNA cleanup kit</td>
		</tr>
		<tr>
			<td>Serological Pipettes 25 ml</td>
			<td>Thermo Fisher</td>
			<td>170357N</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipettes 5 ml</td>
			<td>Thermo Fisher</td>
			<td>170355N</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipettes10 ml</td>
			<td>Thermo Fisher</td>
			<td>170356N</td>
			<td></td>
		</tr>
		<tr>
			<td>Single strand RNA Marker 0.2-10 kb</td>
			<td>Merck</td>
			<td>R7020</td>
			<td></td>
		</tr>
		<tr>
			<td>Skim milk&#160;</td>
			<td>HiMedia</td>
			<td>M530</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide 0.1 M solution</td>
			<td>Merck</td>
			<td>8591</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript III Reverse Transcriptase&#160;</td>
			<td>Invitrogen</td>
			<td>18080044</td>
			<td>reverse transcriptase</td>
		</tr>
		<tr>
			<td>T100 Thermal Cycler</td>
			<td>BioRad</td>
			<td>1861096</td>
			<td></td>
		</tr>
		<tr>
			<td>T175 cell culture flask&#160;</td>
			<td>Tarsons</td>
			<td>159910</td>
			<td></td>
		</tr>
		<tr>
			<td>T25 cell culture flask&#160;</td>
			<td>Tarsons</td>
			<td>950040</td>
			<td></td>
		</tr>
		<tr>
			<td>T7 RiboMax Express Large Scale RNA Production System&#160;</td>
			<td>Promega</td>
			<td>P1320</td>
			<td>&#160;Large Scale RNA Production System&#160;</td>
		</tr>
		<tr>
			<td>T75 cell culture flask&#160;</td>
			<td>Tarsons</td>
			<td>950050</td>
			<td></td>
		</tr>
		<tr>
			<td>Taq DNA Polymerase</td>
			<td>Genetix Biotech (Puregene)</td>
			<td>PGM040</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan RNA-to-CT 1-Step Kit</td>
			<td>Applied Biosystems</td>
			<td>4392653</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan RNA-to-CT&#160;1-Step&#160;Kit</td>
			<td>Thermo Fisher</td>
			<td>4392653</td>
			<td>commercial qRT-PCR kit&#160;</td>
		</tr>
		<tr>
			<td>TOPO-XL--2 Complete PCR Cloning Kit</td>
			<td>Thermo Fisher</td>
			<td>K8050-10</td>
			<td>kit for cloning of long-PCR product&#160;</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>HiMedia</td>
			<td>TC072</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>HiMedia</td>
			<td>TCL007</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>HiMedia</td>
			<td>MB067</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum Concentrator&#160;</td>
			<td>Eppendorf, Concentrator Plus</td>
			<td>100248</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath&#160;</td>
			<td>GRANT</td>
			<td>JBN-18</td>
			<td></td>
		</tr>
		<tr>
			<td>Xba1</td>
			<td>NEB</td>
			<td>R0145S</td>
			<td></td>
		</tr>
	</tbody>
</table>

reverse genetics to engineer positive-sense rna virus variants

The lack of a convenient method for the iterative generation of diverse full-length viral variants has impeded the study of directed evolution in RNA viruses. By integrating a full RNA genome error-prone PCR and reverse genetics, random genome-wide substitution mutagenesis can be induced. We have developed a method using this technique to synthesize diverse libraries to identify viral mutants with phenotypes of interest. This method, called full-length mutant RNA synthesis (FL-MRS), offers the following advantages: (i) the ability to create a large library via a highly efficient one-step error-prone PCR; (ii) the ability to create groups of libraries with varying levels of genetic diversity by manipulating the fidelity of DNA polymerase; (iii) the creation of a full-length PCR product that can directly serve as a template for mutant RNA synthesis; and (iv) the ability to create RNA that can be delivered into host cells as a non-selected input pool to screen for viral mutants of the desired phenotype. We have found, using a reverse genetics approach, that FL-MRS is a reliable tool to study viral-directed evolution at all stages in the life cycle of the hepatitis C virus, JFH1 isolate. This technique appears to be an invaluable tool to employ directed evolution to understand adaptation, replication, and the role of viral genes in pathogenesis and antiviral resistance in positive-sense RNA viruses.

A plethora of full-length HCV variants can be generated and screened for drug-resistant phenotypes of interest following the procedures described in Figure 1. Full genome mutant libraries were synthesized using clonal-pJFH1 in decreasing amounts (100-10 ng), as shown in Figure 2. Average yields of ep-PCR products (mutant libraries) ranged from 3.8-12.5 ng/&#181;L. Figure 3 shows the viral transcripts synthesized from the clonal-pJFH...

Watch this Scientific Journal Video about Reverse Genetics to Engineer Positive-Sense RNA Virus Variants at JoVE.com

Reverse Genetics to Engineer Positive-Sense RNA Virus Variants

The protocol describes a method for introducing controllable genetic diversity in the hepatitis C virus genome by combining full-length mutant RNA synthesis using error-prone PCR and reverse genetics. The method provides a model for phenotype selection and can be used for 10 kb long positive-sense RNA virus genomes.

The lack of a convenient method for the iterative generation of diverse full-length viral variants has impeded the study of directed evolution in RNA ...

reverse-genetics-to-engineer-positive-sense-rna-virus-variants

Research

JoVE Journal

Genetics

1.2K Views.  Shiv Nadar University. The protocol describes a method for introducing controllable genetic diversity in the hepatitis C virus genome by combining full-length mutant RNA synthesis using error-prone PCR and reverse genetics. The method provides a model for phenotype selection and can be used for 10 kb long positive-sense RNA virus genomes.

Video: Reverse Genetics to Engineer Positive-Sense RNA Virus Variants

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA interactions such as microRNA-mRNA interactions have been shown to regulate gene expression, the full extent to which RNA interactions occur in the cell is still unknown. Previous methods to study RNA interactions have primarily focused on subsets of RNAs that are interacting with a particular protein or RNA species. Here, we detail a method named Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH) that allows genome-wide capture of RNA interactions in vivo in an unbiased manner. SPLASH utilizes in vivo crosslinking, proximity ligation, and high throughput sequencing to identify intramolecular and intermolecular RNA base-pairing partners globally. SPLASH can be applied to different organisms including bacteria, yeast and human cells, as well as diverse cellular conditions to facilitate the understanding of the dynamics of RNA organization under diverse cellular contexts. The entire experimental SPLASH protocol takes about 5 days to complete and the computational workflow takes about 7 days to complete.

Here, we detail the method of Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH), which enables genome-wide mapping of intramolecular and intermolecular RNA-RNA interactions in vivo. SPLASH can be applied to study RNA interactomes of organisms including yeast, bacteria and humans.

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase activity. Although TERT is named as a reverse transcriptase, structural and phylogenetic analyses of TERT demonstrate that TERT is a member of right-handed polymerases, and relates to viral RNA-dependent RNA polymerases (RdRPs) as well as viral reverse transcriptase. We firstly identified RdRP activity of human TERT that generates complementary RNA stand to a template non-coding RNA and contributes to RNA silencing in cancer cells. To analyze this non-canonical enzymatic activity, we developed RdRP assay with recombinant TERT in 2009, thereafter established in vitro RdRP assay for endogenous TERT. In this manuscript, we describe the latter method. Briefly, TERT immune complexes are isolated from cells, and incubated with template RNA and rNTPs including radioactive rNTP for RdRP reaction. To eliminate single-stranded RNA, reaction products are treated with RNase I, and the final products are analyzed with polyacrylamide gel electrophoresis. Radiolabeled RdRP products can be detected by autoradiography after overnight exposure.

Human telomerase reverse transcriptase (TERT) synthesizes not only telomeric DNA but also double-stranded RNA through RNA-dependent RNA polymerase activity. Here, we describe a newly established assay to detect RNA-dependent RNA polymerase activity of endogenous TERT.

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase ...

RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding RNA&#39;s family. Although their biological functions remain largely unknown, the number of these lncRNAs has steadily increased and it is now estimated that humans may have more than 10,000 such transcripts. Some of these are known to be involved in important regulatory pathways of gene expression which take place at the transcriptional level, but also at different steps of RNA co- and post-transcriptional maturation. In the latter cases, RNAs that are targeted by the lncRNA have to be identified. That&#39;s the reason why it is useful to develop a method enabling the identification of RNAs associated directly or indirectly with a lncRNA of interest.
This protocol, which was inspired by previously published protocols allowing the isolation of a lncRNA together with its associated chromatin sequences, was adapted to permit the isolation of associated RNAs. We determined that two steps are critical for the efficiency of this protocol. The first is the design of specific anti-sense DNA oligonucleotide probes able to hybridize to the lncRNA of interest. To this end, the lncRNA secondary structure was predicted by bioinformatics and anti-sense oligonucleotide probes were designed with a strong affinity for regions that display a low probability of internal base pairing. The second crucial step of the procedure relies on the fixative conditions of the tissue or cultured cells that have to preserve the network between all molecular partners. Coupled with high throughput RNA sequencing, this RNA pull-down protocol can provide the whole RNA interactome of a lncRNA of interest.

This RNA pull-down method allows identifying the RNA targets of a long non-coding RNA (lncRNA). Based on the hybridization of home-made, designed anti-sense DNA oligonucleotide probes specific to this lncRNA in an appropriately fixed tissue or cell line, it efficiently allows the capture of all RNA targets of the lncRNA.

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding ...

CRISPR Guide RNA Cloning for Mammalian Systems

The outlined protocol describes streamlined methods for the efficient and cost-effective generation of Cas9-associated guide RNAs. Two alternative strategies for guide RNA (gRNA) cloning are outlined based on the usage of the Type IIS restriction enzyme BsmBI in combination with a set of compatible vectors. Outside of the access to Sanger sequencing services to validate the generated vectors, no special equipment or reagents are required aside from those that are standard to modern molecular biology laboratories. The outlined method is primarily intended for cloning one single gRNA or one paired gRNA-expressing vector at a time. This procedure does not scale well for the generation of libraries containing thousands of gRNAs. For those purposes, alternative sources of oligonucleotide synthesis such as oligo-chip synthesis are recommended. Finally, while this protocol focuses on a set of mammalian vectors, the general strategy is plastic and is applicable to any organism if the appropriate gRNA vector is available.

Here, a simple, efficient, and cost-effective method of sgRNA cloning is outlined.

The outlined protocol describes streamlined methods for the efficient and cost-effective generation of Cas9-associated guide RNAs. Two alternative ...

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near full-length (intact and defective), HIV-1 proviruses. FLIPS allows determination of the genetic composition of integrated HIV-1 within a cell population. Through identifying defects within HIV-1 proviral sequences that arise during reverse transcription, such as large internal deletions, deleterious stop codons/hypermutation, frameshift mutations, and mutations/deletions in cis acting elements required for virion maturation, FLIPS can identify integrated proviruses incapable of replication. The FLIPS assay can be utilized to identify HIV-1 proviruses that lack these defects and are&#160;therefore potentially replication-competent. The FLIPS protocol involves: lysis of HIV-1 infected cells, nested PCR of near full-length HIV-1 proviruses (using primers targeted to the HIV-1 5&#39; and 3&#39; LTR), DNA purification and quantification, library preparation for Next-generation Sequencing (NGS), NGS, de novo assembly of proviral contigs, and a simple process of elimination for identifying replication-competent proviruses. FLIPS provides advantages over traditional methods designed to sequence integrated HIV-1 proviruses, such as single-proviral sequencing. FLIPS amplifies and sequences near full-length proviruses enabling replication competency to be determined, and also uses fewer amplification primers, preventing the consequences of primer mismatches. FLIPS is a useful tool for understanding the genetic landscape of integrated HIV-1 proviruses, especially within the latent reservoir, however, its utilization can extend to any application in which the genetic composition of integrated HIV-1 is required.

Full-length individual proviral sequencing (FLIPS) provides an efficient and high-throughput method for the amplification and sequencing of single, near full-length (intact and defective) HIV-1 proviruses and allows for determination of their potential replication-competency. FLIPS overcomes limitations of previous assays designed to sequence the latent HIV-1 reservoir.

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near ...

Reverse Transcription-Loop-mediated Isothermal Amplification (RT-LAMP) Assay for Zika Virus and Housekeeping Genes in Urine, Serum, and Mosquito Samples

Infection with Zika virus (ZIKV) can be asymptomatic in adults, however, infection during pregnancy can lead to miscarriage and severe neurological birth defects. The goal of this protocol is to quickly detect ZIKV in both human and mosquito samples. The current gold standard for ZIKV detection is quantitative reverse transcription PCR (qRT-PCR); reverse transcription loop-mediated isothermal amplification (RT-LAMP) may allow for a more efficient and low-cost testing without the need for expensive equipment. In this study, RT-LAMP is used for ZIKV detection in various biological samples within 30 min, without first isolating the RNA from the sample. This technique is demonstrated using ZIKV infected patient urine and serum, and infected mosquito samples. 18S ribosomal ribonucleic acid and actin are used as controls in human and mosquito samples, respectively.

Reverse Transcription-Loop-mediated Isothermal Amplification RT-LAMP Assay for Zika Virus and Housekeeping Genes in Urine, Serum, and Mosquito Samples

This protocol provides an efficient and low-cost method to detect Zika virus or control targets in human urine and serum samples or in mosquitoes by reverse transcription loop-mediated isothermal amplification (RT-LAMP). This method does not require RNA isolation and can be done within 30 min.

Infection with Zika virus (ZIKV) can be asymptomatic in adults, however, infection during pregnancy can lead to miscarriage and severe neurological birth ...

Evaluation of a Universal Nested Reverse Transcription Polymerase Chain Reaction for the Detection of Lyssaviruses

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription polymerase chain reaction (nested RT-PCR) was developed to detect the conserved region of the nucleoprotein (N) gene of lyssaviruses. The method applies reverse transcription (RT) using viral RNA as template and oligo (dT)15 and random hexamers as primers to synthesize the viral complementary DNA (cDNA). Then, the viral cDNA is used as a template to amplify an 845 bp N gene fragment in first-round PCR using outer primers, followed by second-round nested PCR to amplify the final 371 bp fragment using inner primers. This method can detect different genetic clades of rabies viruses (RABV). The validation, using 9,624 brain specimens from eight domestic animal species in 10 years of clinical rabies diagnoses and surveillance in China, showed that the method has 100% sensitivity and 99.97% specificity in comparison with the direct fluorescent antibody test (FAT), the gold standard method recommended by the World Health Organization (WHO) and the World Organization for Animal Health (OIE). In addition, the method could also specifically amplify the targeted N gene fragment of 15 other approved and two novel lyssavirus species in the 10th Report of the International Committee on Taxonomy of Viruses (ICTV) as evaluated by a mimic detection of synthesized N gene plasmids of all lyssaviruses. The method provides a convenient alternative to FAT for rabies diagnosis and has been approved as a National Standard (GB/T36789-2018) of China.

A pan-lyssavirus nested reverse transcription polymerase chain reaction has been developed to detect specifically all known lyssaviruses. Validation using rabies brain samples of different animal species showed that this method has a sensitivity and specificity equivalent to the gold standard fluorescent antibody test and could be used for routine rabies diagnosis.

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription ...

Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA polymerase II and co-transcriptional capping and splicing of the precursor RNA to form the mature mRNA. During mRNA synthesis, the RNA polymerase II elongation complex is a target for regulation by a large collection of transcription factors that control its catalytic activity, as well as the capping, splicing, and 3&#8217;-processing enzymes that create the mature mRNA. Because of the inherent complexity of mRNA synthesis, simpler experimental systems enabling isolation and investigation of its various co-transcriptional stages have great utility.
In this article, we describe one such simple experimental system suitable for investigating co-transcriptional RNA capping. This system relies on defined RNA polymerase II elongation complexes assembled from purified polymerase and artificial transcription bubbles. When immobilized via biotinylated DNA, these RNA polymerase II elongation complexes provide an easily manipulable tool for dissecting co-transcriptional RNA capping and mechanisms by which the elongation complex recruits and regulates capping enzyme during co-transcriptional RNA capping. We anticipate this system could be adapted for studying recruitment and/or assembly of proteins or protein complexes with roles in other stages of mRNA maturation coupled to the RNA polymerase II elongation complex.

Here, we describe the assembly of RNA polymerase II (Pol II) elongation complexes requiring only short synthetic DNA and RNA oligonucleotides and purified Pol II. These complexes are useful for studying mechanisms underlying co-transcriptional processing of transcripts associated with the Pol II elongation complex.

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA ...

Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational steps are used to regulate specific genes. Sequence-specific nucleic acid-binding proteins target specific sequences to control spatial or temporal gene expression. The binding sites in nucleic acids are typically characterized by mutational analysis. However, numerous proteins of interest have no known binding site for such characterization. Here we describe an approach to identify previously unknown binding sites for RNA-binding proteins. It involves iterative selection and amplification of sequences starting with a randomized sequence pool. Following several rounds of these steps-transcription, binding, and amplification-the enriched sequences are sequenced to identify a preferred binding site(s). Success of this approach is monitored using in vitro binding assays. Subsequently, in vitro and in vivo functional assays can be used to assess the biological relevance of the selected sequences. This approach allows identification and characterization of a previously unknown binding site(s) for any RNA-binding protein for which an assay to separate protein-bound and unbound RNAs exists.

Sequence specificity is critical for gene regulation. Regulatory proteins that recognize specific sequences are important for gene regulation. Defining functional binding sites for such proteins is a challenging biological problem. An iterative approach for identification of a binding site for an RNA-binding protein is described here and is applicable to all RNA-binding proteins.

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational ...

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human genetics research. Although a number of in silico algorithms have been developed to predict the pathogenicity of variants identified in these datasets, functional studies are critical to determining how specific genomic variants affect protein function, especially for missense variants. In the Undiagnosed Diseases Network (UDN) and other rare disease research consortia, model organisms (MO) including Drosophila, C. elegans, zebrafish, and mice are actively used to assess the function of putative human disease-causing variants. This protocol describes a method for the functional assessment of rare human variants used in the Model Organisms Screening Center Drosophila Core of the UDN. The workflow begins with gathering human and MO information from multiple public databases, using the MARRVEL web resource to assess whether the variant is likely to contribute to a patient&#39;s condition as well as design effective experiments based on available knowledge and resources. Next, genetic tools (e.g., T2A-GAL4 and UAS-human cDNA lines) are generated to assess the functions of variants of interest in Drosophila. Upon development of these reagents, two-pronged functional assays based on rescue and overexpression experiments can be performed to assess variant function. In the rescue branch, the endogenous fly genes are &#34;humanized&#34;&#160;by replacing the orthologous Drosophila gene with reference or variant human transgenes. In the overexpression branch, the reference and variant human proteins are exogenously driven in a variety of tissues. In both cases, any scorable phenotype (e.g., lethality, eye morphology, electrophysiology) can be used as a read-out, irrespective of the disease of interest. Differences observed between reference and variant alleles suggest a variant-specific effect, and thus likely pathogenicity. This protocol allows rapid, in vivo assessments of putative human disease-causing variants of genes with known and unknown functions.

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

The goal of this protocol is to outline the design and performance of in vivo experiments in Drosophila melanogaster to assess the functional consequences of rare gene variants associated with human diseases.

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human ...