To form the dCas9-Alexa549 roadblock complex, we initially prepared a tr-crRNA duplex by mixing universal 67-nucleotide oligomer tracerRNA (trRNA) and a custom-designed crRNA (table S1) in a duplex buffer (Integrated DNA Technologies) to a final concentration of 10 μM each. The mixture was incubated at 95°C for 5 min and then slowly cooled to 4°C by decreasing the temperature for 5°C every 5 min over the course of 1.5 hours. We next incubated the tr-crRNA solution with the dCas9 in the “binding buffer” [2 μM tr-crRNA complex, 1 μM dCas9-SNAP (New England Biolabs), and 1× NEB3.1 buffer] for 10 min at 37°C before placing it on ice. Following the tr-crRNA-dCas9 complex formation, we bound it to the DNAparS by incubating the DNAparS:tr-crRNA-dCas9 in molar ratio of 1:50 for 60 min at 37°C. We labeled the dCas9-SNAP by adding Alexa546-O6-benzylguanine (BG) (or Alexa Fluor 647-BG for photobleaching estimation) to a final concentration of 1 μM for 30 min at room temperature before flowing it into the flow cell.
Duplex buffer
Manufactured by Integrated DNA Technologies
Sourced in United States
Duplex buffer is a solution used to maintain the stability and integrity of double-stranded DNA molecules in various molecular biology applications. It is designed to provide optimal conditions for the formation and preservation of DNA duplexes.
Automatically generated - may contain errors
Lab products found in correlation
Powerup sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Alt r crispr cas9 tracrrna, by Integrated DNA Technologies (2 mentions)
Alt r crispr cas9 crrna, by Integrated DNA Technologies (2 mentions)
N hydroxysulfosuccinimide sulfo nhs, by Thermo Fisher Scientific (1 mentions)
1 ethyl 3 3 dimethylaminopropyl carbodiimide hydrochloride edc, by Thermo Fisher Scientific (1 mentions)
Xfect transfection reagent, by Takara Bio (1 mentions)
Bright glo luciferase assay system, by Promega (1 mentions)
Quantstudio 3, by Thermo Fisher Scientific (1 mentions)
Phelper, by Agilent Technologies (1 mentions)
Cas9 protein, by Integrated DNA Technologies (1 mentions)
Standard buffer, by New England Biolabs (1 mentions)
Onetaq 2x master mix, by New England Biolabs (1 mentions)
Dsirna 1, by Integrated DNA Technologies (1 mentions)
Dsirna 2, by Integrated DNA Technologies (1 mentions)
Megaruptor 2, by Diagenode (1 mentions)
Hifi cas9, by Integrated DNA Technologies (1 mentions)
Sqk lsk109 kit, by Oxford Nanopore (1 mentions)
Minion system, by Oxford Nanopore (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
Bluepippin system, by Sage Science (1 mentions)
13 protocols using duplex buffer
1
Docking dCas9-Alexa549 Roadblock Complex
2
DNA-Functionalized Magnetic Nanoparticles
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Both kinds of Fe3O4 MNPs with carboxyl acid on the surface were purchased from Ocean Nanotech, LLC. The one with poor magnetic performance was not well crystallized. 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS) were purchased from Thermo Fisher Scientific Inc. The capture DNA oligomers with amino modification in 5′end, the dehydrated DNA oligomers with biotin modification in 5′ end and the duplex buffer were purchased from Integrated DNA Technologies, Inc.
The sequence of ss-DNA modified on MNPs:
1. 5′-AmMC6/ACC TTC CTC CGC AAT ACT CCC CCA GGT-3′
The sequence of ss-DNA dehydrated
2. 5′-Biosg/ACC TGG GGG AGT ATT GCG GAG GAA GGT-3′
The sequence of ss-DNA modified on MNPs:
1. 5′-AmMC6/ACC TTC CTC CGC AAT ACT CCC CCA GGT-3′
The sequence of ss-DNA dehydrated
2. 5′-Biosg/ACC TGG GGG AGT ATT GCG GAG GAA GGT-3′
3
Validating miRNA Regulatory Mechanism
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Wild-type miRNA decoy sequences (Table 7 ) were derived from the binding sites of NFκB or HOXD10 within the miRNA promoters predicted by AlGGEN PROMO software (see below). The sequence-specific binding of the two TFs was tested using mutant decoys (Table 7 ) that had point mutations in their core binding sequences. The forward and reverse oligonucleotides of decoys at 100 μM each were annealed in Duplex buffer (Integrated DNA Technologies, Coralville, IA, USA, CAT#11-05-01-12), and the same group of decoys was pooled. T4-2 cells were plated at 0.5 × 105/12 wells the day before transfection. NFκB decoys (scramble, WT or MT), along with miRNA promoters fused to luciferase (see above), were transfected into control T4-2 cells that had a high endogenous level of NFκB. HOXD10 decoys (scramble, WT or MT), along with promoter constructs, were transfected into T4-2 cells that overexpressed HOXD10. Transfection was performed with 1 μl XFect transfection reagent (Clontech, cat# 631318), 1.5 μg of promoter DNA and 200 nM of decoy oligonucleotides according to the manufacturer’s protocol. Cells were harvested at 48 hr post transfection. The luciferase activity was analyzed using the Bright-Glo Luciferase assay system (E2610, Promega) according to the manufacturer’s protocol, and the activity was normalized using protein concentration.
4
Optimized AAV Packaging and CRISPR RNP
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Donor template plasmids were constructed using a combination of commercial DNA synthesis (GeneWiz Germany GmbH and VectorBuilder GmbH), conventional cloning and Gibson assembly [35 (link)]. The plasmid constructs were then packaged into AAV serotype 1 either by VectorBuilder GmbH or in-house using the packaging plasmids, pAAV2/1 (Addgene #112,862) and pHelper (Agilent Technologies), as previously described [36 (link)], but with the production scaled down to 3 × 10 cm2 dishes. Post-packaging, rAAV titres were determined as previously described [37 (link)], using a QuantStudio3 (Thermo Fisher) using PowerUp SYBRgreen master mix (Thermo Fisher).
CRISPR-Cas9 target sites were designed (Additional File1 : Table S5) and assessed for predicted on-target activity and specificity using the WGE [10 (link)] and the CRISPOR [11 (link)] algorithms. Synthetic RNA reagents were purchased from Integrated DNA Technologies and Merck. When required, 200 μM of crRNA was annealed with 200 μM of tracrRNA with 4 μl of duplex buffer (Integrated DNA Technologies) in a total volume of 10 μl for 5 min at 95 °C and allowed to cool to room temp. RNP complexes were generated using 100 μM of pre-annealed cr/tracrRNA or 100 μM of sgRNA, complexed with 7.7 μM Cas9 protein (Cas9 Alt-R™ Hifi V3, Integrated DNA Technologies or PURedit™, Merck) and stored on ice prior to electroporation.
CRISPR-Cas9 target sites were designed (Additional File
5
CRISPR/Cas9 Genome Editing in C. elegans
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CRISPR/Cas9 genome editing was performed in C. elegans as previously described with some modifications [47 (link)]. In brief, a 20μl CRISPR injection mixture containing 4.2μM tracrRNA, 2.5μM target sgRNA, 1.7μM rol-6 sgRNA, 1.56μM recombinant Alt-RspCas9 protein (~5μg), and 2.5μM of each repair template for rol-6 and the target gene was prepared. All reagents were purchased from Integrated DNA Technologies, Inc. The tracrRNA, sgRNAs, and Cas9 protein were first incubated at 37°C for 10 minutes in duplex buffer (Integrated DNA Technologies, Inc.) for reconstitution, followed by mixing with the repair templates. Prior to injection, the mixture was filtered through a 0.22μm cellulose filter (Corning Life Sciences). Twenty to thirty young adult worms were injected and maintained individually on plates. The F1 rollers were further maintained and their genotypes were checked. The sequences for the sgRNA and repair templates are listed in (S6 Table ).
6
CRISPR-mediated GFP-lin-41 tagging in C. sulstoni
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Caenorhabditis sulstoni GFP-lin-41 was generated using CRISPR/Cas9 methods adapted from Paix et al. (2014 (link), 2015 (link)) and Dokshin et al. (2018) (link). The germlines of young adult females were injected with a mix of CRISPR RNA (crRNA) that targeted the 5′ end of the lin-41 coding sequence and the “co-CRISPR” marker dpy-10, tracrRNA (Supplementary Table S6), a PCR-derived dsDNA HR template, Cas9 protein (Integrated DNA Technologies), and Integrated DNA Technologies duplex buffer (30 mM HEPES, pH 7.5; 100 mM potassium acetate). L4 females were picked from plates where F1 animals exhibited the co-CRISPR phenotype and mated to a single male picked from the same plate, allowed to lay eggs, and then genotyped using PCR. F2s with GFP expression were cloned from F1s that scored positively by PCR genotyping for the desired modification. Single male and single female progeny were then mated, and a homozygous line was selected by GFP expression and PCR genotyping and subjected to Sanger sequencing for validation. The mutant was then thrice backcrossed to wild type. Sequence of the GFP::lin-41 allele generated in this study can be found in Supplementary Table S7.
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Caenorhabditis sulstoni GFP-lin-41 was generated using CRISPR/Cas9 methods adapted from Paix et al. (2014 (link), 2015 (link)) and Dokshin et al. (2018) (link). The germlines of young adult females were injected with a mix of CRISPR RNA (crRNA) that targeted the 5′ end of the lin-41 coding sequence and the “co-CRISPR” marker dpy-10, tracrRNA (Supplementary Table S6), a PCR-derived dsDNA HR template, Cas9 protein (Integrated DNA Technologies), and Integrated DNA Technologies duplex buffer (30 mM HEPES, pH 7.5; 100 mM potassium acetate). L4 females were picked from plates where F1 animals exhibited the co-CRISPR phenotype and mated to a single male picked from the same plate, allowed to lay eggs, and then genotyped using PCR. F2s with GFP expression were cloned from F1s that scored positively by PCR genotyping for the desired modification. Single male and single female progeny were then mated, and a homozygous line was selected by GFP expression and PCR genotyping and subjected to Sanger sequencing for validation. The mutant was then thrice backcrossed to wild type. Sequence of the GFP::lin-41 allele generated in this study can be found in Supplementary Table S7.
7
PCR Amplification and CRISPR/Cas9 Cleavage of BsPFR-2 Gene
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We amplified BsPFR‐2 from extracted DNA of wild‐type B. saltans cells. The PCR consisted of Onetaq® 2X master mix with standard buffer (New England BioLabs) and specific primers to amplify a 2 kb region of the BsPFR‐2 gene (Supporting Information Fig. S2 ). Following PCR amplification, we purified the PCR products using the E.N.Z.A. Cycle Pure kit following the manufacturer's instructions (Omega, BIO‐TEK). In a 200 μl PCR tube, we combined 1 μg of each of three sgRNAs with 100 ng of SaCas9. We then incubated this mix in a thermo cycler at 37°C for 5 min. Following this incubation, we added 5 μl of the amplified and purified PCR product from above (amplified 2 kb of BsPFR‐2) to the sgRNA/SaCAS9 mix at a concentration of (300–600 ng μl−1), plus 1 μl of 15X BSA and 1 μl of nuclease‐free Duplex buffer [Cat#11‐05‐01‐12, Integrated DNA Technologies and adjusted the volume to 15 μl using RNase‐free water (Zymo Research, USA]. The tubes were incubated at 37°C for 1 h, followed by 5 min at 80°C to terminate the reaction and inactivate the ribonucleoprotein (RNP) complex. To visualize the cleaved DNA fragments by the sgRNA/Cas9 complex, we ran the entire 15 μl of each sample on a 2% agarose gel along with an appropriate DNA ladder and control samples (PCR products that were not treated by the RNP complex) (Supporting Information Fig. S2 ).
8
CRISPR-Mediated Enrichment of AR and RP2 Genes
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Three CRISPR RNAs (crRNAs) on both sides of each region of interest in the AR and RP2 genes were designed using CHOPCHOP64 (link) and selected according to the previously described instructions (Cas-mediated PCR-free enrichment protocol version: ENR_9084_v109_revD_04Dec2018). All custom designed Alt-R crRNAs (Integrated DNA Technologies, Supplementary Table 2 ) were pooled as an equimolar mix of crRNAs (100 µM) and assembled with trans-activating crRNAs (tracrRNAs) (Integrated DNA Technologies, cat 1073190) using Duplex buffer (Integrated DNA Technologies, cat 11010301) according to manufacturer’s instructions. Cas9 enrichment was performed according to manufacturer’s instructions using on 4–5 μg DNA and Long fragment buffer (Cas-mediated PCR-free enrichment protocol SQK-CS9109). Samples were run on a MinION R9.4.1 flow cell and operated using the MinKNOW software (version 20.10.3).
9
AMPK α-Targeting DsiRNA Oligo Design
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Dicer-substrate short interfering RNA (DsiRNA) oligos targeting the S. mansoni AMPK α sequence (GenBank accession number MH445971 ) at three different sequence positions, 26 nucleotides in length, were prepared by Integrated DNA Technologies (IDT): DsiRNA #1- CD.Ri.195363.13.5 (forward 5′- rGrUrCrArArArGrUrUrGrGrArArUrUrCrArCrArArArUrCTA -3′ and reverse 3′-rUrArGrArUrUrUrGrUrGrArArUrUrCrCrAr ArCrUrUrUrGrArCrUrU-5′) targeting nucleotide positions 120–145, DsiRNA #2- CD.Ri.195363.13.4 (forward 5′-rCrArCr UrGrGrArUrCrUrGrCrUrArGrUrCrCrArArCrCrAAT-3′ and reverse 3′-rArUrUrGrGrUrUrGrGrArCrUrArGrCrArGrArUrC rCrArGrUrGrCrU-5′) targeting nucleotide positions 1805–1830, DsiRNA #3- CD.Ri.195363.13.2 (forward 5′-rArGrUrArUrUrUr ArArArGrCrArArUrGrArArUrUrCrArCTT-3′ and reverse 3′-r ArArGrUrGrArArUrUrCrArUrUrGrCrUrUrUrArArArUrArCr UrUrC-5′) targeting nucleotide positions 1,499–1,524. Scrambled Negative Control DsiRNAs were also supplied by IDT. DsiRNAs were reconstituted in Duplex Buffer (Integrated DNA Technologies) to a final concentration of 100 μM then heated at 95°C for 2–3 min and allowed to cool at room temperature for 1 h before use.
10
Cas9-Mediated PCR-Free Oxford Nanopore Sequencing
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Genomic DNA was sheared to 20 kb fragments using Megaruptor 2 (Diagenode) and size selected using the BluePippin system (Sage Science) with a cut-off at 10 kb. 3-4 µg of sheared and size-selected DNA was prepared using the Cas9-mediated PCR-free protocol provided by Oxford Nanopore technologies with minor modifications. The crRNA and tracrRNA with Alt-R modification (Integrated DNA Technologies) were annealed in Duplex buffer (Integrated DNA Technologies) at 95°C for min and were then allowed to cool down to room temperature.
Ribonucleoproteins (RNPs) were formed by combining the annealed gRNA, HiFi Cas9 (Integrated DNA Technologies) and 1x NEB CutSmart buffer (New England Biolabs) and incubated at room temperature for 30 min. The fragmented and size-selected DNA was dephosphorylated to block all ends from ligation of adapters in a downstream adapter ligation step. Subsequently, the DNA molecules were digested by Cas9 using the previously prepared RNPs and the newly cleaved ends were dA-tailed to enable adapter ligation. The library preparation was completed by ligation of adapters from the SQK-LSK109 kit (Oxford Nanopore Technologies) and cleaned up with AMPure XP beads (Beckman Coulter) before preparation for sequencing. Sequencing was performed using the MinION system (Oxford Nanopore Technologies) with a R9.5.1 flow cell and Guppy v3.3.3 was used for base calling.
Ribonucleoproteins (RNPs) were formed by combining the annealed gRNA, HiFi Cas9 (Integrated DNA Technologies) and 1x NEB CutSmart buffer (New England Biolabs) and incubated at room temperature for 30 min. The fragmented and size-selected DNA was dephosphorylated to block all ends from ligation of adapters in a downstream adapter ligation step. Subsequently, the DNA molecules were digested by Cas9 using the previously prepared RNPs and the newly cleaved ends were dA-tailed to enable adapter ligation. The library preparation was completed by ligation of adapters from the SQK-LSK109 kit (Oxford Nanopore Technologies) and cleaned up with AMPure XP beads (Beckman Coulter) before preparation for sequencing. Sequencing was performed using the MinION system (Oxford Nanopore Technologies) with a R9.5.1 flow cell and Guppy v3.3.3 was used for base calling.
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