AsCas12a CRISPR RNA 5′ handle, a pseudoknot structure that mediates binding to Cas12a. Gene editing in human cells required retention of several native RNA residues corresponding to predicted 2′-hydroxyl contacts. Replacing these RNA residues with a variety of ribose-modified nucleotides revealed 2′-hydroxyl sensitivity. Modified 5′ pseudoknots with as little as six out of nineteen RNA residues, with phosphorothioate linkages at remaining RNA positions, yielded heavily modified pseudoknots with robust cell-based editing. High trans activity was usually preserved with cis activity. We show that the 5′ pseudoknot can tolerate near complete modification when design is guided by structural and chemical compatibility. Rules for modification of the 5′ pseudoknot should accelerate therapeutic development and be valuable for CRISPR-Cas12a diagnostics.CRISPR-Cas12a is a leading technology for development of model organisms, therapeutics, and diagnostics. These applications could benefit from chemical modifications that stabilize or tune enzyme properties. Here we chemically modify ribonucleotides of the  Development of Cas12a for human therapeutics and diagnostics may significantly benefit from, or even require, chemical modification of its guide RNA. Here the authors show that the noncanonical 5′ pseudoknot structure of the AsCas12a crRNA guide can be heavily modified and still retain very high editing activity in cells and trans cleavage activity in vitro. The core enzymatic component is a ribonucleoprotein (RNP) comprised of one or more Cas proteins, with at least one being a nuclease, which are associated with one or two CRISPR RNAs (crRNAs)18. After recognition of a short protospacer adjacent motif (PAM) sequence by the Cas endonuclease, base-pairing of crRNA nucleotides to target DNA then guides sequence-specific and site-specific endonucleolytic cleavage of DNA phosphodiester bonds21.Clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated (Cas) enzymes represent an ancient bacterial immune system evolved to fight invading viral pathogensStreptococcus pyogenes (SpCas9) and Cas12a, also known as Cpf1, from Acidominococcus species (AsCas12a) are among the most heavily co-opted and engineered CRISPR-Cas systems for creation of model organisms and development of human therapeutics33. They both efficiently induce DNA double-strand breaks and guide genome editing in mammalian cells38, utilize a single Cas enzyme and can function with a single-guide RNA (sgRNA), although Cas9 functions as a dual-guide RNA in nature33. While these enzymes can ultimately result in similar gene editing outcomes, such as genetic knockout or donor DNA sequence knock-in, they are quite distinct. Cas12a is a smaller protein that naturally utilizes a single crRNA guide (39 nucleotides), less than half the size of the commonly used artificial Cas9 sgRNA (99 nucleotides)39. Cas12a recognizes thymine (T)-rich PAMs, whereas SpCas9 uses guanine (G)-rich PAMs40. The Cas12a crRNA adopts a pseudoknot structure at its 5′ end that creates a “handle” for Cas12a to bind, as opposed to a series of stem structures for SpCas939.Cas9 from trans activity, which is a non-sequence-specific single-stranded DNA cleavage (ssDNase) activity with high catalytic turnover42. Trans activity requires that the Cas12a RNP first bind to and cut a target DNA strand, which is referred to as cis activity. Trans activity has been exploited to create molecular diagnostics that can detect very small amounts of DNA and amplify the detection signal through cleavage of ssDNA substrates that can be detected, such as by fluorescence, antibodies on test strips, and nanopores44. The digestion of the ssDNA reporter by trans activity is intrinsically tied to the cis cleavage of the specific target DNA because both mechanisms use the same catalytic residues46. Mutation of neighboring amino acids or REC (recognition lobe) linker and lid regions of Cas12a, however, could allow trans but not cis activity when Cas12a was bound to a complementary single-strand DNA target46. However, it appears that cis and trans activity cannot be fully uncoupled for recognition of double-stranded targets via trans activity without cis cleavage of the target strand46.A unique property of Cas12a is 56. To achieve this potential, however, various challenges must be overcome. These include long-term safety, delivery, and efficacy59. Lipid nanoparticles are a leading technology being developed for delivery of Cas enzymes, either as protein or as mRNA that encode the Cas protein, along with their cognate crRNA guides64. In principle, this may allow greater spatial and temporal control over tissue and cell targeting63 and provide a better window of safety when compared to therapeutic delivery approaches that involve genetically encoded expression of the CRISPR-Cas system, such as adeno-associated viruses64. However, RNA is not an ideal drug due to its size and lability—it is quickly degraded in the body. Chemical modification has emerged as a practical necessity to stabilize and control the half-life, bioavailability, and activity of RNA and nucleic acid drugs66. Chemical modification has been a pillar in the growing success of other nucleic acid-based therapeutics, including antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs)67. The lessons learned from these technologies can be applied to CRISPR-based therapeutics. The degree of chemical modification required for crRNA guides is unclear and may depend on delivery or target tissue. Nonetheless, ultimate control over stability, protein interaction, enzyme activity, and pharmacology would ideally be achieved by understanding what chemical modifications are tolerated at every position within a crRNA guide71.CRISPR-Cas systems hold tremendous promise for revolutionizing medicine, particularly through improved gene therapy or gene-targeted approachesO-methyl with phosphorothioate (PS-2′OMe) or 2′-O-methyl-3′-thiophosphonoacetate (2′OMe thioPACE) for Cas973. In the Cas12a system, 3′ end capping has been explored with 2′-F, 2′-O-methyl, unlocked nucleic acid (UNA), locked nucleic acid (LNA), and phosphorothioate (PS)74 while 5′ end capping has been explored by addition of short MS or DNA-PS modified sequences onto the 5′ handle75. Interestingly, extension of the 3′ end with 2′-O-methyl nucleotides76 or the 5′ handle with DNA-PS77 has also been reported to improve editing or increase the kinetics of trans cleavage activity for Cas12a, respectively.Previous investigations of CRISPR-Cas guide RNA chemical modification have primarily focused on improving therapeutic gene editing by increasing nuclease resistance, understanding biochemical limitations, and reducing off-target editing. Minimal addition of modified nucleotides or inter-nucleotide linkages at the guide RNA 5′ and 3′ termini have been shown to maintain or increase editing, presumably through reduced nuclease sensitivity. These have included 2′-80. A broader array of modifications, like 2′-F, 2′-O-methyl, bridged nucleic acid (BNA), UNA, LNA, 2′F-arabinonucleic acid (FANA), and PS have been tested alone or in combination as well84. These studies largely demonstrated that much of the guide could be modified, with moderate to high editing preserved, but particular positions, such as seed regions or individual residues, required RNA nucleotides. Specificity could also be improved with careful placement of modifications in the guide region84. For Cas9, retention of editing activity has been suggested to correlate with conserved polar contacts between the protein and the hydroxyl at the ribose 2′ position and the need to retain A-form-like helical architecture80. For these apparently conserved positions between Cas9 and its guide, RNA cannot be readily replaced.More complete or systematic modification of Cas9 and Cas12a guide RNAs has been explored using a variety of chemistries. DNA (2′-deoxy) has been reported to alter enzyme activity or specificity while helping to probe 2′-hydroxyl requirements of the enzyme81. However, the 5′ pseudoknot handle posed the greatest challenge as it appeared to tolerate very little modification to the ribose, suggesting that successful modification of this unique structure would be the major limitation to greater modification of Cas12a guides for therapeutic development. To address this bottleneck, we took a rational, structure-guided approach with respect to predicted 2′-hydoxyl (2′-OH) polar contacts.For Cas12a, attempts to fully modify the crRNA guide have proven unsuccessful39. The bridging phosphodiester bonds 3′ to these residue positions could further be modified to PS and retain very high cis and trans cleavage activity, as well as gene editing. When crRNAs with chemically modified 5′ pseudoknot handles were screened for trans cleavage activity, some induced differential activities like high trans activity with little or no cis dsDNA target cleavage. These results were relegated to the high catalytic turnover of trans activity even when very modest cis cleavage occurred. Together, these results advance efforts toward chemical modification of AsCas12a guides for therapeutic editing and potential diagnostic applications.In this work, we utilized chemical modifications that specifically explored ribose and 2′-OH properties. We successfully modified the 5′ pseudoknot and obtained very robust gene editing when as few as 6 out of 19 residues remained unmodified. The key positions that remained resistant to modification correlated with predicted 2′-OH polar contacts within the Cas12a-crRNA crystal structureAcidominococcus species Cas12a (AsCas12a) and a 39-nucleotide crRNA were used for this study. The crRNA can be structurally and functionally divided into two domains: a 19-nucleotide 5′ handle with a pseudoknot structure that anchors crRNA binding to Cas12a and a 20-nucleotide 3′ guide region that base-pairs to complementary target strand DNA  groups in the pseudoknot structure, as well as intramolecular 2′-OH RNA-RNA contacts20. The importance of RNA A-form helical structure and 2′-OH chemistry has been partially investigated for pseudoknot stability and protein interaction86, but not in the context of CRISPR-Cas systems. The unique noncanonical nature of the Cas12a 5′ pseudoknot, as well as previous attempts at modification81, suggested that it would be challenging to modify for therapeutic development. To focus on the pseudoknot structure and uncouple it from guide region effects, we maintained an entirely native RNA ribose (2′-OH) guide region throughout most of this study. This was accomplished through splint ligation of a 5′ phosphorylated guide RNA and chemically modified pseudoknot RNAs using T4 DNA ligase87 , 2′-fluoro (2′-F), oxepane (OxN), 2′-arabino (2′-araOH), and 2′-amino (2′-NH2) residues . No sequence-specific (cis) cleavage activity was observed for this design in vitro, indicating that either 2′-OH contacts or A-form helical structure, or both were necessary for Cas12a RNP assembly or enzyme activity  completely rescued in vitro cleavage activity. These results, along with inspection of the Cas12a crystal structure, suggested to us that the 2′-OH at residue -19 may not be a critical polar contact that impacts activity. Conversely, an all-RNA pseudoknot with the same seven potentially critical 2′-OH contact positions (omitting position -19) replaced with DNA (cpEGIP-N2) only provided a quarter of the normal AsCas12a activity. Thus, these results supported the critical role of 2′-OH contacts for either pseudoknot structure or protein interaction. However, the observation that cpEGIP-N2 still conferred cleavage activity with no 2′-OH groups at the predicted critical positions also suggested that A-form helical structure or C3′-endo sugar pucker may play an important, albeit potentially lesser role. Indeed, adding additional DNA nucleotides to cpEGIP-N2 to create cpEGIP-A1, which possessed only six RNA residues, abrogated cleavage activity. The activity of cpEGIP-N2, although low, also suggested that pseudoknot 2′-OH contacts may not be obligatory for activity.While DNA nucleotides can probe the importance of 2′-OH contacts, they introduce conformational flexibility and can therefore also probe the importance of A-form structural preferencesTo further test the role of individual positions and explore RNA–DNA combinations, we synthesized and tested several more crRNAs. Starting with a design that preserved the putative critical 2′-OH contacts and included more RNA residues (cpEGIP-B1), we replaced -19, -18, and -17 positions individually . None of these substitutions were detrimental to activity Fig. . We thenAsCas12a exhibited high activity with all these crRNAs . S.pyogenes80. Critical 2′-OH contact positions lie in the SpCas9 crRNA guide (spacer) seed region and the repeat region proximal to the guide71. Thus, they are localized in the center of the SpCas9 crRNA. Furthermore, RNA–DNA chimeric Cas9 crRNAs composed of DNA with only RNA at the critical 2′-OH positions were highly active in vitro69. To determine if a similar phenomenon existed for the 5′ pseudoknot of Cas12a, we selected several RNA–DNA chimeric pseudoknot crRNAs with high activity in vitro and tested their ability to knock out, and therefore edit, an EGFP gene. We started with HEK293T cells stably expressing EGFP79 and transduced them with a 3xNLS-AsCas12a-expressing lentivector followed by selection  designed to target the integrated EGFP gene  is not necessary for in vitro cleavage but is required for efficient gene editing in cells by CRISPR-Cas9 from endo sugar pucker and strongly favor A-form helical structure93. However, when tested for cell-based editing activity cpEGIP-SJ01 was completely inactive  (cpEGIP-SJ01). This fully modified pseudoknot provided very high cleavage activity, similar to or higher than the native all-RNA control Fig. . The robive Fig. . 2′-F nu94. We synthesized thymidine (OxT) nucleotide replacements with the phosphodiester linkage at three different positions, designated OxT-1, -2, and -3. These were then incorporated into the pseudoknot sequence at putative critical 2′-OH positions -13 and -17 during solid-phase synthesis. The incorporation of OxN  completely abrogated AsCas12a activity, indicating that certain OxN properties, such as the bulkiness of the seven-membered ring or conformational restraints, may simply be incompatible despite providing hydroxyl groups at various positions  and 2′-araOH in putative critical 2′-OH positions. OxN is a seven-membered ring structure that can be synthesized with multiple hydroxyl groups and a phosphodiester linkage at different ring positionsons Fig. . Based oendo (DNA-like) conformation95. When placing 2′-araOH at -13 and -17 positions (cpEGIP-SJ06), some enzyme activity was retained. We therefore chose to explore more 2′-araOH substitutions but in the background of a 2′-F pseudoknot, reasoning that the strong A-form preference of 2′-F might offset structural preferences of 2′-araOH. Incorporating more than two 2′-araOH  severely reduced or eliminated enzyme activity  are stereoisomers of RNA that place the hydroxyl group at the 2′ position on the opposite face of the ribose ring. While arabinonucleosides retain a 2′-OH group, the “up” orientation steers the arabinose sugar to adopt a C2′-ity Fig. . Only cplls Fig. .cis) activity . This design, cpEGIP-SJ14, supported very high in vitro cleavage  as a potentially promising modification. The relatively small amino moiety and its ability to both donate and accept hydrogen bonds made it an attractive replacement for RNA. In addition, the pKa of the 2′-amino should be 6.2, as determined by Eckstein and colleagues96, making the major species non-protonated at physiological pH. 2′-NH2 nucleotides have been used for other technologies, like aptamer and ribozyme development99, but have not been applied to CRISPR guide RNA modification previously. Starting with an all-2′-F pseudoknot, we sought to replace the putative critical positions individually with 2′-NH2 to ensure that in vitro activity was not affected. Because 2′-NH2 is an uncommon modification and only pyrimidine monomers were commercially available, we did not substitute positions -18 and -6. All the crRNAs containing a 2′-NH2 substitution in the 2′-F pseudoknot (cpEGIP-LA-N14-1 through -LA-N14-5) were highly active in vitro . These new designs, cpEGIP-LA-N14-6 through -LA-N14-10, all exhibited robust in vitro cleavage activity . Substitution at positions -13 and -1 resulted in approximately 30% gene editing (cpEGIPe-LA-N14-8 and -LA-N14-10). In contrast, substitutions with 2′-NH2 at positions -10 and -14 (cpEGIPe-LA-N14-9 and -LA-N14-7) provided ~60–70% editing, which is substantial considering that the native RNA pseudoknot typically provides 80% editing. Interestingly, substitution at position -14 resulted in similar activities whether 2′-F (cpEGIPe-SJ14-3) or 2′-NH2 (cpEGIPe-LA-N14-7) were used, suggesting relatively flexible accommodation at this position. 2′-NH2 was not suitable for replacing RNA at positions -17 or -1, which was also the case with 2′-F (cpEGIPe-SJ14-2 and cpEGIPe-SJ14-7). However, position -13 was differentially affected, with 2′-F being very well-tolerated (cpEGIPe-SJ14-4), whereas 2′-NH2 was poorly tolerated (cpEGIPe-LA-N14-8). Amines do not form hydrogen bonds as stably as alcohols and are bulkier at the 2′ position than OH and F, which may explain differential activity effects.To begin with modified pseudoknots more likely to generate gene editing activity, we created the equivalent of cpEGIP-SJ14 (~80% editing) but walked a 2′-NHity Fig. . When tacis cleavage in vitro versus cell-based gene editing prompted us to investigate whether chemically modified 5′ pseudoknots impacted non-sequence-specific trans cleavage activity. We employed a fluorescence-based assay similar to that originally reported by Chen and coworkers41   Fig. . Incubatcis–trans activity was guide-sequence dependent, we ligated the modified 5′ pseudoknot to the cpEGIPe guide sequence used for editing in cells. With this guide sequence, both cpEGIPe-B1d and cpEGIPe-F6 still produced markedly low cis but about 50% trans activity  and target (antisense) strands and performed trans cleavage assays on duplexes using cpEGIPe and cpEGIPe-B1d, -F1, -F6, and -SJ14-7 crRNAs, followed by resolution of reaction products on denaturing polyacrylamide gels  or target (antisense) strand  previously shown to possess robust in vitro cleavage and editing activity  were less tolerant. Although OxN and 2′-araOH were not sufficient substitutes for ribose, it appears that a few 2′-NH2 and 2′-deoxy could be incorporated in the context of 2′-F. Positions most amenable to these modifications are position -19 and more central positions like -6. In summary, removal of all but a handful of ribose sugars is achievable in the 5′ pseudoknot structure of AsCas12a crRNAs while still maintaining very high gene editing activity in cells. In addition, selective placement of PS linkages 3′ to remaining RNA nucleotides further stabilized the RNA while not compromising editing activity. A similar approach succeeded in stabilizing remaining RNA positions in a Cas9 guide RNA, although editing in the final heavily modified RNA guide was significantly reduced70. Previously, full PS modification of the native 5′ pseudoknot of the AsCas12a crRNA resulted in a substantial loss of editing in cells81.Here we focused primarily on 2′-deoxyribose, or DNA, and 2′-F as substitutions and also included modifications designed to potentially retain hydrogen bonding between pseudoknot nucleotides or with O-methyl78. This approach is typically suitable for oligonucleotides that function almost exclusively through Watson–Crick base-pairing, such as ASOs or siRNAs104. However, it resulted in no editing activity, likely due to loss of critical 2′-OH contacts. Overall, these results reinforce the need to incorporate molecular and structural data into modification design and the value of nucleotide analogs that can mimic or substitute the properties of native ribose.Previous attempts to modify the 5′ pseudoknot sequence replaced entire blocks of contiguous nucleotides, several at a time, with DNA, 2′-F, or 2′-O-methyl, 2′-F, DNA, LNA, UNA, and PS linkages81. The general conclusion from these studies was that modification at or near the 3′ end of the guide region is relatively well-tolerated for most modifications. However, modification at the 5′ end of the guide, and therefore within or near the “seed” region, were very poorly tolerated. A notable feature of the seed region of both Cas12a and Cas9 CRISPR systems is the concentrated presence of conserved 2′-OH contacts with the Cas protein20. In addition, the ability for Cas9 to recognize and bind DNA targets, as well as catalyze cleavage, has been shown to be dependent on the formation of an A-form-like duplex between the guide and target DNA71. Together, the 2′-OH and A-form-like architectural requirements of crRNAs reflect on the unique co-evolution of bacterial CRISPR-Cas systems to use RNA as guides106. Complete chemical modification of guide RNAs, such as for therapeutic CRISPR-based editing, will require addressing the 2′-OH bottleneck that has finally become apparent for Cas9 and Cas12a, and probably for other Cas enzymes. Nonetheless, our results combined with previous studies now provide a roadmap to near-complete chemical modification across the entire Cas12a crRNA. We propose that a primarily 2′-F and 2′-O-methyl oligonucleotide, with some DNA or 2′-NH2 nucleotides, combined with selective PS modification, may be sufficient to provide drug-like profiles with favorable editing activity. Exploring a greater diversity of modifications at well-placed positions may further unlock efficient editing with little or no native RNA residues. In addition, other properties of Cas12a will need to be investigated, such as the impact of chemical modification schemes on off-target editing, as heavier modification is incorporated.Ultimately, complete ribose chemical modification of the entire crRNA would be desirable for precise control over therapeutic development. Attempts to modify the guide region of Cas12a crRNAs explored 2′-80. For example, all RNA nucleotides can be replaced with 2′-F in the Cas9 crRNA71, and now the Cas12a pseudoknot, and provide very high cleavage in vitro. However, these crRNAs provide little or no editing in cells. Converting predicted 2′-OH polar contact positions back to RNA usually rescues gene editing71. Thus, it appears that intrinsic biochemical activity is not dependent on 2′-OH contacts in bulk reactions in vitro but these become critical within cells, perhaps due to low biochemical RNP concentrations, more complex chromosomal DNA targets, or other unexplored biochemical or cellular factors107. Experiments like precision biochemical, thermodynamic, and structural studies, as well as sophisticated single-molecule or gene editing assays in cells, may be necessary to fully understand this phenomenon and unlock “RNA-free” CRISPR-Cas editing for advanced therapeutic control.A recurring theme in crRNA chemical modification studies, especially with Cas9, is that heavy modification schemes successful for in vitro cleavage often do not translate into high editing efficiency in cellstrans activity, by Cas12a was quickly converted into an amplification-based diagnostic method41. After sequence-specific binding and cutting (cis cleavage) of a DNA target, the catalytic domain of Cas12a is proposed to become solvent exposed such that ssDNA is accessible to the active site46. This results in high turnover degradation of ssDNA. Once DNA target paired to the guide RNA is released, ssDNase cleavage activity should cease as Cas12a returns to a pre-catalytic structural state46. Thus, trans cleavage activity of Cas12a appears to be intrinsically tied to its cis cleavage activity, turnover of cis cleavage products, and conformational-state transitions.The discovery of non-sequence-specific ssDNase cleavage, or cis versus trans cleavage activity for a few crRNAs possessing chemically modified pseudoknots. Certain modification patterns or schemes contributed to this effect. Ultimately, the differential cis–trans activity observed here appears to be a consequence of variable cis cleavage compared to catalytic trans turnover rates for certain guides. This can result in variable ratios of cis to trans activity at time points within a certain activity regime. However, trans activity by modified crRNAs still required cleavage of the target antisense DNA strand in a dsDNA target. Thus, the overall mechanism of trans activity was unaffected. The pseudoknot is proposed to influence catalysis by modulation of AsCas12a conformational transitions46, which may explain how various activity profiles were achieved by modified pseudoknots. If differential cis–trans cleavage activity could be controlled predictably, it might prove useful for some applications. For example, binding to a specific on-target DNA without cleaving (cis activity) but still allowing trans activity, could provide trans activity for long durations and possibly improve diagnostics. Conversely, cis cleavage without activation of trans cleavage might improve the specificity or safety of other applications, like gene editing108. Recently, it was reported that extending the 5′ or 3′ ends of the LbCas12a crRNA could increase on-target activity, and especially enhance trans cleavage rates77. Thus, multiple types of crRNA modification may be useful for modulating cis specificity and trans turnover.In this study, we initially found what appeared to be differences in DNA oligonucleotides, crRNAs, and DNA–RNA chimeric 5′-handle RNAs were synthesized and purified by Integrated DNA Technologies (IDT). Full-length crRNAs, including DNA–RNA and chemically modified crRNA, were ligated using splint ligation . A solution of 3% trichloroacetic acid in THF, delivered over 95 s, was used to deprotect DMTr groups.Oligonucleotide chemical syntheses were carried out using either an ABI 3400 DNA synthesizer (Applied Biosystems) or a MerMade 12 Oligonucleotide synthesizer (BioAutomation) on a Unylinker CPG (ChemGenes) solid support at a 1 µmol scale. Conventional 2′-tert-butyl-dimethylsilyl (TBDMS) ribonucleoside, 2′-fluoro-ribonucleoside (2′-FRNA), and 2′-trifluoroacetyl amino phosphoramidites (ChemGenes) were used, along with newly synthesized oxepane phosphoramidites. Phosphoramidites were dissolved in MeCN (0.15 M) and activated with 5-ethylthio-1H-tetrazole (0.25 M in MeCN). Capping of failed couplings was carried out by the simultaneous delivery of acetic anhydride in pyridine/THF and N-methylimidazole (16% in THF) and contacting the solid support for 6 s. Oxidation of the phosphite triester intermediates was affected with 0.1 M iodine in pyridine/H3N/TREAT-HF  for 90 min at 65 °C (samples in the SJ series were desilylated by exposure to neat TREAT-HF for 48 h), followed by quenching with 3 M NaOAc buffer  and precipitation of the crude oligonucleotide from cold butanol . Samples were chilled on dry ice for 30 min and then centrifuged. After removing the supernatant, the remaining pellet was evaporated to dryness, taken up in autoclaved Milli-Q water (1 mL), and filtered.Deprotection and cleavage of oligonucleotides from the solid support was achieved by treatment with 1 mL of cold 29% aqueous ammonia/ethanol  for 16 h at 65 °C (or 48 h at room temperature for oxepane-containing oligonucleotides). The samples were centrifuged and the supernatant was transferred to a clean 1.5 mL microcentrifuge tube and vented for 30 min, chilled on dry ice, and evaporated to dryness. Removal of the 2′-TBDMS protecting groups for the RNA-containing oligonucleotides was achieved by treatment with a 300 µL solution of NMP/Et4, 10 mM NaOAc, 20% MeCN in Milli-Q water) was used for analysis and purification. Using a gradient of 0–50% LiClO4 (buffer B) over 50 min , the desired compounds eluted at around 30 min. cpEGIP-SJ oligonucleotides were purified on an Agilent 1200 Series Instrument using a Protein-Pak DEAE 5PW column (7.5 × 75 mm) at a flow rate of 1 mL/min, with a buffer system consisting of buffer A (Milli-Q water) and buffer B (1 M LiClO4 in Milli-Q water) using a method of 0–60% buffer B over 37 min at 60 °C. Following collection of the desired peaks, excess LiClO4 salts were removed using Gel Pak 2.5 size exclusion columns (Glen Research). Purified oligonucleotides were characterized by electrospray ionization-mass spectrometry . Extinction coefficients for RNA were used for oligonucleotides containing oxepane modifications.Crude oligonucleotides were purified by ion exchange (IE) HPLC. cpEGIP-LA oligonucleotides were purified on a Waters 1525 instrument using a Protein-Pak DEAE 5PW anion exchange column (21.5 mm × 150 mm). A buffer system consisting of buffer A  and buffer B , and 0.5 µL of SUPERase-In (Invitrogen) in a final volume of 100 μL. The reaction was incubated for 90 min at 37 °C and stopped by adding 1 µL of 0.5 M EDTA pH 8 and phenol-chloroform extracted. Ligation products were visualized by resolving on 15% denaturing polyacrylamide gels and staining with methylene blue. Full-length ligation products were gel-purified by crush-and-soak elution, phenol-chloroform extracted, ethanol precipitated, and quantified by measuring absorbance at 260 nm and calculated extinction coefficients using nearest neighbor approximations with Beer’s Law. Electrospray ionization-mass spectrometry was used to confirm the mass of a few full-length, ligated crRNAs to ensure ligation and gel-purification was proceeding properly.crRNA guide sequence (200 pmol), synthesized with a 5′ phosphate, was ligated to 200 pmol of 5′-handle RNA (synthesized with a 3′-OH) using T4 DNA ligaseAsCas12a was obtained from Addgene (79007). AsCas12a proteins were prepared similarly to that previously described for SpCas9109. Briefly, protein expression was induced in Rosetta (DE3) cells grown in Luria-Bertani (LB) broth with 0.2 mM isopropyl thiogalactopyranoside (IPTG) at 18 °C for 16 h. Cell pellets were resuspended in 6 mL chilled binding buffer  per 0.5 L of culture. Resuspended cells were sonicated and clarified by centrifugation. For the 0.5 L of culture, 5 mL His-Pur Cobalt-CMA resin (Thermo Scientific) was equilibrated with binding buffer and the supernatant added to the equilibrated resin and incubated at 4 °C for 1 h with inversion to mix every 15 min. The column was washed sequentially with at least 10 bed volumes of increasing concentrations of NaCl in wash buffer . Protein was eluted with 130 mM imidazole buffer . Purified AsCas12a enzyme was concentrated and buffer exchanged using centrifugal concentrators  into 2x storage buffer . Then one volume of glycerol added to obtain a final of 50% glycerol. Protein stocks were then stored at −80 °C. Concentration of AsCas12a was determined by UV absorbance at 280 nm using a calculated extinction coefficient and Beer’s law.Plasmid encoding of an AsCas12a  and crRNA  in 1x cleavage buffer  supplemented with 0.1 mg/mL of purified yeast tRNA. Molar excess of the AsCas12a over crRNA allows RNP concentration approximation by using crRNA concentration. Although a fraction of the Cas enzyme remains unbound, we have previously found this approach to be an accurate method for predicting the concentration of actual assembled RNP complexes71. The 40 µL reaction was incubated at 37 °C for 2 h. The reaction was then treated with 10 µg of RNase A (Thermo Scientific) for 15 min followed by 20 µg of Proteinase K (Thermo Scientific) for 15 min at room temperature. The reaction products were then precipitated in 10 volumes of 2% LiClO4 in acetone for >1 h at −20 °C. Precipitated reactions were centrifuged and washed with acetone, air dried, and resuspended in gel-loading dye  and resolved on 1.5% agarose gels. Agarose gels were stained with ethidium bromide and visualized using a UV imager. The fractions of target cleaved versus uncleaved were quantified using ImageJ software (v1.43u). Dot plot data points represent experimental replicates, not technical replicates.A 1 kb fragment of the target EGFP gene was PCR-amplified using “cpEGIP_vitro targ” primers (Supplementary Table AsCas12a and crRNA (cpEGIP or cpEGIP-SJ14) were assembled at room temperature for 10 min. The RNP was then added to a target DNA in at 37 °C in a final of 1x cleavage buffer and reactions stopped at indicated time points by precipitation with 2% LiClO4 in acetone and incubated at −20 °C. Samples were pelleted by centrifugation then washed with acetone. After samples were air dried, they were resuspended in 1x loading dye , treated with RNase A and proteinase K as described above, and cleavage products resolved on 1.5% agarose gels. Quantification was performed as described above.For cleavage time-course assays, 61. We subcloned AsCas12a from pY010 (pcDNA3.1-hAsCpf1)  into pLJM1-EGFP lentivector110  such that the EGFP gene was replaced with AsCas12a. Subcloning was performed with NheI and EcoRI restriction enzymes and resulted in retention of the C-terminal NLS and HA tag on AsCpf1 in the new vector, pLJM1-AsCas12a , and pCMV-dR8.2 dvpr 111 using the calcium phosphate technique. Medium was replaced 18 h after transfection. Lentiviral containing medium was collected 48 h later and centrifuged at 1000g for 5 min. Hexadimethrine bromide was added to the medium (8 µg/mL) and the medium was used to infect HEK293T-EGFP cells on 6-well plates for 8 h.HEK293T cells stably expressing EGFP were a kind gift from Dr. Wen XueAsCas12a and staining with anti-HA  revealed primarily cytoplasmic localization  with 1x non-essential amino acids (NEAA), 5% cosmic calf serum (CCS) and 2.5% fetal bovine serum (FBS) without antibiotics. Cells were reverse-transfected  in six experimental replicates in 96-well plates with 20 pmol of crRNA and 0.3 µL RNAiMAX lipid (Invitrogen) in a final reaction of 200 μL of OptiMEM. After 8 h, one volume of media containing 5% FBS and 5% CCS was added to cells and further incubated overnight. Media was then replaced with full media and cells grown for an additional 4 days. For flow cytometry, cells were washed with 200 μL phosphate-buffered saline (PBS) and trypsinized by adding 70 μL of trypsin-EDTA solution. Then, 100 μL of media was added to the cells. The cells were spun for 5 min at 300g at room temperature. Cells were washed again with 200 μL PBS, resuspended in 200 μL PBS and counted in an Attune flow cytometer. EGFP was detected using the blue laser (BL1 channel). At least 20,000 events were collected and analyzed by Attune software (v3.12). The cells were gated based on forward and side scattering (FSC-A/SSC-A) to remove cell debris, gated to select single cells, and gated to select EGFP-positive cells. The quadrant gate was established using the signal from non-EGFP-expressing control cells. Untreated HEK293T cells expressing EGFP and AsCas12a contained ∼5% non-fluorescent cells  on ice for 15 min. Certain experiments either omitted target DNA, crRNA, or used PS-modified target at the same concentrations as indicated above. Reactions were initiated by adding FQ ssDNA substrate to a final of 3 µM and placing the plate in a Bio-Rad CFX96 instrument with the block set at 4 °C. The block was cycled to 37 °C and the plate was read using the SYBR-only channel every 53 s (reading takes 7 s) such that fluorescence readings were collected at 1 min intervals. Fluorescence was collected for up to 150 min. Maximum fluorescence values at 1 h were used to compare non-sequence-specific trans cleavage activity of varying crRNAs containing modified 5′ pseudoknots. Error was calculated by standard deviation, representing three or more experimental replicates.32P-γ-ATP as previously described112. Radiolabeled DNA strands were then gel-purified from a 15% denaturing Urea-PAGE by crush-and-soak elution after visualizing radioactive bands by autoradiography. Duplexes with differentially radiolabeled sense (non-target) or antisense (target) strands were annealed and slow-cooled with a ~1.2-fold molar excess of unlabeled complementary strand. Reaction conditions then utilized those of a standard trans cleavage assay but lacking a FQ-labeled ssDNA substrate. Reactions proceeded for 1 h before being stopped by phenol-chloroform extraction. Cleavage products were resolved on a 15% denaturing PAGE and exposed to phosphorimager for visualization.For strand-nicking cleavage assays utilizing radiolabeled target DNA strands, individual DNA strands (cpEGIPe target sequence) were 5′ radiolabeled with T4 polynucleotide kinase and AsCas12a in the presence and absence of crRNA and double-stranded target DNA (1:1.5:2 molar ratio)113. The RNP was incubated at room temperature for 5 min. The resulting samples were incubated with Trypsin-EDTA solution  at a mass ratio of 100:1 and the partial proteolysis was conducted at 37 °C for 15 min. The reaction was stopped by the addition of SDS-PAGE loading buffer and heating the samples for 5 min at 95 °C. The reaction products were analyzed by 12% SDS-PAGE and stained with Coomassie brilliant blue G-250 in 50% (v/v) methanol and 10% (v/v) acetic acid then destained in the same solution without dye.Trypsin proteolysis was performed using 30 μg of 4, and resolved on a 15% TBE-buffered denaturing (7 M urea) polyacrylamide gel. Gels were stained with methylene blue to visualize digestion patterns.Serum stability test was performed using 300 pmol of pseudoknot or crRNA and 5 µL of 10% FBS in 1x PBS in a total reaction volume of 50 µL. Reactions were incubated at 37 °C and then stopped after 2 h by adding 1 µL of 0.5 M EDTA. RNA was extracted by phenol-chloroform extraction, precipitated with 2% LiClOFurther information on research design is available in the Supplementary information.Reporting summary.


Increasing evidence suggests that post-transcriptional ribonucleic acid (RNA) modifications regulate essential biomolecular functions and are related to the pathogenesis of various diseases. Precise identification of RNA modification sites is essential for understanding the regulatory mechanisms of RNAs. To date, many computational approaches for predicting RNA modifications have been developed, most of which were based on strong supervision enabled by base-resolution epitranscriptome data. However, high-resolution data may not be available.We propose WeakRM, the first weakly supervised learning framework for predicting RNA modifications from low-resolution epitranscriptome datasets, such as those generated from acRIP-seq and hMeRIP-seq. Evaluations on three independent datasets (corresponding to three different RNA modification types and their respective sequencing technologies) demonstrated the effectiveness of our approach in predicting RNA modifications from low-resolution data. WeakRM outperformed state-of-the-art multi-instance learning methods for genomic sequences, such as WSCNN, which was originally designed for transcription factor binding site prediction. Additionally, our approach captured motifs that are consistent with existing knowledge, and visualization of the predicted modification-containing regions unveiled the potentials of detecting RNA modifications with improved resolution.https://github.com/daiyun02211/WeakRMThe source code for the WeakRM algorithm, along with the datasets used, are freely accessible at: Bioinformatics online. Increasing evidence suggests that post-transcriptional ribonucleic acid (RNA) modifications regulate essential biological processes and are related to the pathogenesis of various diseases including multiple cancers  sequences as well as other predictive genomic features. Among them, SRAMP is one of the earliest and widely applied predictive approaches for m6A RNA methylation based on the Random Forests method from RNA sequences .To date, many 5C) and N4-acetylcytidine (ac4C). These two modifications can be detected by enrichment-based sequencing approaches, such as hMeRIP-seq and acRIP-seq, respectively  or very narrow applicable scope -resolution data, but usually overlook the weakly supervised information of RNA sequences when applied to low-resolution datasets, such as 5-hydroxymethylcytidine  as input and predicts the sub-regions that are most likely to contain the modification of interest. To the best of our knowledge, this is the first time that RNA modification prediction was formulated under the framework of weakly supervised learning. Additionally, compared to existing MIL algorithms, which were originally developed for transcription factor binding site (TFBS) prediction, our model achieved better performance in RNA modification site prediction with major improvements, i.e. using the gated attention  demonstrated the general effectiveness of our approach in predicting RNA modifications from low-resolution data. Our approach outperformed state-of-the-art MIL algorithms for genomic sequences, such as WSCNN, which was originally designed for TFBS prediction. Our approach captured motifs that are consistent with existing knowledge. Visualization of the predicted modification-containing regions unveiled the potentials of detecting RNA modifications with improved resolution. WeakRM should make a powerful and useful tool for learning RNA modifications with only low-resolution epitranscriptome data.4C, hm5C and m7G) and their respective sequencing technologies  (see The proposed WeakRM framework described below was tested on three independent epitranscriptome datasets of low-resolution (around 100 nt), which corresponded to three distinct RNA modifications (acseq) see . All thr7G sites from m7GHub  runs over each bag (length l) to capture different portions of it with stride s, resulting in total c and the step-wise shift s are two tunable hyperparameters. Reducing c and s may allow us to locate modifications at a higher resolution but can increase computational load or decrease the prediction accuracy. The difference between those two parameters (c–s) reflects the number of nucleotides shared by two adjacent instances.MIL framework treats each RNA sequence as a ‘bag’ with more than one ‘instance’. The target label (with or without RNA modification) is associated with the bag rather than with each instance, indicating whether the RNA modification of interest has occurred within a piece of sequence. In practice, the algorithm divides the entire sequence into multiple overlapping sub-sequences as the ‘instances’ contained within the bag  of around 100–300 nt long, depending on the peak calling algorithm and the fragment length in the protocol. Very wide peaks, in theory, reflect multiple modifiable nucleotides located in proximity. This property allows us to break up the labeled long sequence into shorter pieces and still ensure a reliable label .Starting from the input layer, this sub-section presents a detailed description of the proposed weakly supervised learning framework. The first step in feeding RNA sequences into the WeakRM model is to numerically represent the nucleotides. One-hot encoding is a common way in deep learning-based models which maps each nucleotide into a vector of size 4  and outputs instance-level features.As referred to earlier, in the case of the MIL problem, only an overall binary label associated with the input bag can be assessed. Therefore, how to obtain bag-level probabilities from instance-level features without instance-level labels becomes the key to the MIL framework. Generally, we can divide existing label probability modeling methods into two categories:The instance score merging approach: this method requires the construction of an instance-level classifier to estimate the score of each instance. A chosen pooling method then aggregates all instance scores and returns the bag-level class (probability of at least one RNA modification site contained within the input sequence).The instance feature merging approach: this method aims at obtaining bag-level feature representations using weighted summation along instance embeddings. The bag-level features are subsequently fed into the final classifier.Maximum and average are the two most common fusion methods for score merging. However, max-pooling only extracts information concerning the most favored instance, which overlooks other valuable instances and may suffer from outliers. This weakness can even be amplified when our subsequence instances overlap with each other and are therefore highly correlated. Average pooling, on the other hand, assigns equal weights to all instances, which ignores the fact that our instances are sparsely distributed. Other score merging approaches such as, log-sum-exp pooling  and noisNoisy-and  and contribute most (highest attention weight) to bag-level prediction.5C and ac4C, their transcriptome map of base-resolution is not yet available. Therefore, we developed a validation approach based on low-resolution data. For each peak, we obtained the two marginal areas connecting the negative and positive regions. Specifically, 600 nt of sequences were extracted from both the Aside from distinguishing the RNA modification-containing and non-containing sequences, a key purpose of our model is to identify the sub-regions containing RNA modifications from a long input sequence. Unfortunately, for hmpeak see . We can 7G RNA internal modification data for further validation. Both non-base and single-base techniques are available for m7G, which allows us to train our model using peak data and validate using ground truth base-resolution sites. To visually display the results, we placed the known modifiable sites in the center and extracted 300 flanking regions on both sides to obtain a set of 601 nt sequences. For both cases, we picked the most important instance from each true positive bag, recorded their distance to the middle point and plotted their distributions.To demonstrate the effectiveness of our model more convincingly, we select mInterpretability of predictive models is often highly desired for biological systems. In the case of RNA modification prediction, this refers to finding the recurring sequence patterns preferred by the model and elucidating the difference between the high-weight and low-weight instances.Existing motif discovery methods for neural networks can generally be divided into two types. One method is to extract the weights of convolutional kernels in the first network layers, count the occurrence of nucleotides that activate the kernels, and visualize them as position weight matrices  method  has traditionally been considered a cap modification of mRNAs. Recent studies identified its widespread internal existence and pivotal roles in translation control . Since we previously established benchmark datasets and developed a base-resolution m7G predictor (7G data. The availability of base-resolution profiling (m7G-seq) also enabled a more reliable validation using data produced from an independent biotechnology .N7-methylguanine . For negative data, only the sequences that appeared as negative in both two cell lines were used. The model performance of each cell line data and two-way cross cell line evaluation were also provided in 7G-seq sites as our ground truth data and extracted the sequences from the 300 nt flanking region on both sides to form a testing sequence of 601 nt. It was expected that the central instance covering the known m7G site should have greater attention weights.To reduce the number of false-positive samples, we extracted the sequences that appear as peaks in both cell lines (HeLa and HepG2) as the positive samples, as in the case of the original study (Zhang 7G datasets (m7G-MeRIP-seq) to produce a reliable comparison. The data were evenly divided into 10 parts, each with the same amount of positive and negative peaks. For all models, an instance length of 50 nt and a stride size of 10 nt were chosen because they generally have better performance on the m7G dataset . Given the instance length (50) and stride (10), there exist five instances containing the m7G site detected using base-resolution technology in each bag. The instances with the m7G site near their centers are likely to produce the highest attention weights. There are still some high-weight instances that do not contain known m7G sites. Among them, those near the center area showed a higher probability to be high-weight instances. This may indicate that WeakRM has captured some sequence patterns that are not immediately close to the m7G site, or there existed previously undetected m7G sites and the modification exhibits a clustering effect, as previously observed in the case of N6-methyladenosine (m6A)  and dinucleotide shuffled references to obtain attribution maps on the input instances. For each test sequence, we simulated 50 shuffled references and used 20 steps in calculating IG values. As our input data were all one-hot encoded, such scores of contribution can be easily transformed to the importance of each nucleotide.7G, is a modification that happens on nucleotide G. By observing the distribution of the G contribution score, we observe that not all guanines have received high values, and some may even have a negative impact on the positive peak prediction. Therefore, such interpretable scores can be a potential way to further narrow the range of predicted modified sites.As shown in 7G-containing region. For visualization purposes, we individually normalized the contribution scores for each instance, which resulted in comparable score ranges between the high-weight and low-weight instances. However, the former is usually much larger than the latter in the absolute sense.On the other hand, continuous cytosine (C) and uracil (U) patterns were abundant in the low weight instances , which ok-mers selection, clustering of similar patterns, and multiple sequence alignment for obtained motifs in each cluster. In our work, we use TF-MoDISco to extract consensus motif from instances with higher than average weights. A great advantage of TF-MoDISco is that it provides the continuous Jaccard similarity calculation to carry out alignment directly based on contribution scores instead of only selecting the most important bases. By allowing three gaps and two mismatches and trimming using overall letter frequencies, we found one consensus motif, given in the 7G-MeRIP-seq using HOMER software and reported in data source paper , we found the motif learned by WeakRM can be matched to the Top-1 known motif with a P-value of 8.71e-03. The P-value here represents the probability that a random motif of the same width has the same or better matching score as the target. The value 8.71e-03 is sufficient to infer a high similarity between the motifs.Attribution maps show only the model preference of nucleotides in every single test. To summarize the recurring motifs captured by WeakRM, the current general pipeline carries out high-weight 5C) in fly and mouse brains . Due to the existence of multiple Cytosines within such regions, it is not exactly clear which specific Cytosine can be modified by hydroxylmethylation. When WeakRM was applied to hm5C, all sequences under the hm5C peaks from Drosophila S2 cells hMeRIP-seq data were used as the positive samples. Negative samples were collected from negative regions that do not intersect with the positive peaks in the same cell line. These samples were further selected and randomly trimmed to fit the number and width of the positive sequences.Recent studies have revealed a relatively high abundance of 5-hydroxymethylcytidine , average precision (AP) and accuracy with 0.5 threshold were selected as the main evaluation metrics during performance evaluation.5C data, and the improvement is more obvious than that of m7G. This may be because the peak of hm5C has a larger width overall. Furthermore, through data augmentation, the training is more stable.As shown in et al., 2020) and iRNA5hmC-PS  was unavailable. As such, we developed a new peak margin-based method to demonstrate the effectiveness of our model in identifying the sub-regions containing hm5C out of a long input sequence. Specifically, the peaks from hMeRIP-seq data naturally have two margins, one toward the 5C-containing sub-regions against the rest of the sequences.A potentially useful application of the trained WeakRM model is to detect the sub-regions containing hm4C) were identified on poly(A) RNA isolated from a variety of human cells , indicating a better spatial accuracy in detecting RNA-modification containing sub-regions. It may be because compared with hm5C, the peaks generated from ac4C sites are narrower and have more accurate boundaries. This may be related to the experiment protocol (e.g. fragment size) and peak calling algorithms (e.g. sliding window size) used in their original studies.To demonstrate the ability of the proposed WeakRM algorithm in detecting sub-regions containing achm5C see , the mos4C and hm5C. We proposed here the first weakly supervised learning framework WeakRM for learning RNA modifications from low-resolution epitranscriptome datasets, such as those generated from hMeRIP-seq and acRIP-seq.Existing computational approaches for decoding the RNA modifications are mostly based on strong supervision and ideally require epitranscriptome data of base-resolution. Due to technical limitations, such data may not be available for some modifications, such as ac7G, hm5C and ac4C) and their respective sequencing technologies . We demonstrated that WeakRM substantially improved the prediction performance and applicable scope compared with existing approaches that were based on strong supervision. Importantly, our model captured sequence patterns that are consistent to the known motif detected by HOMOR software, and can vaguely identify regions containing the RNA modifications of interest. These results together demonstrated the generality and effectiveness of our approach for learning from low-resolution epitranscriptome data.We validated the proposed WeakRM method on three independent datasets, corresponding to three different RNA modification types  through XJTLU Key Programme Special Fund (KSF-P-02).Conflict of interest: none declared.btab278_Supplemenatry_DataClick here for additional data file.


The startle reflex in larval zebrafish describes a C-bend of the body occurring in response to sudden, unexpected, stimuli of different sensory modalities. Alterations in the startle reflex habituation (SRH) have been reported in various human and animal models of neurological and psychiatric conditions and are hence considered an important behavioural marker of neurophysiological function. The amplitude, offset and decay constant of the auditory SRH in larval zebrafish have recently been characterised, revealing that the measures are affected by variation in vibratory frequency, intensity, and interstimulus-interval. Currently, no study provides a model-based analysis of the effect of physical properties of light stimuli on the visual SRH. This study assessed the effect of incremental light-stimulus intensity on the SRH of larval zebrafish through a repeated-measures design. Their total locomotor responses were normalised for the time factor, based on the behaviour of a (non-stimulated) control group. A linear regression indicated that light intensity positively predicts locomotor responses due to larger SRH decay constants and offsets. The conclusions of this study provide important insights as to the effect of light properties on the SRH in larval zebrafish. Our methodology and findings constitute a relevant reference framework for further investigation in translational neurophysiological research. It can be induced by different sensory modalities, including the visual, auditory, and somatosensory9 and it is largely mediated by Mauthner interneurons8, although not exclusively10. Brief light flashes can cause C-starts with average latency of ~ 183 ms4, and their frequency  is proportional to contrast to baseline lighting levels4. Startle responses have also been probed using looming visual stimuli11. Dark light flashes can likewise trigger reflex turns (O-turns), although of prolonged latency (mean =  ~ 408 ms) and larger bend angle4 compared to classic visually evoked C-starts. Endogenous modulating factors of the startle reflex include arousal and stress levels13, which can be increased by sensitisation or fear-potentiation14 and decreased by pre-pulse inhibition16.The startle reflex in larval zebrafish is a widely studied evolutionary-preserved escape response to sudden/unexpected—potentially threatening—stimuli18. It has been studied in several animal species23 and humans24. Larval zebrafish SRH is reliably inducible, although not stereotypic , short-lasting (hours), or long-lasting (days), depending on how prolonged the exposure to stimulation is10. The SRH is comprised of peripheral and central components. The first is a sensory adaptation that involves the saturation of transducing cells after repeated exposure to the same sensory stimulus30. The central component of SRH is a habituation sensu stricto that reduces the motor reaction to sudden sensory stimuli, if regarded as non-threatening/relevant, despite being still processed at primary cortical level34. Both peripheral and central components of larval zebrafish SRH are induced by high-frequency stimuli35, while the peripheral component, i.e., sensory adaptation, may fail when longer inter-stimuli intervals ((ISIs), ≥ of 3 s) are utilised35. Alterations in startle reflex amplitude and SRH characterise several neuropsychiatric conditions41 and hence constitute important markers of neurophysiological function used in translational research .Startle reflex habituation (SRH) constitutes a basic, non-associative form of learning whereby repeated or continued exposure to an unexpected stimulus results in reduced motor responsivity4, although Beppi et al. in a recent study using the same stimuli found no such effect35. However, Burgess and Granato4 quantified the escape responses distinguishing ‘scoot’ (< 30–40°) and ‘R-turn’ (> 30–40°) initiations, observing that only the latter increase in frequency with stronger light intensities. In contrast, Beppi et al.35 did not distinguish scoot and R-turn initiations in their SRH quantifications and hence their global effect size might have been deflated. Moreover, Burgess and Granato4 pre-adapted the fish at ~ 135 Lux and tested them with light stimuli of ~ 1366 Lux , while Beppi et al.35 pre-adapted the fish at ~ 330 Lux and tested them with a step-increase in lighting ranging from 35 to 64%. Therefore, the authors suggested reapplying their experimental protocol with stronger light intensities to test whether this would result in findings consistent with the literature.Evidence suggests that startle response frequency of larval zebrafish to single white-light-flash stimuli depends on the magnitude-change in lighting35, reproducing their visual SRH study protocol using stronger lighting conditions (from 0.5 to 16 kLux), to test the effect of different luminance levels on the SRH of larval zebrafish of the same age. We hypothesised that the stimulated group would show a significantly larger mean total  distance travelled (TDT20) relative to the control group, increasing along with higher lux levels. We further hypothesised that such increase in TDT20 would be underlain by changes of exponential SRH parameters .This study aimed at following-up the study of Beppi et al.20 values for each group, at each lux level, are reported in Table 20 of the stimulated group was normalised for the effect of time, subtracting the free locomotor distance travelled by a control group, which was tested concomitantly to the experimental group at all the runs in the absence of additional light stimulation. The TDT20 distribution of both groups at all test-times approximately met the assumptions for single linear regression analysis, which was therefore performed to predict the change in TDT20 (relative to control) as an effect of light intensity (lux). The lux level significantly explained 5.1% of variance in mean TDT20, with F = 38.326, p < 0.001. A one-unit increase in lux causes a control-relative positive change in mean TDT20 of 0.794 . The predictive model is shown in Fig. A simple visual SRH task was applied repeatedly over 10 runs during which the larvae were stimulated at incrementing lux levels from 0.5 to 16 kLux 1 run = 20 stimuli). Plots of the groups’ mean distance travelled in each lux-level condition, cumulatively over successive stimuli, are shown in Fig. 0 stimuli11. The SRH is a basic non-associative form of learning, evidenced by a reduction in motor responsivity after repeated or continued exposure to a startling stimulus18. The SRH in larval zebrafish, although non-stereotypic, is reliably inducible25. Recently, Beppi et al.35 have proposed a first-order exponential model description of SRH in larval zebrafish, showing that its three main descriptive measures—amplitude, decay constant and offset—are sensitive to variation in vibratory frequency and intensity as well as ISI duration. However, no variability in such SRH measures was found as an effect of light stimulation within 0.4 kLux.The startle reflex in larval zebrafish is an evolutionary preserved escape response to potential threat, occurring in different sensory modalities35. A simple visual SRH task was conducted repeatedly in 10 runs stimulating at incremental kLux levels. We predicted an increase in TDT20 and associated changes in SRH parameters—amplitude, decay constant or offset—along with higher stimulatory lux levels. In line with our first hypothesis, the linear regression analysis revealed that light intensity was a significant positive predictor of locomotor responses. The stimulated group showed a modest—but highly significant—increase in TDT20 relative to the control group, along with higher light-stimuli intensity. The mean TDT20 value of the fish stimulated at the lowest 3 kLux levels was comparable to those of the control group, while an increase in TDT20 relative to control started to be visible at 4 kLux (~ 19%), increasing up to > 80% in the highest lux levels. Noticeably, when stimulated at 0.5 kLux, the fish showed a markedly lower amplitude of the exponential SRH fit compared to when tested at higher lux levels. This suggests that 0.5 kLux might not be sufficient for inducing a reliable SRH. This is further confirmed by unrealistically large decay constant for the 0.5 and 1 kLux levels, relative to the higher ones. It is hence recommendable for future SRH experiments in larval zebrafish  to stimulate with light flashes of at least 2 kLux.This study aimed at assessing the effect of light-stimulus intensities ≥ 0.5 kLux (with a step-increase in contrast from baseline lighting > 64%) on SRH of 72 larval zebrafish, based on the model-based analytical approach of Beppi et al.bleaching effect—generally defined as a reduction in light sensitivity of the photoreceptors  after extensive rhodopsin photoconversion45. Specifically to our case, if the fish are stimulated with high kLux levels , the retinal representation of the image might last longer (stronger sensory memory), presumably beyond the ISI duration, as if the fish were exposed to “permanent light”. This would cause an increasing (perceived) magnitude of subsequent stimuli—as a putative linear or asymptotic trend—in turns leading to a fictitious increase of offset and decay constant of the single exponential fit, as we could observe in our plots.The fitting of the locomotor data with a single exponential—with parameters amplitude, decay constant and offset—highlights a consistent trend of enlargement of the decay constants starting from 2000 towards higher lux levels. This effect is concomitant to an increase in offset along with higher lux levels  and a general reduction  in amplitude in the kLux levels beyond 4. The increasing of both offset and decay constant might be product of the retinal 47) would be valuable to monitor the visual function during the SRH task and for testing the validity of these assumptions. We therefore suggest its complementary use in future light-induced SRH studies in larval zebrafish.Our data did not allow the fitting of more complex functions than first-order exponentials. However, one could consider adding a low-pass filter on the fitted data to model the retinal receptor property causing subsequent stimuli to be perceived with increasing magnitude . The study and methods were conducted also in compliance with the ARRIVE guidelines of animal research.Danio rerio adult zebrafish were bred and maintained in accordance with standard protocols48. Their eggs were raised in transparent breeding tanks of 1.7 L water, incubated at 28 °C, under a 14:10 light–dark cycle. Upon reaching 3–4 DPF, they were moved into 35 × 12 mm cell-culture plates filled with E3 medium  and kept in the same location until the test day.Wild Indian Karyotype (WIK) The Viewpoint Zebrabox  behavioural recording system and software was used to track the locomotor responses of the animals in response to light stimulation. The light source consisted of a rectangular LED emitting white whole-field light flashes from beneath the 24-well plates.On the test day, zebrafish larvae of 92–96 h past fertilization (N = 144) were moved from the cell-culture plates and distributed in six 24-well plates filled with fresh E3 medium—half of which were randomly assigned to the control condition , and the remaining half to the experimental condition . The samples’ size provided sufficient statistical power. After an acclimatisation time of 90 min in the new plates, the testings took place. The experimental group performed the behavioural task at a given kLux level, while the control group was always tested at baseline  lighting level (~ 0.33 kLux) without additional light stimulation, for the same time duration of the experimental group’s task. Each of the six plates was tested consecutively, in its respective condition . The plates were tested with 1–2 min of time-lag between one another. All the groups performed the task ten times (10 runs). Each run consisted of a series of 20 identical white light flashes lasting 500 ms of a given kLux level with instantaneous onset/offset (step pattern). Each stimulus was interleaved by ISIs of 1000 ms, where the illuminance remained at baseline level. At each successive run, the kLux level of the experimental group incremented , while the control group always performed the no-stimulation protocol. The first run began at ~ 11 a.m. Each of the following runs were interleaved by 10–12 min from the previous. During all testings, the light exposure of the fish in the ZebraBox was kept at environmental levels for the purpose of maintaining their normal circadian rhythm and avoid affecting their arousal state, which could result in unwanted motility changes.35 in terms of locomotor distance travelled [mm] within the time frame of each stimulus (500 ms), for each fish of both groups. Movements that occurred during each ISI (1000 ms) were not quantified. The total distance travelled at all 20 stimuli (TDT20) was then computed for each fish of both groups. Lastly, the control-relative change in TDT20—e.g., mean of the control group subtracted from that of the stimulated group, for each respective kLux level—was linearly regressed to model its relationship with the illuminance level. Data bootstrapping  was applied—at the individual fish level—on the distance travelled over the stimuli by the experimental group (N = 72), at each test-time . This was done to obtain a “population-level” description of behaviour. Based on the SRH model described by Beppi et al.35, a first-order exponential (in-built MATLAB function: lsqcurvefit) was fitted into each individual bootstrap (N = 500), to extract individual offset, amplitude, and decay constant—the three main descriptive measures of startle habituation . These measures where then averaged, to obtain group-level mean measures.Startle responses were quantified based on Beppi et al.20 (outcome variable). The startle responses of 72 larval zebrafish were assessed and quantified in 10 runs of the experimental paradigm using incrementing kLux levels , against an age-matched control group (N = 72) that was not stimulated with additional (baseline-relative) light . A single linear regression was performed to model the relationship of the stimuli lighting (predictor variable) with the (baseline-relative) change in TDTe) light .Supplementary Information.


Germinal vesicle (GV) stage is a critical transition point from growth to maturation in mammalian oocyte development. During the following meiotic maturation, active RNA degradation and absence of transcription significantly reprofile the oocyte transcriptome to determine oocyte quality. Oocyte RNA-seq has revealed transcriptome differences between two defined phases of GV stage, namely non-surrounded nucleolus (NSN) and surrounded nucleolus (SN) phases. In addition, oocyte RNA-seq has identified a variety of dysregulated genes upon genetic mutation or environmental perturbation. Historically, due to the low amount of RNA per oocyte, a few (20–200) oocytes were needed for a regular library construction in bulk RNA-seq. In recent years, development of single cell sequencing allows detailing the transcriptome of individual oocytes. Here in this study, different RNA-seq datasets from single and bulk of mouse oocytes are compared, and single oocyte RNA-seq (soRNA-seq) shows higher reproducibility. In addition, soRNA-seq better illustrates developmental progression of GV oocytes, revealing more complex gene changes than traditional views. Specially, an elevated level of ribosomal RNA 5′-ETS  has been shown to highly correlate with SN property. This study further demonstrates that UMI (unique molecular identifiers) based and other deduplication methods are limited in their ability to improve the precision of the soRNA-seq datasets. Finally, this study proposes that external spike-in molecules are useful for normalizing samples of different transcriptome sizes. A list of stable genes has been identified during oocyte maturation that are comparable to external spike-in molecules. These findings highlight the advantage of soRNA-seq, and have established ways for better clustering and cross-stage normalization, which can provide more insight into the biological features of oocyte maturation. Oocyte meiotic maturation immediately determines oocyte quality, during which around 70% of oocyte RNA undergoes degradation . AblatioSingle cell RNA-seq has revolutionized investigations of transcriptomes that transform cell fate during development. As a single cell, the mammalian fully-grown oocyte is unique in its large size, uniform morphology, high nucleic acid content and numerous types of RNA. Since single oocyte RNA-seq (soRNA-seq) was introduced, it has become increasingly popular for multi-omic studies in mice . These aThe current study firstly aimed to do a thorough comparison among single and bulk oocyte RNA-seq datasets. By pairwise analyses of several published RNA-seq datasets, soRNA-seq exhibited higher reproducibility. Secondly, the variation of soRNA-seq data were shown to reveal more complex transcriptome differences of morphologically similar GV oocytes  than previous views. Interestingly, a strong correlation of 5′-ETS elevation with potential SN phase has been discovered. Thirdly, by performing multiple deduplication strategies, there was high similarity in downstream analyses either with or without deduplication. Finally, it was demonstrated that external spike-in molecules, such as ERCC , can effectively account for transcriptome size changes during oocyte maturation, during which active RNA degradation takes place without transcription. For situations when ERCC is not available, a group of stably transcribed genes (constGenes) during oocyte maturation was extracted, which provided high similarity to ERCC for cross-stage normalization. This normalization allows greater appreciation of oocyte heterogeneity at GV stage and across maturation. These observations shed light on future oocyte transcriptomic studies.Ovaries were dissected in PBS, and transferred into M2 medium  plus milrinone (2.5 μM). The ovaries were pierced mechanically with a 30-gauge needle to release oocytes and only fully-grown oocytes (nuclear envelope-intact oocytes) were collected for further experiments.F1 female at 12 weeks old were collected and transferred individually into 2.5 μl RLT Plus (Qiagen) and stored at −80°C. A single GV oocyte lysis was diluted 1:2, 1:4 and 1:8 which represented fractions of oocytes. In total, eight oocytes were used for all libraries, including six for different amplification cycles and two for fractional oocytes. To prepare libraries for sequencing, 1 μl of the 105-fold diluted ERCC spike-in mix  was added to 2.5 μl of each single or fractional oocyte sample. Poly(A) RNA was isolated by oligo (dT) beads, reverse transcribed, amplified and purified  of mouse GV oocyte RNA-seq libraries were prepared according to a published pipeline with minor modifications . Brieflypurified . Differe15 published mouse oocyte RNA-seq datasets, including 121 libraries (containing bulk and single) are used in the current study . Only wiMus musculus GRCm38 genome plus ERCC. fasta using STAR (v2.7.8a) to get the Bam files.SRA files of all datasets  were dowThe Bam files were processed differently for downstream analyses in Original, Dedup and Picard groups. For the Original group, reads were counted using HTSeq (v0.11.4) with default parameters. For the Dedup reads group, before reads counting, the Bam files were processed for deduplication using the UMI index reads and alignments according to the Ovation Solo RNA-seq manual v4 (python nudup. py). For the Picard reads group, the Bam files were pre-processed by Picard tools (v2.25.7) to mark and remove the duplicated reads without implementing the UMI index reads. The deduplicated Bam files were then counted using HTSeq (v0.11.4) with default parameters. Count files were used for differential analysis.P-adjust < 0.01. Principal component analysis and k-means clustering were performed using the factoextra R package (v1.0.3). In all k-means clustering, the value of nstart was set as 25 to get the best one among 25 initial configurations.Differential expression between groups was analyzed using R (v3.5.1) with DESeq2 (v1.24.0) using default parameters. A gene/ERCC was considered valid to be included in differential analysis when it had at least five reads in at least two libraries. In all cases, significantly changed genes are defined as When ERCC molecules or constGenes were used for normalization, the counts of the ERCC molecules or constGenes were provided as the “controlGenes” for estimating the size factors. Otherwise, the default estimation method (median ratio) was used.The regression analyses of gene expression levels and ERCC molecules were performed by R (v3.5.1). For example, in GSE141190  containsTo estimate the reproducibility of mouse oocyte RNA-seq, I obtained all published datasets from single or bulk mouse oocytes at GV  stage, which is the beginning of oocyte maturation  Supplem. All rawFully grown mouse oocytes are highly uniform in their morphology and size. However, there are two different populations regarding nuclear configuration, developmental potential and transcriptional activity, namely non-surrounded nucleolus (NSN) and surrounded nucleolus (SN) oocytes . DefinitTo document this, I analyzed all published soRNA-seq datasets -based soRNA-seq datasets  to allow more powerful analysis . Based oThen I sought to perform k-means clustering by defining cluster number as 3. Interestingly, the original SN-High cluster could be further divided into two subsets, which were named as SN-High2 and SN-Mid according to the level of SN-featured genes . PresumaIn eukaryotic cells, a primary rRNA transcript contains both mature rRNA subunit fragments, namely 18, 5.8 and 28S rRNA, and the Internal/External Transcribed Spacers, namely ITS1, ITS2 and 5′-ETS, 3′-ETS  Henras . MultiplMaterials and Methods), both of which contain EXOSC10 depleted oocytes and control oocytes. For both datasets, I extracted all reads that are mapped to rDNA genomic sequence and ranked the samples according to the 5′-ETS coverage  soRNA-seq . In each soRNA-seq dataset, I profiled the 5′-ETS coverage of all libraries in the PCA plot (I then made use of the four poly (A) soRNA-seq datasets . Due to the non-specific spike signals in the 5′-ETS, a region  was selected to calculate the 5′-ETS coverage, which in most datasets represent the residual 5′-ETS fragments  when comparing Original and Dedup at the same stage  were sensitive to deduplication , five from GSE96638 poly (A) and 11 from GSE141190 RiboMinus . When coTo better illustrate ERCC normalization, I performed poly (A) RNA-seq of GV oocytes using different fractional amounts  of whole oocytes. The differential analysis also confirmed the overall smaller library sizes of the fractional GV oocytes compared to the intact GV oocyte . Thus, ITo allow cross-stage comparison of sequencing samples when ERCC is unavailable, I have extracted a set of 147 constant genes (constGenes) during oocyte maturation. These 147 genes exhibit little change (less than 50%) in transcript abundance from GV to MII whether obtained by poly(A) RNA-seq or RiboMinus RNA-seq . The conIn summary, based on higher reproducibility and known genetic/developmental heterogeneity, single cell RNA-seq appears to be a better method for transcriptome analyses of mouse oocytes. When an overall change of transcriptome size is anticipated, external spike-in or the constGenes are recommended for normalization to provide better insight into biological changes of the transcriptome.This study has compared currently available mouse GV oocyte RNA-seq datasets and concluded that soRNA-seq has high reproducibility and reveals better details of developmental progression. In addition, this study has also established external spike-in or constant genes normalization method for cross-stage comparison, and suggests that deduplication of the single oocyte RNA-seq datasets does not significantly alter the downstream analyses.A fully grown oocyte has much larger size and more RNA content compared to a somatic cell. This feature makes a single oocyte a unique type of single cell, which is more like a “bulk” status. For example, a regular somatic cell could capture around a few thousand genes using 10x genomic sequencing , or arouUnlike different cell types detected in a tissue-derived single cell RNA-seq, single oocytes have almost identical morphology and size. Thus, the heterogeneity comes mostly from developmental variation or potential quality difference, which has been largely neglected in bulk RNA-seq. By integrated analysis of all soRNA-seq datasets, genes that undergo increase or decrease during early or late phases of GV oocytes have been discovered, which provide more information than the NSN and SN-featured genes identified by bulk RNA-seq. Presumably, under conditions that could cause more variation, such as chemical treatment or genetic perturbation, employing soRNA-seq can better illustrate the spectrum of changes in oocytes. In addition, I observed rRNA 5′-ETS level exhibits high correlation with SN property, which may be caused by both rRNA transcription during meiotic prophase I and the different processing mechanisms of individual rRNA fragments. Given the extremely low level of rRNA 5′-ETS in a cell, it is hard to setup a threshold to define SN phase. However, when multiple single oocytes are compared, the earlier and later stages could be very well aligned based on their rRNA 5′-ETS levels.In summary, this study demonstrates the advantage of soRNA-seq. I have also proposed rRNA 5′-ETS as a new marker for SN oocytes and established constant genes-mediated cross-stage normalization.


Deltavirus. HDV is a satellite virus and depends on the hepatitis B virus (HBV) for packaging, release, and transmission. Extracellular HDV virions contain the genomic HDV RNA, a single-stranded negative-sense and covalently closed circular RNA molecule, which is associated with the HDV-encoded delta antigen forming a ribonucleoprotein complex, and enveloped by the HBV surface antigens. Replication occurs in the nucleus and is mediated by host enzymes and assisted by cis-acting ribozymes allowing the formation of monomer length molecules which are ligated by host ligases to form unbranched rod-like circles. Recently, meta-transcriptomic studies investigating various vertebrate and invertebrate samples identified RNA species with similarities to HDV RNA. The delta-like agents may be representatives of novel subviral agents or satellite viruses which share with HDV, the self-complementarity of the circular RNA genome, the ability to encode a protein, and the presence of ribozyme sequences. The widespread distribution of delta-like agents across different taxa with considerable phylogenetic distances may be instrumental in comprehending their evolutionary history by elucidating the transition from transcriptome to cellular circular RNAs to infectious subviral agents.Hepatitis delta virus (HDV) is a human pathogen, and the only known species in the genus  Deltavirus  is a unique human pathogen, and has been the only known species in the genus ltavirus , but wasozyviria . Due to The existence of HDV was discovered in 1977 by the identification of a new antigen, the delta antigen (HDAg), in liver biopsies and sera from patients with a severe form of hepatitis B . Experimde novo synthesized genomic and antigenomic RNA molecules are processed into monomers by intrinsic ribozymes, which are formed by nested double pseudoknot structures  contain additional N-terminal extensions of the S-domain. In particular, the dual topology of the preS1 domain of HBsAg-L is essential for hepatitis B virion assembly and attachment to the host cell. The preS1 domain of newly synthesized HBsAg-L proteins are on the cytosolic side and interact with the hepatitis B nucleocapsid allowing virion assembly. During the HBV budding process, the HBsAg-L preS1 domain translocates across the viral lipid layer to be surface exposed , which iThe dynamics of HDV infections following orthotopic liver transplantations provided evidence that HDV can cause subclinical helper-independent or mono-infections  but HDV A cell culture based study showed that HDV can be pseudotyped with envelope proteins derived from various viruses, including vesicular stomatitis virus (VSV), hepatitis C virus (HCV), and Dengue virus. The VSV and HCV envelope proteins supported the release and assembly of genomic HDV RNA, also depending on HDAg-L farnesyl-mediated targeting of cell membranes similar to the assembly process supported by HBV envelope proteins . The HDVOrthohepadnavirus), such as the woodchuck hepatitis B virus (WHV) as alternative helper viruses for HDV assembly and transmission allowed the establishment of animal models to study HDV replication and pathogenesis , canary (Serinus canaria), Zebra finch (Taeniopygia guttata) and white-tailed deer (Odocoileus virginianus), the read depths of predicted transcribed regions (the coding region for the delta-like antigen) were greater than those of other genomic regions indicating that most delta reads were derived from delta mRNAs suggesting that the novel delta-like agents replicate in their hosts  is the host for the woodchuck hepatitis B virus (WHV) which is an efficient helper virus providing the envelope proteins for the assembly of infectious HDV in the woodchuck animal model , woodchuck (Marmota monax), and one of the bat (Desmodus rotundus) genotypes of delta-like agents were found to form a sister clade with the human HDV (Desmodus rotundus) genotype, rodent (Proechimys semispinosus), snake (Boa constrictor) and duck delta-like agents, while the remaining delta-like agents formed a more distal clade . However, despite the low level of identity between the HDAg protein sequences of delta-like agents from different taxa (14–67%)  , no clea(14–67%) .(i) Delta-Like Agents and Cellular Circular RNAs as Potential Precurors of Subviral Agents.Viroids were the first circular RNA discovered , then foProechimys semispinosus)   and in cpinosus)  contain pinosus) . Importapinosus)  but the pinosus) . In contpinosus) . The sDLpinosus) . Rodent pinosus) . It was pinosus) . Hepatitpinosus) , and imppinosus) . Similarpinosus) , and HBVpinosus) . CircRNApinosus) , and henpinosus) . The exapinosus) .Both the sDLAs and rDLAs studies demonstrated the expression of the corresponding DL-Ags confirming that the ORF is translated . Transfe(ii) HDV and Its RibozymeCPEB3) gene and is possibly involved in co-transcriptional processing of CPEB3 primary RNA transcripts   ; 2011. Tctively) . The prectively) . Translactively) . The intctively) . Similaronic RNA ; 2011. Ionic RNA  and henconic RNA , 2011. Sonic RNA . The HDVonic RNA . The widonic RNA , the exi(iii) HDV and ViroidsPospiviroidae) or Y-shaped or branched structures (viroids of the family Avsunviroidae). The viroids replicate in two different cellular compartments, the nucleus for the Pospiviroidae, and the chloroplast for the Asunviroidae. HDV replication is restricted to the nucleus (Avsunviridae replicate by a symmetric rolling circle cycle generating (+) and complement (-) circles, similar to the HDV replication cycle producing circular genomic and antigenomic HDV RNA molecules. This is in contrast to the asymmetric replication cycle of viroids of the family Pospiviroidae, which generate only (+) circles followed by cleavage of oligomeric strands by a host RNase III  circular RNA species are abundant in eukaryotic cells, (ii) HDV-like ribozymes are present in highly divergent organisms with important roles in many molecular pathways, and (iii) delta-like circular RNA agents are found in vertebrates and invertebrates, suggest that HDV is derived from the cellular transcriptome. Delta-like agents have not yet been identified in non-human primates strongly indicating that a transspecies transmission event of a delta-like agent into a HBV-infected human host allowed a host shift and the emergence of HDV. The HDV sequence diversity is possibly a consequence of human migration and geographic vicariance.The author HJN of this publication has an equity interest in, and serves as a consultant to ClearB Therapeutics. ClearB Therapeutics had no role in the writing of the article.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The author HJN has an equity interest in, and serves as a consultant to ClearB Therapeutics. ClearB Therapeutics had no role in the writing of the article. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.