Double-stranded DNA Cleavage Research Articles

Enhancing Salivary DNA Preservation via DNase I Inactivation: Role of Temperature and EDTA

Background Deoxyribonuclease I (DNase I) catalyzes the cleavage of double-stranded DNA into its end products. This enzyme requires cations for its activity. In addition, DNase activity is influenced by indirect DNase inhibitors (i.e. EDTA) and temperature. EDTA is an indirect DNase I inhibitor. Therefore, DNase inactivation is crucial for DNA preservation, especially for salivary samples, and the downstream application of genetic techniques using non-blood samples. Saliva is a complex mixture contaminated with oral microbes and contains DNase which is harmful for DNA preservation. This study aims to preserve salivary DNA through temperature and EDTA treatment. Methods This is an experimental study. Salivary DNA was extracted using the spin-column method. DNA degradation assays were carried out using either 5 µL of salivary supernatant or 2.5 mg/mL of DNase I which were incubated at -20°C, 2-8°C, room temperature, 40°C, and 50°C; and added with 0.125 mM, 0.25 mM, 0.5 mM, 1 mM, and 2 mM EDTA for 60 minutes. After incubation, the results were visualized using agarose gel electrophoresis. Results The average concentration of salivary DNA was 61.70 µg/mL (30.50-109.05) and the purity was 1.893 µg/mL (1.800-2.005). Incubation at -20°C, 2-8°C, and room temperature degraded salivary DNA but incubation at 40 and 50°C did not completely degrade DNA. Addition 0.125 mM, 0.25 mM, 0.5 mM, 1 mM, and 2 mM EDTA to 5 µL of salivary supernatant or 2.5 mg/mL DNase I preserved salivary DNA. Conclusions Temperature of 40 and 50°C, and 0.125 mM EDTA can inactivate DNase and preserve salivary DNA.

Interactions between Zoliflodacin and Neisseria gonorrhoeae Gyrase and Topoisomerase IV: Enzymological Basis for Cellular Targeting.

Gyrase and topoisomerase IV are the cellular targets for fluoroquinolones, a critically important class of antibacterial agents used to treat a broad spectrum of human infections. Unfortunately, the clinical efficacy of the fluoroquinolones has been curtailed by the emergence of target-mediated resistance. This is especially true for Neisseria gonorrhoeae, the causative pathogen of the sexually transmitted infection gonorrhea. Spiropyrimidinetriones (SPTs), a new class of antibacterials, were developed to combat the growing antibacterial resistance crisis. Zoliflodacin is the most clinically advanced SPT and displays efficacy against uncomplicated urogenital gonorrhea in human trials. Like fluoroquinolones, the primary target of zoliflodacin in N. gonorrhoeae is gyrase, and topoisomerase IV is a secondary target. Because unbalanced gyrase/topoisomerase IV targeting has facilitated the evolution of fluoroquinolone-resistant bacteria, it is important to understand the underlying basis for the differential targeting of zoliflodacin in N. gonorrhoeae. Therefore, we assessed the effects of this SPT on the catalytic and DNA cleavage activities of N. gonorrhoeae gyrase and topoisomerase IV. In all reactions examined, zoliflodacin displayed higher potency against gyrase than topoisomerase IV. Moreover, zoliflodacin generated more DNA cleavage and formed more stable enzyme-cleaved DNA-SPT complexes with gyrase. The SPT also maintained higher activity against fluoroquinolone-resistant gyrase than topoisomerase IV. Finally, when compared to zoliflodacin, the novel SPT H3D-005722 induced more balanced double-stranded DNA cleavage with gyrase and topoisomerase IV from N. gonorrhoeae, Escherichia coli, and Bacillus anthracis. This finding suggests that further development of the SPT class could yield compounds with a more balanced targeting against clinically important bacterial infections.

TracrRNA reprogramming enables direct PAM-independent detection of RNA with diverse DNA-targeting Cas12 nucleases

Many CRISPR-Cas immune systems generate guide (g)RNAs using trans-activating CRISPR RNAs (tracrRNAs). Recent work revealed that Cas9 tracrRNAs could be reprogrammed to convert any RNA-of-interest into a gRNA, linking the RNA’s presence to Cas9-mediated cleavage of double-stranded (ds)DNA. Here, we reprogram tracrRNAs from diverse Cas12 nucleases, linking the presence of an RNA-of-interest to dsDNA cleavage and subsequent collateral single-stranded DNA cleavage—all without the RNA necessarily encoding a protospacer-adjacent motif (PAM). After elucidating nuclease-specific design rules, we demonstrate PAM-independent RNA detection with Cas12b, Cas12e, and Cas12f nucleases. Furthermore, rationally truncating the dsDNA target boosts collateral cleavage activity, while the absence of a gRNA reduces background collateral activity and enhances sensitivity. Finally, we apply this platform to detect 16 S rRNA sequences from five different bacterial pathogens using a universal reprogrammed tracrRNA. These findings extend tracrRNA reprogramming to diverse dsDNA-targeting Cas12 nucleases, expanding the flexibility and versatility of CRISPR-based RNA detection.

Why Does the E1219V Mutation Expand T-Rich PAM Recognition in Cas9 from Streptococcus pyogenes?

Popular RNA-guided DNA endonuclease Cas9 from Streptococcus pyogenes (SpCas9) recognizes the canonical 5'-NGG-3' protospacer adjacent motif (PAM) and triggers double-stranded DNA cleavage activity. Mutations in SpCas9 were demonstrated to expand the PAM readability and hold promise for therapeutic and genome editing applications. However, the energetics of the PAM recognition and its relation to the atomic structure remain unknown. Using the X-ray structure (precatalytic SpCas9:sgRNA:dsDNA) as a template, we calculated the change in the PAM binding affinity in response to SpCas9 mutations using computer simulations. The E1219V mutation in SpCas9 fine-tunes the water accessibility in the PAM binding pocket and promotes new interactions in the SpCas9:noncanonical T-rich PAM, thus weakening the PAM stringency. The nucleotide-specific interaction of two arginine residues (i.e., R1333 and R1335 of SpCas9) ensured stringent 5'-NGG-3' PAM recognition. R1335A substitution (SpCas9R1335A) completely disrupts the direct interaction between SpCas9 and PAM sequences (canonical or noncanonical), accounting for the loss of editing activity. Interestingly, the double mutant (SpCas9R1335A,E1219V) boosts DNA binding affinity by favoring protein:PAM electrostatic contact in a desolvated pocket. The underlying thermodynamics explain the varied DNA cleavage activity of SpCas9 variants. A direct link between the energetics, structures, and activity is highlighted, which can aid in the rational design of improved SpCas9-based genome editing tools.

Biofouling mitigation of Nb2AlC and Mo3AlC2 MXene-precursors doped polyether sulfone mixed matrix membranes for pathogen microorganisms

This study explores the incorporation of Nb2AlC and Mo3AlC2 MAX phases, known for their nano-layered structure, into polyether sulfone (PES) membranes to enhance their antifouling and permeability properties for pathogen microorganism filtration against bovine serum albumin (BSA) and Escherichia coli (E. coli). The composite membranes were characterized for their structural and morphological properties, and their performance in mitigating biofouling was evaluated. The structural characterizations have been performed for all the prepared MAX phases and corresponding composite membranes. The antioxidant ability of Nb2AlC and Mo3AlC2 MAX phases was defined by the DPPH radical scavenging assay, and the highest antioxidant ability was found to be 59.35 %, while 53.69 % scavenging potential was recorded at 100 mg/L. The percentage scavenging ability was raised with an increase in concentrations. The antimicrobial properties of MAX phases, evaluated as the minimum inhibitory concentration, were stated against several pathogen microorganisms. The tested compounds of Nb2AlC and Mo3AlC2 composites containing MAX phases exhibited excellent chemical nuclease activity, and it was determined that Nb2AlC caused double strand DNA cleavage activity while Mo3AlC2 induced the complete fragmentation of the DNA molecule. Biofilm inhibition of Nb2AlC and Mo3AlC2 MAX phases was studied against Staphylococcus aureus, and Pseudomonas aeruginosa and the maximum biofilm inhibition of Nb2AlC and Mo3AlC2 MAX phases was found to be 77.15 % and 69.07 % against S. aureus and also 69.74 % and 65.01 % against P. aeruginosa. Furthermore, Nb2AlC and Mo3AlC2 MAX phases demonstrated excellent E. coli growth inhibition of 100 % at 125 and 250 mg/L.

C-to-G editing generates double-strand breaks causing deletion, transversion and translocation.

Base editors (BEs) introduce base substitutions without double-strand DNA cleavage. Besides precise substitutions, BEs generate low-frequency 'stochastic' byproducts through unclear mechanisms. Here, we performed in-depth outcome profiling and genetic dissection, revealing that C-to-G BEs (CGBEs) generate substantial amounts of intermediate double-strand breaks (DSBs), which are at the centre of several byproducts. Imperfect DSB end-joining leads to small deletions via end-resection, templated insertions or aberrant transversions during end fill-in. Chromosomal translocations were detected between the editing target and off-targets of Cas9/deaminase origin. Genetic screenings of DNA repair factors disclosed a central role of abasic site processing in DSB formation. Shielding of abasic sites by the suicide enzyme HMCES reduced CGBE-initiated DSBs, providing an effective way to minimize DSB-triggered events without affecting substitutions. This work demonstrates that CGBEs can initiate deleterious intermediate DSBs and therefore require careful consideration for therapeutic applications, and that HMCES-aided CGBEs hold promise as safer tools.

CRISPR-Cas9 Activities with Truncated 16-Nucleotide RNA Guides Are Tuned by Target Duplex Stability Beyond the RNA/DNA Hybrid.

CRISPR-Cas9 has been adapted as a readily programmable genome manipulation agent, and continuing technological advances rely on an in-depth mechanistic understanding of Cas9 target discrimination. Cas9 interrogates a target by unwinding the DNA duplex to form an R-loop, where the RNA guide hybridizes with one of the DNA strands. It has been shown that RNA guides shorter than the normal length of 20-nucleotide (-nt) support Cas9 cleavage activity by enabling partial unwinding beyond the RNA/DNA hybrid. To investigate whether DNA segment beyond the RNA/DNA hybrid can impact Cas9 target discrimination with truncated guides, Cas9 double-stranded DNA cleavage rates (kcat) were measured with 16-nt guides on targets with varying sequences at +17 to +20 positions distal to the protospacer-adjacent-motif (PAM). The data reveal a log-linear inverse correlation between kcat and the PAM+(17-20) DNA duplex dissociation free energy (ΔGNN(17-20)0), with sequences having smaller ΔGNN(17-20)0 showing faster cleavage and a higher degree of unwinding. The results indicate that, with a 16-nt guide, "peripheral" DNA sequences beyond the RNA/DNA hybrid contribute to target discrimination by tuning the cleavage reaction transition state through the modulation of PAM-distal unwinding. The finding provides mechanistic insights for the further development of strategies that use RNA guide truncation to enhance Cas9 specificity.

Actions of a Novel Bacterial Topoisomerase Inhibitor against Neisseria gonorrhoeae Gyrase and Topoisomerase IV: Enhancement of Double-Stranded DNA Breaks.

Novel bacterial topoisomerase inhibitors (NBTIs) are an emerging class of antibacterials that target gyrase and topoisomerase IV. A hallmark of NBTIs is their ability to induce gyrase/topoisomerase IV-mediated single-stranded DNA breaks and suppress the generation of double-stranded breaks. However, a previous study reported that some dioxane-linked amide NBTIs induced double-stranded DNA breaks mediated by Staphylococcus aureus gyrase. To further explore the ability of this NBTI subclass to increase double-stranded DNA breaks, we examined the effects of OSUAB-185 on DNA cleavage mediated by Neisseria gonorrhoeae gyrase and topoisomerase IV. OSUAB-185 induced single-stranded and suppressed double-stranded DNA breaks mediated by N. gonorrhoeae gyrase. However, the compound stabilized both single- and double-stranded DNA breaks mediated by topoisomerase IV. The induction of double-stranded breaks does not appear to correlate with the binding of a second OSUAB-185 molecule and extends to fluoroquinolone-resistant N. gonorrhoeae topoisomerase IV, as well as type II enzymes from other bacteria and humans. The double-stranded DNA cleavage activity of OSUAB-185 and other dioxane-linked NBTIs represents a paradigm shift in a hallmark characteristic of NBTIs and suggests that some members of this subclass may have alternative binding motifs in the cleavage complex.

P07 Optimizing DNA specificity and applicability of base and prime editors on COL7A1 variants causing recessive dystrophic epidermolysis bullosa

Abstract Base and prime editors are novel CRISPR variants that reduce the risk of off-target effects by avoiding double-stranded DNA cleavage, bypassing the need for homology-directed repair. These tools offer great therapeutic potential for inherited skin diseases such as recessive dystrophic epidermolysis bullosa (RDEB). Biallelic loss-of-function mutations in COL7A1 cause RDEB, resulting from loss of functional type VII collagen (C7) in the basement membrane zone. Current therapies for RDEB are limited, but ex vivo and in vivo COL7A1 base or prime editing therapies can potentially restore normal protein production with a single intervention treatment. This study compared DNA editing specificity, safety and efficiency on RDEB fibroblasts harbouring a homozygous c.4448 G>A (p.Gly1483Asp) mutation in COL7A1. Firstly, transfected adenine base editor (ABE8e) mRNA together with single guide RNA (sgRNA) were introduced into patient primary fibroblasts with Lipofectamine MessengerMax. By varying concentrations and ratios of guide RNA and ABE8e mRNA, we have achieved 100% biallelic correction without introducing any bystander mutations using 200 ng ABE8e and 28 ng sgRNA by transfecting 100 000 cells. Initial prime editing experiments are underway with PEmax plasmid and engineered prime editing guide RNAs (epegRNA) to optimize the guide design and to compare PE2max and PE4max editing systems. The functionality of the 100% biallelic correction using ABE8e is being evaluated with Western blot and in three-dimensional skin models. These early experiments demonstrate the potential value of base editing as a gene-editing tool in RDEB therapeutics; however, more functional work and off-target analyses are needed.

Exogenous DNA enhances DUOX2 expression and function in human pancreatic cancer cells by activating the cGAS-STING signaling pathway

Pro-inflammatory cytokines upregulate the expression of the H2O2-producing NADPH oxidase dual oxidase 2 (DUOX2)2 which, when elevated, adversely affects survival from pancreatic ductal adenocarcinoma (PDAC). Because the cGAS-STING pathway is known to initiate pro-inflammatory cytokine expression following uptake of exogenous DNA, we examined whether activation of cGAS-STING could play a role in the generation of reactive oxygen species by PDAC cells. Here, we found that a variety of exogenous DNA species markedly increased the production of cGAMP, the phosphorylation of TBK1 and IRF3, and the translocation of phosphorylated IRF3 into the nucleus, leading to a significant, IRF3-dependent enhancement of DUOX2 expression, and a significant flux of H2O2 in PDAC cells. However, unlike the canonical cGAS-STING pathway, DNA-related DUOX2 upregulation was not mediated by NF-κB. Although exogenous IFN-β significantly increased Stat1/2-associated DUOX2 expression, intracellular IFN-β signaling that followed cGAMP or DNA exposure did not itself increase DUOX2 levels. Finally, DUOX2 upregulation subsequent to cGAS-STING activation was accompanied by the enhanced, normoxic expression of HIF-1α and VEGF-A as well as DNA double strand cleavage, suggesting that cGAS-STING signaling may support the development of an oxidative, pro-angiogenic microenvironment that could contribute to the inflammation-related genetic instability of pancreatic cancer.

Powerful Microbial Base-Editing Toolbox: From Optimization Strategies to Versatile Applications.

Base editors (BE) based on CRISPR systems are practical gene-editing tools which continue to drive frontier advances of life sciences. BEs are able to efficiently induce point mutations at target sites without double-stranded DNA cleavage. Hence, they are widely employed in the fields of microbial genome engineering. As applications of BEs continue to expand, the demands for base-editing efficiency, fidelity, and versatility are also on the rise. In recent years, a series of optimization strategies for BEs have been developed. By engineering the core components of BEs or adopting different assembly methods, the performance of BEs has been well optimized. Moreover, series of newly established BEs have significantly expanded the base-editing toolsets. In this Review, we will summarize the current efforts for BE optimization, introduce several novel BEs with versatility, and look forward to the broadened applications for industrial microorganisms.

In vitro characterization of a pAgo nuclease TtdAgo from Thermococcus thioreducens and evaluation of its effect in vivo.

In spite of the development of genome-editing tools using CRISPR-Cas systems, highly efficient and effective genome-editing tools are still needed that use novel programmable nucleases such as Argonaute (Ago) proteins to accelerate the construction of microbial cell factories. In this study, a prokaryotic Ago (pAgo) from a hyperthermophilic archaeon Thermococcus thioreducens (TtdAgo) was characterized in vitro. Our results showed that TtdAgo has a typical DNA-guided DNA endonuclease activity, and the efficiency and accuracy of cleavage are modulated by temperature, divalent ions, and the phosphorylation and length of gDNAs and their complementarity to the DNA targets. TtdAgo can utilize 5'-phosphorylated (5'-P) or 5'- hydroxylated (5'-OH) DNA guides to cleave single-stranded DNA (ssDNA) at temperatures ranging from 30°C to 95°C in the presence of Mn2+ or Mg2+ and displayed no obvious preference for the 5'-end-nucleotide of the guide. In addition, single-nucleotide mismatches had little effects on cleavage efficiency, except for mismatches at position 4 or 8 that dramatically reduced target cleavage. Moreover, TtdAgo performed programmable cleavage of double-stranded DNA at 75°C. We further introduced TtdAgo into an industrial ethanologenic bacterium Zymomonas mobilis to evaluate its effect in vivo. Our preliminary results indicated that TtdAgo showed cell toxicity toward Z. mobilis, resulting in a reduced growth rate and final biomass. In conclusion, we characterized TtdAgo in vitro and investigated its effect on Z. mobilis in this study, which lays a foundation to develop Ago-based genome-editing tools for recalcitrant industrial microorganisms in the future.

The effect of DNA tension on CRISPR/Cas12a cleavage can be reversible.

CRISPR/Cas12a (known as Cpf1) is a class 2 type V endonuclease which offers a two-component system for double stranded DNA cleavage. Cas12a is the enzymatic component with an active nuclease domain (RuvC) for cleaving the non-target and target DNA strands in order. CRISPR RNA (crRNA) is the RNA component which is initially processed by Cas12a and later associates with the Cas12a enzyme for the target detection. After Cas12a and crRNA are associated, they form a stable Cas12a/crRNA complex to interrogate the DNA target. Once the complementarity of the guide RNA and the target DNA is maintained, DNA cleavage occurs. Like other CRISPR enzymes, Cas12a is mainly derived from bacteria. However, unlike prokaryotic DNA, eukaryotic DNA faces mechanical stress. For example, DNA stretching and torsional stress can occur during various cellular processes that involve DNA compaction and extraction. As previously reported, the mechanical tension on the DNA reduces or inhibits the CRISPR/Cas12a cleavage reaction. Yet, it remains unknown that if Cas12a cleavage inhibition by tension is reversible. Here I show that the inhibited DNA cleavage can be rescued when the tension is relieved. I employed a high-throughput single-molecule technique to monitor the effect of stretching on hundreds of the target DNA in the same microchamber. The target DNA molecules were tethered to the surface and labeled with magnetic beads. Cas12a/crRNA complexes were introduced when the target DNA was stretched. Later, the target DNA was relaxed by removing the magnetic field. Shortly after this step, I observed the cleavage process of the relaxed DNA tethers. This observation has many implications for the CRISPR/Cas12a gene-editing technique. As it can explain the heterogenous targeting efficiencies reported among the cell cultures with the same guide RNA and targeting strategy.

Cas12a2 elicits abortive infection through RNA-triggered destruction of dsDNA

Bacterial abortive-infection systems limit the spread of foreign invaders by shutting down or killing infected cells before the invaders can replicate1,2. Several RNA-targeting CRISPR–Cas systems (that is, types III and VI) cause abortive-infection phenotypes by activating indiscriminate nucleases3–5. However, a CRISPR-mediated abortive mechanism that leverages indiscriminate DNase activity of an RNA-guided single-effector nuclease has yet to be observed. Here we report that RNA targeting by the type V single-effector nuclease Cas12a2 drives abortive infection through non-specific cleavage of double-stranded DNA (dsDNA). After recognizing an RNA target with an activating protospacer-flanking sequence, Cas12a2 efficiently degrades single-stranded RNA (ssRNA), single-stranded DNA (ssDNA) and dsDNA. Within cells, the activation of Cas12a2 induces an SOS DNA-damage response and impairs growth, preventing the dissemination of the invader. Finally, we harnessed the collateral activity of Cas12a2 for direct RNA detection, demonstrating that Cas12a2 can be repurposed as an RNA-guided RNA-targeting tool. These findings expand the known defensive abilities of CRISPR–Cas systems and create additional opportunities for CRISPR technologies.

Two distinct rotations of bithiazole DNA intercalation revealed by direct comparison of crystal structures of Co(III)•bleomycin A2 and B2 bound to duplex 5′-TAGTT sites

Bleomycins constitute a family of anticancer natural products that bind DNA through intercalation of a C-terminal tail/bithiazole moiety and hydrogen-bonding interactions between the remainder of the drug and the minor groove. The clinical utility of the bleomycins is believed to result from single- and double-strand DNA cleavage mediated by the HOO-Fe(III) form of the drug. The bleomycins also serve as a model system to understand the nature of complex drug-DNA interactions that may guide future DNA-targeted drug discovery. In this study, the impact of the C-terminal tail on bleomycin-DNA interactions was investigated. Toward this goal, we determined two crystal structures of HOO-Co(III)•BLMA2 “green” (a stable structural analogue of the active HOO-Fe(III) drug) bound to duplex DNA containing 5′-TAGTT, one in which the entire drug is bound (fully bound) and a second with only the C-terminal tail/bithiazole bound (partially bound). The structures reported here were captured by soaking HOO-Co(III)•BLMA2 into preformed host–guest crystals including a preferred DNA-binding site. While the overall structure of DNA-bound BLMA2 was found to be similar to those reported earlier at the same DNA site for BLMB2, the intercalated bithiazole of BLMB2 is “flipped” 180˚ relative to DNA-bound BLMA2. This finding highlights an unidentified role for the C-terminal tail in directing the intercalation of the bithiazole. In addition, these analyses identified specific bond rotations within the C-terminal domain of the drug that may be relevant for its reorganization and ability to carry out a double-strand DNA cleavage event.

Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases.

Programmable genome integration of large, diverse DNA cargo without DNA repair of exposed DNA double-strand breaks remains an unsolved challenge in genome editing. We present programmable addition via site-specific targeting elements (PASTE), which uses a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase for targeted genomic recruitment and integration of desired payloads. We demonstrate integration of sequences as large as ~36 kilobases at multiple genomic loci across three human cell lines, primary T cells and non-dividing primary human hepatocytes. To augment PASTE, we discovered 25,614 serine integrases and cognate attachment sites from metagenomes and engineered orthologs with higher activity and shorter recognition sequences for efficient programmable integration. PASTE has editing efficiencies similar to or exceeding those of homology-directed repair and non-homologous end joining-based methods, with activity in non-dividing cells and in vivo with fewer detectable off-target events. PASTE expands the capabilities of genome editing by allowing large, multiplexed gene insertion without reliance on DNA repair pathways.

283 Optimising specificity and applicability of base editing for COL7A1 mutations in recessive dystrophic epidermolysis bullosa

Base and prime editors are novel CRISPR variants that reduce the risk of off-target effects by avoiding double-stranded DNA cleavage, bypassing the need for homology-directed repair. As such, these tools offer great therapeutic potential for inherited skin diseases such as recessive dystrophic epidermolysis bullosa (RDEB), a severe blistering skin disorder caused by bi-allelic loss-of-function mutations in COL7A1, which encodes type VII collagen (C7) the major component of anchoring fibrils in skin basement membrane.

Treatment of Individuals with Severe Sickle Cell Disease with OTQ923, an Autologous, Ex Vivo, CRISPR/Cas9-Edited, CD34+ Hematopoietic Stem and Progenitor Cell Product, Leads to Durable Engraftment and Fetal Hemoglobin Induction

Introduction: Increased levels of fetal hemoglobin (HbF; encoded by γ-globin [HBG1/HBG2]) are protective against complications of sickle cell disease (SCD). BCL11A is a transcription factor that binds to HBG1/HBG2 promoters and produces a γ-to-β globin (fetal-to-adult hemoglobin) switch. Inhibiting BCL11A binding to its target can reverse the switch and induce HbF expression in adult red blood cells (RBCs). OTQ923 is an autologous, ex vivo, CRISPR/Cas9-edited, CD34+ cellular product with a targeted disruption of the HBG1/HBG2 promoters on chromosome 11. Targeting this region in preclinical models led to HbF induction, phenotypically recapitulating hereditary persistence of fetal hemoglobin (a naturally occurring condition whose co-inheritance ameliorates SCD severity). This approach is more targeted than complete elimination of BCL11A expression in erythrocyte precursors for treating hemoglobinopathies. Additionally, unlike lentiviral vector gene addition, induction of endogenous γ-globin transcription could concur with reduction of βS-globin expression to avoid α- and β-like globin chain imbalance. Methods: Subjects with severe SCD, defined by a history of stroke, recurrent vaso-occlusive events (VOE), acute chest syndrome (ACS), chronic transfusions, recurrent priapism or red cell alloimmunization, were eligible to participate in the adult cohort (≥18-≤40 years old) of this clinical trial (NCT04443907). OTQ923 was manufactured from cryopreserved, peripheral blood derived CD34+ cells, collected after plerixafor mobilization. Prior to infusion, participants received myeloablative busulfan. Primary endpoints are to evaluate engraftment, long-term safety, and efficacy of HbF expression. Results: As of the data cut-off (July 8, 2022), 2 participants had received OTQ923, with follow-ups of 9 months (mos) and 3 mos, respectively. Here, we present initial clinical data from these 2 participants; additional data will be presented at the meeting. Both participants were homozygous for the sickle mutation. Participant 1 is a 22-year-old male with a history of silent cerebral infarcts, ACS, and priapism. At study entry, he was receiving chronic transfusions. He received 2.8x106 CD34+ cells/kg of OTQ923 (2 combined manufacturing batches). Participant 2 is a 21-year-old male with a history of ACS, and a silent cerebral infarct and was receiving hydroxyurea. He received 5.99x106 CD34+ cells/kg of OTQ923 (3 combined manufacturing batches). Time to neutrophil engraftment (the first of 3 consecutive days of absolute neutrophil count above 500 cells/mL after cell infusion) was 26 and 20 days for Participants 1 and 2, respectively. No OTQ923-related adverse events (AEs) were reported in either participant. All observed AEs were consistent with busulfan conditioning. Participants received RBC transfusions around conditioning and have received none after engraftment. For Participant 1, HbF level increased in the 6 mos after infusion and was stable at data cut-off (22.1% HbF at 9 mos after infusion). Induction of HbF was pancellular, with an F-cell percentage around 91% at 6 mos after infusion (Fig. 1A). Participant 2 had achieved an HbF level of 15.9% at 3 mos after infusion, the latest evaluable timepoint at data cut-off. Neither participant had any SCD-related VOEs after infusion of the cellular product. The proportion of edited alleles in the peripheral blood of Participant 1 was maintained at over 60% until Mo 6, which was the last evaluable timepoint (Fig. 1B). The indels were diverse, except for a predominant, large 5 kb deletion arising from simultaneous double-stranded DNA cleavage at HBG1 and HBG2 on-target sites, similar to previous reports. Conclusion: Preliminary data demonstrate clinically meaningful increases in HbF and evidence of benefit for both Participants 1 and 2 after OTQ923 infusion. Edited alleles were stable and maintained in the peripheral blood for >6 mos. The safety profile of OTQ923 was as expected after myeloablative busulfan and autologous hematopoietic stem cell transplantation. Taken together, these data suggest that this approach is safe and offers a potentially curative option for patients with severe SCD. Using cryopreserved, CD34+ cells as a starting material offers the advantage of centralizing manufacturing, scaling up the process to allow for improved patient access around the globe. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Evolution and Diversity of the TopoVI and TopoVI-like Subunits With Extensive Divergence of the TOPOVIBL subunit.

Type II DNA topoisomerases regulate topology by double-stranded DNA cleavage and ligation. The TopoVI family of DNA topoisomerase, first identified and biochemically characterized in Archaea, represents, with TopoVIII and mini-A, the type IIB family. TopoVI has several intriguing features in terms of function and evolution. TopoVI has been identified in some eukaryotes, and a global view is lacking to understand its evolutionary pattern. In addition, in eukaryotes, the two TopoVI subunits (TopoVIA and TopoVIB) have been duplicated and have evolved to give rise to Spo11 and TopoVIBL, forming TopoVI-like (TopoVIL), a complex essential for generating DNA breaks that initiate homologous recombination during meiosis. TopoVIL is essential for sexual reproduction. How the TopoVI subunits have evolved to ensure this meiotic function is unclear. Here, we investigated the phylogenetic conservation of TopoVI and TopoVIL. We demonstrate that BIN4 and RHL1, potentially interacting with TopoVIB, have co-evolved with TopoVI. Based on model structures, this observation supports the hypothesis for a role of TopoVI in decatenation of replicated chromatids and predicts that in eukaryotes the TopoVI catalytic complex includes BIN4 and RHL1. For TopoVIL, the phylogenetic analysis of Spo11, which is highly conserved among Eukarya, highlighted a eukaryal-specific N-terminal domain that may be important for its regulation. Conversely, TopoVIBL was poorly conserved, giving rise to ATP hydrolysis-mutated or -truncated protein variants, or was undetected in some species. This remarkable plasticity of TopoVIBL provides important information for the activity and function of TopoVIL during meiosis.

Cas12a-Capture: A Novel, Low-Cost, and Scalable Method for Targeted Sequencing.

Targeted sequencing remains a valuable technique for clinical and research applications. However, many existing technologies suffer from pervasive guanine-cytosine (GC) sequence content bias, high input DNA requirements, and high cost for custom panels. We have developed Cas12a-Capture, a low-cost and highly scalable method for targeted sequencing. The method utilizes preprogrammed guide RNAs to direct CRISPR-Cas12a cleavage of double-stranded DNA in vitro and then takes advantage of the resulting four to five nucleotide overhangs for selective ligation with a custom sequencing adapter. Addition of a second sequencing adapter and enrichment for ligation products generates a targeted sequence library. We first performed a pilot experiment with 7176 guides targeting 3.5 Mb of DNA. Using these data, we modeled the sequence determinants of Cas12a-Capture efficiency, then designed an optimized set of 11,438 guides targeting 3.0 Mb. The optimized guide set achieves an average 64-fold enrichment of targeted regions with minimal GC bias. Cas12a-Capture variant calls had strong concordance with Illumina Platinum Genome calls, especially for single nucleotide variants, which could be improved by applying basic variant quality heuristics. We believe Cas12a-Capture has a wide variety of potential clinical and research applications and is amendable for selective enrichment for any double-stranded DNA template or genome.