Biology subjects

Watts, J. K.

Publications and source records attributed to Watts, J. K..

13 recordsLinked to original sources

bioRxiv2026

Fully Modified SpyCas9 Guide RNAs Enable Robust Genome Editing In Cells and In Vivo

Precision engineering of CRISPR/Cas9 components has advanced genome editing toward therapeutic applications. Completely chemically stabilized guide RNAs (gRNAs) have the potential to improve in vivo editing efficacy while enabling greater flexibility in delivery strategies. However, previous generations of fully modified guides have been associated with reduced Cas9 activity. Here, we employed an iterative, structure-guided optimization strategy to systematically introduce chemical modifications at each position of SpyCas9 gRNAs. Extending beyond commonly used nucleotide modifications, we incorporated 2-amino-RNA, 4-thio-RNA, and extended nucleic acid (exNA) to generate gRNA designs in which 90-100% of the nucleotides are sugar- or backbone-modified. Although certain modification patterns exhibit sequence-dependent variability, we have established a growing repertoire of guides that consistently maintain or enhance editing efficacy when applied both in vitro and in vivo. Collectively, our heavily and fully modified gRNAs hold potential for applications in nuclease editing, base editing, and other genome editing tools. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/725424v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1118f08org.highwire.dtl.DTLVardef@1c57069org.highwire.dtl.DTLVardef@1572148org.highwire.dtl.DTLVardef@14a111e_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

CRYPTID-exon: streamlined detection of cryptic exons from RNA-seq data

Cryptic splicing has emerged as a pervasive feature of mammalian gene expression, with recent studies uncovering thousands of previously unannotated splice sites. Despite its prevalence, the functional consequences of this hidden layer of splicing remain largely unknown due to challenges in identifying the exact exonic regions introduced into mRNA transcripts. Here, we introduce a novel computational approach, CRYPTID-exon, that accurately predicts exon boundaries by modeling RNA-seq read coverage around empirically derived splice sites. We use CRYPTID-exon to identify and characterize thousands of cryptic exons in nascent and mature RNA from human cells. Additionally, we demonstrate that CRYPTID-exon is well powered to identify exons that are sensitive to translation-mediated degradation processes. Finally, given the growing interest in leveraging cryptic exons to modulate gene expression levels, we use our approach to identify cryptic exons in disease-relevant genes. We see that targeting these cryptic exons with splice-switching antisense oligonucleotides (ASOs) can alter gene expression and splicing patterns of the parent genes. Our study provides a framework to systematically identify and characterize cryptic exons, which will enable downstream insights into their impact on mRNA stability and translation.

genomics↗

bioRxiv2025

Pervasive noise in human splice site selection

RNA splicing has historically been thought to be highly efficient and accurate, with little opportunity for deviation from regulated alternative splicing decisions. This dogma has been challenged by recent observations that suggest that biological noise may contribute substantially to transcriptome diversity. However, quantitative understanding of stochastic variations in splicing is challenging because these transcripts are likely subject to rapid degradation. Here, we use ultra-deep sequencing across RNA compartments to track splicing intermediates in human cells and see abundant cryptic splicing associated with genomic features that promote splicing noise. We observe pervasive usage of low-fidelity splice sites, likely due to stochasticity in recruitment or binding of the spliceosome. These sites are most likely degraded in the nucleus rather than targeted by translation-dependent degradation processes, suggesting widespread surveillance and rapid quality control of non-productive RNA transcripts. Our findings provide unprecedented insights into the propensity for error in RNA processing mechanisms and the regulation of alternative splice sites across a gene.

genomics↗

bioRxiv2024

Preventing acute neurotoxicity of CNS therapeutic oligonucleotides with the addition of Ca2+ and Mg2+ in the formulation

Oligonucleotide therapeutics (ASOs and siRNAs) have been explored for modulation of gene expression in the central nervous system (CNS), with several drugs approved and many in clinical evaluation. Administration of highly concentrated oligonucleotides to the CNS can induce acute neurotoxicity. We demonstrate that delivery of concentrated oligonucleotides to the CSF in awake mice induces acute toxicity, observable within seconds of injection. Electroencephalography (EEG) and electromyography (EMG) in awake mice demonstrated seizures. Using ion chromatography, we show that siRNAs can tightly bind Ca2+ and Mg2+ up to molar equivalents of the phosphodiester (PO)/phosphorothioate (PS) bonds independently of the structure or phosphorothioate content. Optimization of the formulation by adding high concentrations (above biological levels) of divalent cations (Ca2+ alone, Mg2+ alone, or Ca2+ and Mg2+) prevents seizures with no impact on the distribution or efficacy of the oligonucleotide. The data here establishes the importance of adding Ca2+ and Mg2+ to the formulation for the safety of CNS administration of therapeutic oligonucleotides.

neuroscience↗

bioRxiv2023

Addressing the dNTP bottleneck restricting prime editing activity

Prime editing efficiency is modest in cells that are quiescent or slowly proliferating where intracellular dNTP levels are tightly regulated. MMLV-reverse transcriptase - the prime editor polymerase subunit - requires high intracellular dNTPs levels for efficient polymerization. We report that prime editing efficiency in primary cells and in vivo is increased by mutations that enhance the enzymatic properties of MMLV-reverse transcriptase and can be further complemented by targeting SAMHD1 for degradation.

biochemistry↗

bioRxiv2023

Engineering Nme2Cas9 Adenine Base Editors with Improved Activity and Targeting Scope

Nme2Cas9 has been established as a genome editing platform with compact size, high accuracy, and broad targeting range, including single-AAV-deliverable adenine base editors. Here, we have engineered Nme2Cas9 to further increase the activity and targeting scope of compact Nme2Cas9 base editors. We first used domain insertion to position the deaminase domain nearer the displaced DNA strand in the target-bound complex. These domain-inlaid Nme2Cas9 variants exhibited shifted editing windows and increased activity in comparison to the N-terminally fused Nme2-ABE. We next expanded the editing scope by swapping the Nme2Cas9 PAM-interacting domain with that of SmuCas9, which we had previously defined as recognizing a single-cytidine PAM. We used these enhancements to correct two common MECP2 mutations associated with Rett syndrome with little or no bystander editing. Finally, we validated domain-inlaid Nme2-ABEs for single-AAV delivery in vivo.

molecular biology↗

bioRxiv2023

Self-delivering CRISPR RNAs for AAV Co-delivery and Genome Editing in vivo

Guide RNAs offer programmability for CRISPR-Cas9 genome editing but also add challenges for delivery. Chemical modification, which has been key to the success of oligonucleotide therapeutics, can enhance the stability, distribution, cellular uptake, and safety of nucleic acids. Previously, we engineered heavily and fully modified SpyCas9 crRNA and tracrRNA, which showed enhanced stability and retained activity when delivered to cultured cells in the form of the ribonucleoprotein complex. In this study, we report that a short, fully stabilized oligonucleotide (a "protecting oligo"), which can be displaced by tracrRNA annealing, can significantly enhance the potency and stability of a heavily modified crRNA. Furthermore, protecting oligos allow various bioconjugates to be appended, thereby improving cellular uptake and biodistribution of crRNA in vivo. Finally, we achieved in vivo genome editing in adult mouse liver and central nervous system via co-delivery of unformulated, chemically modified crRNAs with protecting oligos and AAV vectors that express tracrRNA and either SpyCas9 or a base editor derivative. Our proof-of-concept establishment of AAV/crRNA co-delivery offers a route towards transient editing activity, target multiplexing, guide redosing, and vector inactivation.

molecular biology↗

bioRxiv2021

Quantification of Antisense Oligonucleotides by Splint Ligation and Quantitative Polymerase Chain Reaction

Reliable detection and quantification of antisense oligonucleotides (ASOs) in experimental and clinical specimens is essential to understand the biological function of novel oligonucleotide-based therapeutics. In this study, we describe a method to detect and quantify ASOs in biological samples, whereby the ASO acts as a splint to direct the ligation of complementary probes and quantitative real-time PCR was used to monitor ligation products. Low levels of 2'-O-MOE gapmer ASO in serum, liver, kidney, lung, heart, muscle, and brain tissues can be detected over a 6-log linear range for detection using this method. This method allows quantification of various types of chemically modified ASOs, including PS linkage, 2'-OMe, 2'-O-MOE, locked nucleic acid (LNA), and siRNA. This method does not require probe modifications, and can be performed using standard laboratory equipment; making it a fast, sensitive, and reliable technique that can be widely applied. This detection method may find potential applications in detection of therapeutic oligonucleotides in biological samples.

molecular biology↗

bioRxiv2021

Quantifying and Mitigating Motor Phenotypes Induced by Antisense Oligonucleotides in the Central Nervous System

Antisense oligonucleotides (ASOs) are emerging as a promising class of therapeutics for neurological diseases. When injected directly into the cerebrospinal fluid, ASOs distribute broadly across brain regions and exert long-lasting therapeutic effects. However, many phosphorothioate (PS)-modified gapmer ASOs show transient motor phenotypes when injected into the cerebrospinal fluid, ranging from reduced motor activity to ataxia or acute seizure-like phenotypes. The effect of sugar and phosphate modifications on these phenotypes has not previously been systematically studied. Using a behavioral scoring assay customized to reflect the timing and nature of these effects, we show that both sugar and phosphate modifications influence acute motor phenotypes. Among sugar analogues, PS-DNA induces the strongest motor phenotype while 2-substituted RNA modifications improve the tolerability of PS-ASOs. This helps explain why gapmer ASOs have been more challenging to develop clinically relative to steric blocker ASOs, which have a reduced tendency to induce these effects. Reducing the PS content of gapmer ASOs, which contain a stretch of PS-DNA, improves their toxicity profile, but in some cases also reduces their efficacy or duration of effect. Reducing PS content improved the acute tolerability of ASOs in both mice and sheep. We show that this acute toxicity is not mediated by the major nucleic acid sensing innate immune pathways. Formulating ASOs with calcium ions before injecting into the CNS further improved their tolerability, but through a mechanism at least partially distinct from the reduction of PS content. Overall, our work identifies and quantifies an understudied aspect of oligonucleotide toxicology in the CNS, explores its mechanism, and presents platform-level medicinal chemistry approaches that improve tolerability of this class of compounds.

pharmacology and toxicology↗

bioRxiv2020

CASCADES, a novel SOX2 super-enhancer associated long noncoding RNA, regulates cancer stem cell specification and differentiation in glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults, with a median survival of just over one year. The failure of available treatments to achieve remission in patients with GBM has been attributed to the presence of cancer stem cells (CSCs), which are thought to play a central role in tumor development and progression and serve as a treatment-resistant cell repository capable of driving tumor recurrence; in fact, the property of "stemness" itself may be responsible for treatment resistance. In this study, we identify a novel lncRNA, Cancer stem cell associated distal enhancer of SOX2 (CASCADES) that functions as an epigenetic regulator in glioma CSCs (GSCs). CASCADES is expressed in IDH-wild type GBM and significantly enriched in GSCs. Knockdown of CASCADES in GSCs results in differentiation towards a neuronal lineage in a cell- and cancer-specific manner. Bioinformatics analysis reveals that CASCADES functions as a super-enhancer associated lncRNA epigenetic regulator of SOX2. Our findings identify CASCADES as a critical regulator of stemness in GSCs and represent a novel epigenetic and therapeutic target for disrupting the cancer stem cell compartment in GBM.

cancer biology↗

bioRxiv2020

Detecting chromatin interactions along and between sister chromatids with SisterC

Accurate chromosome segregation requires chromosome compaction with concordant disentanglement of the two sister chromatids. This process has been studied extensively by microscopy but has remained a challenge for genomic methods, such as Hi-C, because sister chromatids have identical DNA sequences. Here we describe SisterC, a chromosome conformation capture assay that can distinguish interactions between and within sister chromatids. The assay is based on BrdU incorporation during S-phase, which labels the newly replicated strands of the sister chromatids. This is followed by Hi-C, e.g. during different stages of mitosis, and the selective destruction of BrdU containing strands by UV/Hoechst treatment. After PCR amplification and sequencing of the remaining intact strands, this allows for the assignment of Hi-C products as inter- and intra-sister interactions by read orientation. We performed SisterC on mitotically arrested S. cerevisiae cells. As expected, we find prominent interactions and alignment of sister chromatids at their centromeres. Along the arms, sister chromatids are less precisely aligned with inter-sister connections every ~35kb. In many instances, inter-sister interactions do not involve the interaction of two identical loci but occur between cohesin binding sites that can be offset by 5 to 25kb. Along sister chromatids, extruding cohesin forms loops up to 50kb. Combined, SisterC allows the observation of the complex interplay between sister chromatid compaction and sister chromatid segregation as the cell transitions from late S-phase to mitosis. SisterC should be applicable to study mitotic events in a wide range of organisms and cell types.

genomics↗

bioRxiv2018

5′ Modifications Improve Potency and Efficacy of DNA Donors for Precision Genome Editing

Nuclease-directed genome editing is a powerful tool for investigating physiology and has great promise as a therapeutic approach to correct mutations that cause disease. In its most precise form, genome editing can use cellular homology-directed repair (HDR) pathways to insert information from an exogenously supplied DNA repair template (donor) directly into a targeted genomic location. Unfortunately, particularly for long insertions, toxicity and delivery considerations associated with repair template DNA can limit HDR efficacy. Here, we explore chemical modifications to both double-stranded and single-stranded DNA-repair templates. We describe 5'-terminal modifications, including in its simplest form the incorporation of triethylene glycol (TEG) moieties, that consistently increase the frequency of precision editing in the germlines of three animal models (Caenorhabditis elegans, zebrafish, mice) and in cultured human cells.

molecular biology↗

bioRxiv2018

Heavily and Fully Modified RNAs Guide Efficient SpyCas9-Mediated Genome Editing

RNA-based drugs depend on chemical modifications to increase potency and nuclease stability, and to decrease immunogenicity in vivo. Chemical modification will likely improve the guide RNAs involved in CRISPR-Cas9-based therapeutics as well. Cas9 orthologs are RNA-guided microbial effectors that cleave DNA. No studies have yet explored chemical modification at all positions of the crRNA guide and tracrRNA cofactor. Here, we have identified several heavily-modified versions of crRNA and tracrRNA that are more potent than their unmodified counterparts. In addition, we describe fully chemically modified crRNAs and tracrRNAs (containing no 2-OH groups) that are functional in human cells. These designs demonstrate a significant breakthrough for Cas9-based therapeutics since heavily modified RNAs tend to be more stable in vivo (thus increasing potency). We anticipate that our designs will improve the use of Cas9 via RNP and mRNA delivery for in vivo and ex vivo purposes.

biochemistry↗