A model of heritable genome editing for complex traits
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Prime editing: therapeutic advances and mechanistic insights
We are often confronted with a simple question, “which gene editing technique is the best?”; the simple answer is “there isn’t one”. In 2021, a year after prime editing first made its mark, we evaluated the landscape of this potentially transformative advance in genome engineering towards getting treatments to the clinic [1]. Nearly 20% of the papers we cited were still in pre-print at the time which serves to indicate how early-stage the knowledge base was at that time. Now, three years later, we take a look at the landscape and ask what has been learnt to ensure this tech is broadly accessible, highlighting some key advances, especially those that push this towards the clinic. A big part of the appeal of prime editing is its ability to precisely edit DNA without double stranded breaks, and to install any of the 12 possible single-nucleotide conversion events as well as small insertions and/or deletions, or essentially any combination thereof. Over the last few decades, other transformative and Nobel prize-winning technologies that rely on Watson-Crick base-pairing such as PCR, site-directed mutagenesis, RNA interference, and one might say, “classic” CRISPR, were swiftly adopted across labs around the world because of the speed with which mechanistic rules governing their efficiency were determined. Whilst this perspective focuses on the context of gene therapy applications of prime editing, we also further look at the recent studies which have increased our understanding of the mechanism of PEs and simultaneously improved the efficiency and diversity of the PE toolbox.
Taking control with RNA
RNA roles RNA seized the global limelight when a modified RNA was used in the vaccine against the virus SARS-CoV-2 that caused a global pandemic.…
SEED-Selection enables high-efficiency enrichment of primary T cells edited at multiple loci
Engineering T cell specificity and function at multiple loci can generate more effective cellular therapies, but current manufacturing methods produce heterogenous mixtures of partially engineered cells. Here we develop a one-step process to enrich unlabeled cells containing knock-ins at multiple target loci using a family of repair templates named synthetic exon expression disruptors (SEEDs). SEEDs associate transgene integration with the disruption of a paired target endogenous surface protein while preserving target expression in nonmodified and partially edited cells to enable their removal (SEED-Selection). We design SEEDs to modify three critical loci encoding T cell specificity, coreceptor expression and major histocompatibility complex expression. The results demonstrate up to 98% purity after selection for individual modifications and up to 90% purity for six simultaneous edits (three knock-ins and three knockouts). This method is compatible with existing clinical manufacturing workflows and can be readily adapted to other loci to facilitate production of complex gene-edited cell therapies.
Genome-wide association study reveals multiple loci for nociception and opioid consumption behaviors associated with heroin vulnerability in outbred rats
The increased prevalence of opioid use disorder (OUD) makes it imperative to disentangle the biological mechanisms contributing to individual differences in OUD vulnerability. OUD shows strong heritability, however genetic variants contributing to vulnerability remain poorly defined. We performed a genome-wide association study using over 850 male and female heterogeneous stock (HS) rats to identify genes underlying behaviors associated with OUD such as nociception, as well as heroin-taking, extinction and seeking behaviors. By using an animal model of OUD, we were able to identify genetic variants associated with distinct OUD behaviors while maintaining a uniform environment, an experimental design not easily achieved in humans. Furthermore, we used a novel non-linear network-based clustering approach to characterize rats based on OUD vulnerability to assess genetic variants associated with OUD susceptibility. Our findings confirm the heritability of several OUD-like behaviors, including OUD susceptibility. Additionally, several genetic variants associated with nociceptive threshold prior to heroin experience, heroin consumption, escalation of intake, and motivation to obtain heroin were identified. Tom1, a microglial component, was implicated for nociception. Several genes involved in dopaminergic signaling, neuroplasticity and substance use disorders, including Brwd1, Pcp4, Phb1l2 and Mmp15 were implicated for the heroin traits. Additionally, an OUD vulnerable phenotype was associated with genetic variants for consumption and break point, suggesting a specific genetic contribution for OUD-like traits contributing to vulnerability. Together, these findings identify novel genetic markers related to the susceptibility to OUD-relevant behaviors in HS rats.
Phenotypic divergence between individuals with self-reported autistic traits and clinically ascertained autism
While allowing for rapid recruitment of large samples, online research relies heavily on participants’ self-reports of neuropsychiatric traits, foregoing the clinical characterizations available in laboratory settings. Autism spectrum disorder (ASD) research is one example for which the clinical validity of such an approach remains elusive. Here we compared 56 adults with ASD recruited in person and evaluated by clinicians to matched samples of adults recruited through an online platform (Prolific; 56 with high autistic traits and 56 with low autistic traits) and evaluated via self-reported surveys. Despite having comparable self-reported autistic traits, the online high-trait group reported significantly more social anxiety and avoidant symptoms than in-person ASD participants. Within the in-person sample, there was no relationship between self-rated and clinician-rated autistic traits, suggesting they may capture different aspects of ASD. The groups also differed in their social tendencies during two decision-making tasks; the in-person ASD group was less perceptive of opportunities for social influence and acted less affiliative toward virtual characters. These findings highlight the need for a differentiation between clinically ascertained and trait-defined samples in autism research.
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