Prime editors (PEs) mediate genome modification without utilizing double-stranded DNA breaks or exogenous donor DNA as a template. PEs facilitate nucleotide substitutions or local insertions or ...deletions within the genome based on the template sequence encoded within the prime editing guide RNA (pegRNA). However, the efficacy of prime editing in adult mice has not been established. Here we report an NLS-optimized SpCas9-based prime editor that improves genome editing efficiency in both fluorescent reporter cells and at endogenous loci in cultured cell lines. Using this genome modification system, we could also seed tumor formation through somatic cell editing in the adult mouse. Finally, we successfully utilize dual adeno-associated virus (AAVs) for the delivery of a split-intein prime editor and demonstrate that this system enables the correction of a pathogenic mutation in the mouse liver. Our findings further establish the broad potential of this genome editing technology for the directed installation of sequence modifications in vivo, with important implications for disease modeling and correction.
Adeno-associated virus (AAV) vectors are important delivery platforms for therapeutic genome editing but are severely constrained by cargo limits. Simultaneous delivery of multiple vectors can limit ...dose and efficacy and increase safety risks. Here, we describe single-vector, ~4.8-kb AAV platforms that express Nme2Cas9 and either two sgRNAs for segmental deletions, or a single sgRNA with a homology-directed repair (HDR) template. We also use anti-CRISPR proteins to enable production of vectors that self-inactivate via Nme2Cas9 cleavage. We further introduce a nanopore-based sequencing platform that is designed to profile rAAV genomes and serves as a quality control measure for vector homogeneity. We demonstrate that these platforms can effectively treat two disease models type I hereditary tyrosinemia (HT-I) and mucopolysaccharidosis type I (MPS-I) in mice by HDR-based correction of the disease allele. These results will enable the engineering of single-vector AAVs that can achieve diverse therapeutic genome editing outcomes.
Clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) have recently opened a new avenue for gene therapy. Cas9 nuclease guided by a single-guide ...RNA (sgRNA) has been extensively used for genome editing. Currently, three Cas9 orthologs have been adapted for in vivo genome engineering applications: Streptococcus pyogenes Cas9 (SpyCas9), Staphylococcus aureus Cas9 (SauCas9), and Campylobacter jejuni (CjeCas9). However, additional in vivo editing platforms are needed, in part to enable a greater range of sequences to be accessed via viral vectors, especially those in which Cas9 and sgRNA are combined into a single vector genome.
Here, we present in vivo editing using Neisseria meningitidis Cas9 (NmeCas9). NmeCas9 is compact, edits with high accuracy, and possesses a distinct protospacer adjacent motif (PAM), making it an excellent candidate for safe gene therapy applications. We find that NmeCas9 can be used to target the Pcsk9 and Hpd genes in mice. Using tail-vein hydrodynamic-based delivery of NmeCas9 plasmid to target the Hpd gene, we successfully reprogram the tyrosine degradation pathway in Hereditary Tyrosinemia Type I mice. More importantly, we deliver NmeCas9 with its sgRNA in a single recombinant adeno-associated vector (rAAV) to target Pcsk9, resulting in lower cholesterol levels in mice. This all-in-one vector yielded > 35% gene modification after two weeks of vector administration, with minimal off-target cleavage in vivo.
Our findings indicate that NmeCas9 can enable the editing of disease-causing loci in vivo, expanding the targeting scope of RNA-guided nucleases.
RNA-based drugs depend on chemical modifications to increase potency 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. Here, we explore chemical modifications at all positions of the crRNA guide and tracrRNA cofactor. We identify 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 will contribute to 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.
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 (
, zebrafish, mice) and in cultured human cells.
The hammerhead ribozyme is a self-cleaving RNA broadly dispersed across all kingdoms of life. Although it was the first of the small, nucleolytic ribozymes discovered, the mechanism by which it ...catalyzes its reaction remains elusive. The nucleobase of G12 is well positioned to be a general base, but it is unclear if or how this guanine base becomes activated for proton transfer. Metal ions have been implicated in the chemical mechanism, but no interactions between divalent metal ions and the cleavage site have been observed crystallographically. To better understand how this ribozyme functions, we have solved crystal structures of wild-type and G12A mutant ribozymes. We observe a pH-dependent conformational change centered around G12, consistent with this nucleotide becoming deprotonated. Crystallographic and kinetic analysis of the G12A mutant reveals a Zn2+ specificity switch suggesting a direct interaction between a divalent metal ion and the purine at position 12. The metal ion specificity switch and the pH–rate profile of the G12A mutant suggest that the minor imino tautomer of A12 serves as the general base in the mutant ribozyme. We propose a model in which the hammerhead ribozyme rearranges prior to the cleavage reaction, positioning two divalent metal ions in the process. The first metal ion, positioned near G12, becomes directly coordinated to the O6 keto oxygen, to lower the pK a of the general base and organize the active site. The second metal ion, positioned near G10.1, bridges the N7 of G10.1 and the scissile phosphate and may participate directly in the cleavage reaction.
The development of CRISPR genome editing has transformed biomedical research. Most applications reported thus far rely upon the Cas9 protein from Streptococcus pyogenes SF370 (SpyCas9). With many RNA ...guides, wildtype SpyCas9 can induce significant levels of unintended mutations at near-cognate sites, necessitating substantial efforts toward the development of strategies to minimize off-target activity. Although the genome-editing potential of thousands of other Cas9 orthologs remains largely untapped, it is not known how many will require similarly extensive engineering to achieve single-site accuracy within large genomes. In addition to its off-targeting propensity, SpyCas9 is encoded by a relatively large open reading frame, limiting its utility in applications that require size-restricted delivery strategies such as adeno-associated virus vectors. In contrast, some genome-editing-validated Cas9 orthologs are considerably smaller and therefore better suited for viral delivery.
Here we show that wildtype NmeCas9, when programmed with guide sequences of the natural length of 24 nucleotides, exhibits a nearly complete absence of unintended editing in human cells, even when targeting sites that are prone to off-target activity with wildtype SpyCas9. We also validate at least six variant protospacer adjacent motifs (PAMs), in addition to the preferred consensus PAM (5'-N
GATT-3'), for NmeCas9 genome editing in human cells.
Our results show that NmeCas9 is a naturally high-fidelity genome-editing enzyme and suggest that additional Cas9 orthologs may prove to exhibit similarly high accuracy, even without extensive engineering.
In their natural settings, CRISPR-Cas systems play crucial roles in bacterial and archaeal adaptive immunity to protect against phages and other mobile genetic elements, and they are also widely used ...as genome engineering technologies. Previously we discovered bacteriophage-encoded Cas9-specific anti-CRISPR (Acr) proteins that serve as countermeasures against host bacterial immunity by inactivating their CRISPR-Cas systems (A. Pawluk, N. Amrani, Y. Zhang, B. Garcia, et al., Cell 167:1829-1838.e9, 2016, https://doi.org/10.1016/j.cell.2016.11.017). We hypothesized that the evolutionary advantages conferred by anti-CRISPRs would drive the widespread occurrence of these proteins in nature (K. L. Maxwell, Mol Cell 68:8-14, 2017, https://doi.org/10.1016/j.molcel.2017.09.002; A. Pawluk, A. R. Davidson, and K. L. Maxwell, Nat Rev Microbiol 16:12-17, 2018, https://doi.org/10.1038/nrmicro.2017.120; E. J. Sontheimer and A. R. Davidson, Curr Opin Microbiol 37:120-127, 2017, https://doi.org/10.1016/j.mib.2017.06.003). We have identified new anti-CRISPRs using the same bioinformatic approach that successfully identified previous Acr proteins (A. Pawluk, N. Amrani, Y. Zhang, B. Garcia, et al., Cell 167:1829-1838.e9, 2016, https://doi.org/10.1016/j.cell.2016.11.017) against
Cas9 (NmeCas9). In this work, we report two novel anti-CRISPR families in strains of
and
, both of which harbor type II-C CRISPR-Cas systems (A. Mir, A. Edraki, J. Lee, and E. J. Sontheimer, ACS Chem Biol 13:357-365, 2018, https://doi.org/10.1021/acschembio.7b00855). We characterize the type II-C Cas9 orthologs from
and
, show that the newly identified Acrs are able to inhibit these systems, and define important features of their inhibitory mechanisms. The
Acr is the most potent NmeCas9 inhibitor identified to date. Although inhibition of NmeCas9 by anti-CRISPRs from
and
reveals cross-species inhibitory activity, more distantly related type II-C Cas9s are not inhibited by these proteins. The specificities of anti-CRISPRs and divergent Cas9s appear to reflect coevolution of their strategies to combat or evade each other. Finally, we validate these new anti-CRISPR proteins as potent off-switches for Cas9 genome engineering applications.
As one of their countermeasures against CRISPR-Cas immunity, bacteriophages have evolved natural inhibitors known as anti-CRISPR (Acr) proteins. Despite the existence of such examples for type II CRISPR-Cas systems, we currently know relatively little about the breadth of Cas9 inhibitors, and most of their direct Cas9 targets are uncharacterized. In this work we identify two new type II-C anti-CRISPRs and their cognate Cas9 orthologs, validate their functionality
and in bacteria, define their inhibitory spectrum against a panel of Cas9 orthologs, demonstrate that they act before Cas9 DNA binding, and document their utility as off-switches for Cas9-based tools in mammalian applications. The discovery of diverse anti-CRISPRs, the mechanistic analysis of their cognate Cas9s, and the definition of Acr inhibitory mechanisms afford deeper insight into the interplay between Cas9 orthologs and their inhibitors and provide greater scope for exploiting Acrs for CRISPR-based genome engineering.
CRISPR-Cas9 systems provide powerful tools for genome editing. However, optimal employment of this technology will require control of Cas9 activity so that the timing, tissue specificity, and ...accuracy of editing may be precisely modulated. Anti-CRISPR proteins, which are small, naturally occurring inhibitors of CRISPR-Cas systems, are well suited for this purpose. A number of anti-CRISPR proteins have been shown to potently inhibit subgroups of CRISPR-Cas9 systems, but their maximal inhibitory activity is generally restricted to specific Cas9 homologs. Since Cas9 homologs vary in important properties, differing Cas9s may be optimal for particular genome-editing applications. To facilitate the practical exploitation of multiple Cas9 homologs, here we identify one anti-CRISPR, called AcrIIA5, that potently inhibits nine diverse type II-A and type II-C Cas9 homologs, including those currently used for genome editing. We show that the activity of AcrIIA5 results in partial in vivo cleavage of a single-guide RNA (sgRNA), suggesting that its mechanism involves RNA interaction.
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•Anti-CRISPR AcrIIA5 potently inhibits all Cas9 homologs used in genome editing•AcrIIA5 functions well in a variety of mammalian cell genome-editing applications•The AcrIIA5 functional mechanism leads to sgRNA cleavage
Garcia et al. show that anti-CRISPR protein AcrIIA5 strongly inhibits all of the CRISPR-Cas9 homologs that are commonly used for genome editing. They show that it functions effectively in bacterial and mammalian cells. This anti-CRISPR will be useful for a wide variety of biotechnological applications.
Cognitive reappraisal intends to study the significance of an event concerning any emotional reaction. Understanding the efficacy of cognitive reappraisal in emotion regulation requires reliable and ...valid techniques for quantifying emotions. Sentiments and emotions are complex mental states that play a significant role in every aspect of human life. The analysis of relevant literature published from 2012 till June 2023 indicated the limited investigation into predicting quantifiable emotional states. The existing emotion recognition techniques use static labels for emotion quantization. This study investigates prominent techniques for dynamic emotion modelling along with associated challenges, shortcomings, and research gaps. Subsequently, a generic framework is proposed that may help researchers explore dynamic emotion modelling techniques and future directions.
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