Consensus Guidelines for the Design and In Vitro Preclinical Efficacy Testing N-of-1 Exon Skipping Antisense Oligonucleotides.

Antisense oligonucleotides (ASOs) can modulate pre-mRNA splicing. This offers therapeutic opportunities for numerous genetic diseases, often in a mutation-specific and sometimes even individual-specific manner. Developing therapeutic ASOs for as few as even a single patient has been shown feasible with the development of Milasen for an individual with Batten disease. Efforts to develop individualized ASOs for patients with different genetic diseases are ongoing globally. The N = 1 Collaborative (N1C) is an umbrella organization dedicated to supporting the nascent field of individualized medicine. N1C recently organized a workshop to discuss and advance standards for the rigorous design and testing of splice-switching ASOs. In this study, we present guidelines resulting from that meeting and the key recommendations: (1) dissemination of standardized experimental designs, (2) use of standardized reference ASOs, and (3) a commitment to data sharing and exchange.

The miniature CRISPR-Cas12m effector binds DNA to block transcription.

CRISPR-Cas are prokaryotic adaptive immune systems. Cas nucleases generally use CRISPR-derived RNA guides to specifically bind and cleave DNA or RNA targets. Here, we describe the experimental characterization of a bacterial CRISPR effector protein Cas12m representing subtype V-M. Despite being less than half the size of Cas12a, Cas12m catalyzes auto-processing of a crRNA guide, recognizes a 5'-TTN' protospacer-adjacent motif (PAM), and stably binds a guide-complementary double-stranded DNA (dsDNA). Cas12m has a RuvC domain with a non-canonical catalytic site and accordingly is incapable of guide-dependent cleavage of target nucleic acids. Despite lacking target cleavage activity, the high binding affinity of Cas12m to dsDNA targets allows for interference as demonstrated by its ability to protect bacteria against invading plasmids through silencing invader transcription and/or replication. Based on these molecular features, we repurposed Cas12m by fusing it to a cytidine deaminase that resulted in base editing within a distinct window.

Engineered Cas12i2 is a versatile high-efficiency platform for therapeutic genome editing.

The CRISPR-Cas type V-I is a family of Cas12i-containing programmable nuclease systems guided by a short crRNA without requirement for a tracrRNA. Here we present an engineered Type V-I CRISPR system (Cas12i), ABR-001, which utilizes a tracr-less guide RNA. The compact Cas12i effector is capable of self-processing pre-crRNA and cleaving dsDNA targets, which facilitates versatile delivery options and multiplexing, respectively. We apply an unbiased mutational scanning approach to enhance initially low editing activity of Cas12i2. The engineered variant, ABR-001, exhibits broad genome editing capability in human cell lines, primary T cells, and CD34+ hematopoietic stem and progenitor cells, with both robust efficiency and high specificity. In addition, ABR-001 achieves a high level of genome editing when delivered via AAV vector to HEK293T cells. This work establishes ABR-001 as a versatile, specific, and high-performance platform for ex vivo and in vivo gene therapy.

Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants.

The number and diversity of known CRISPR-Cas systems have substantially increased in recent years. Here, we provide an updated evolutionary classification of CRISPR-Cas systems and cas genes, with an emphasis on the major developments that have occurred since the publication of the latest classification, in 2015. The new classification includes 2 classes, 6 types and 33 subtypes, compared with 5 types and 16 subtypes in 2015. A key development is the ongoing discovery of multiple, novel class 2 CRISPR-Cas systems, which now include 3 types and 17 subtypes. A second major novelty is the discovery of numerous derived CRISPR-Cas variants, often associated with mobile genetic elements that lack the nucleases required for interference. Some of these variants are involved in RNA-guided transposition, whereas others are predicted to perform functions distinct from adaptive immunity that remain to be characterized experimentally. The third highlight is the discovery of numerous families of ancillary CRISPR-linked genes, often implicated in signal transduction. Together, these findings substantially clarify the functional diversity and evolutionary history of CRISPR-Cas.

CRISPR-Cas in mobile genetic elements: counter-defence and beyond.

The principal function of CRISPR-Cas systems in archaea and bacteria is defence against mobile genetic elements (MGEs), including viruses, plasmids and transposons. However, the relationships between CRISPR-Cas and MGEs are far more complex. Several classes of MGE contributed to the origin and evolution of CRISPR-Cas, and, conversely, CRISPR-Cas systems and their components were recruited by various MGEs for functions that remain largely uncharacterized. In this Analysis article, we investigate and substantially expand the range of CRISPR-Cas components carried by MGEs. Three groups of Tn7-like transposable elements encode 'minimal' type I CRISPR-Cas derivatives capable of target recognition but not cleavage, and another group encodes an inactivated type V variant. These partially inactivated CRISPR-Cas variants might mediate guide RNA-dependent integration of the respective transposons. Numerous plasmids and some prophages encode type IV systems, with similar predicted properties, that appear to contribute to competition among plasmids and between plasmids and viruses. Many prokaryotic viruses also carry CRISPR mini-arrays, some of which recognize other viruses and are implicated in inter-virus conflicts, and solitary repeat units, which could inhibit host CRISPR-Cas systems.

Functionally diverse type V CRISPR-Cas systems.

Type V CRISPR-Cas systems are distinguished by a single RNA-guided RuvC domain-containing effector, Cas12. Although effectors of subtypes V-A (Cas12a) and V-B (Cas12b) have been studied in detail, the distinct domain architectures and diverged RuvC sequences of uncharacterized Cas12 proteins suggest unexplored functional diversity. Here, we identify and characterize Cas12c, -g, -h, and -i. Cas12c, -h, and -i demonstrate RNA-guided double-stranded DNA (dsDNA) interference activity. Cas12i exhibits markedly different efficiencies of CRISPR RNA spacer complementary and noncomplementary strand cleavage resulting in predominant dsDNA nicking. Cas12g is an RNA-guided ribonuclease (RNase) with collateral RNase and single-strand DNase activities. Our study reveals the functional diversity emerging along different routes of type V CRISPR-Cas evolution and expands the CRISPR toolbox.

Cas13d Is a Compact RNA-Targeting Type VI CRISPR Effector Positively Modulated by a WYL-Domain-Containing Accessory Protein.

Bacterial class 2 CRISPR-Cas systems utilize a single RNA-guided protein effector to mitigate viral infection. We aggregated genomic data from multiple sources and constructed an expanded database of predicted class 2 CRISPR-Cas systems. A search for novel RNA-targeting systems identified subtype VI-D, encoding dual HEPN domain-containing Cas13d effectors and putative WYL-domain-containing accessory proteins (WYL1 and WYL-b1 through WYL-b5). The median size of Cas13d proteins is 190 to 300 aa smaller than that of Cas13a-Cas13c. Despite their small size, Cas13d orthologs from Eubacterium siraeum (Es) and Ruminococcus sp. (Rsp) are active in both CRISPR RNA processing and targeting, as well as collateral RNA cleavage, with no target-flanking sequence requirements. The RspWYL1 protein stimulates RNA cleavage by both EsCas13d and RspCas13d, demonstrating a common regulatory mechanism for divergent Cas13d orthologs. The small size, minimal targeting constraints, and modular regulation of Cas13d effectors further expands the CRISPR toolkit for RNA manipulation and detection.

CRISPR-Cas9-based treatment of myocilin-associated glaucoma.

Primary open-angle glaucoma (POAG) is a leading cause of irreversible vision loss worldwide, with elevated intraocular pressure (IOP) a major risk factor. Myocilin (MYOC) dominant gain-of-function mutations have been reported in ∼4% of POAG cases. MYOC mutations result in protein misfolding, leading to endoplasmic reticulum (ER) stress in the trabecular meshwork (TM), the tissue that regulates IOP. We use CRISPR-Cas9-mediated genome editing in cultured human TM cells and in a MYOC mouse model of POAG to knock down expression of mutant MYOC, resulting in relief of ER stress. In vivo genome editing results in lower IOP and prevents further glaucomatous damage. Importantly, using an ex vivo human organ culture system, we demonstrate the feasibility of human genome editing in the eye for this important disease.

A machine learning approach for predicting CRISPR-Cas9 cleavage efficiencies and patterns underlying its mechanism of action.

The adaptation of the CRISPR-Cas9 system as a genome editing technique has generated much excitement in recent years owing to its ability to manipulate targeted genes and genomic regions that are complementary to a programmed single guide RNA (sgRNA). However, the efficacy of a specific sgRNA is not uniquely defined by exact sequence homology to the target site, thus unintended off-targets might additionally be cleaved. Current methods for sgRNA design are mainly concerned with predicting off-targets for a given sgRNA using basic sequence features and employ elementary rules for ranking possible sgRNAs. Here, we introduce CRISTA (CRISPR Target Assessment), a novel algorithm within the machine learning framework that determines the propensity of a genomic site to be cleaved by a given sgRNA. We show that the predictions made with CRISTA are more accurate than other available methodologies. We further demonstrate that the occurrence of bulges is not a rare phenomenon and should be accounted for in the prediction process. Beyond predicting cleavage efficiencies, the learning process provides inferences regarding patterns that underlie the mechanism of action of the CRISPR-Cas9 system. We discover that attributes that describe the spatial structure and rigidity of the entire genomic site as well as those surrounding the PAM region are a major component of the prediction capabilities.

Engineered Cpf1 variants with altered PAM specificities.

The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells. However, the utility of the commonly used Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) and Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) is limited by their requirement of a TTTV protospacer adjacent motif (PAM) in the DNA substrate. To address this limitation, we performed a structure-guided mutagenesis screen to increase the targeting range of Cpf1. We engineered two AsCpf1 variants carrying the mutations S542R/K607R and S542R/K548V/N552R, which recognize TYCV and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Genome-wide assessment of off-target activity using BLISS indicated that these variants retain high DNA-targeting specificity, which we further improved by introducing an additional non-PAM-interacting mutation. Introducing the identified PAM-interacting mutations at their corresponding positions in LbCpf1 similarly altered its PAM specificity. Together, these variants increase the targeting range of Cpf1 by approximately threefold in human coding sequences to one cleavage site per ∼11 bp.

BLISS is a versatile and quantitative method for genome-wide profiling of DNA double-strand breaks.

Precisely measuring the location and frequency of DNA double-strand breaks (DSBs) along the genome is instrumental to understanding genomic fragility, but current methods are limited in versatility, sensitivity or practicality. Here we present Breaks Labeling In Situ and Sequencing (BLISS), featuring the following: (1) direct labelling of DSBs in fixed cells or tissue sections on a solid surface; (2) low-input requirement by linear amplification of tagged DSBs by in vitro transcription; (3) quantification of DSBs through unique molecular identifiers; and (4) easy scalability and multiplexing. We apply BLISS to profile endogenous and exogenous DSBs in low-input samples of cancer cells, embryonic stem cells and liver tissue. We demonstrate the sensitivity of BLISS by assessing the genome-wide off-target activity of two CRISPR-associated RNA-guided endonucleases, Cas9 and Cpf1, observing that Cpf1 has higher specificity than Cas9. Our results establish BLISS as a versatile, sensitive and efficient method for genome-wide DSB mapping in many applications.

Diversity and evolution of class 2 CRISPR-Cas systems.

Class 2 CRISPR-Cas systems are characterized by effector modules that consist of a single multidomain protein, such as Cas9 or Cpf1. We designed a computational pipeline for the discovery of novel class 2 variants and used it to identify six new CRISPR-Cas subtypes. The diverse properties of these new systems provide potential for the development of versatile tools for genome editing and regulation. In this Analysis article, we present a comprehensive census of class 2 types and class 2 subtypes in complete and draft bacterial and archaeal genomes, outline evolutionary scenarios for the independent origin of different class 2 CRISPR-Cas systems from mobile genetic elements, and propose an amended classification and nomenclature of CRISPR-Cas.

In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a devastating disease affecting about 1 out of 5000 male births and caused by mutations in the dystrophin gene. Genome editing has the potential to restore expression of a modified dystrophin gene from the native locus to modulate disease progression. In this study, adeno-associated virus was used to deliver the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system to the mdx mouse model of DMD to remove the mutated exon 23 from the dystrophin gene. This includes local and systemic delivery to adult mice and systemic delivery to neonatal mice. Exon 23 deletion by CRISPR-Cas9 resulted in expression of the modified dystrophin gene, partial recovery of functional dystrophin protein in skeletal myofibers and cardiac muscle, improvement of muscle biochemistry, and significant enhancement of muscle force. This work establishes CRISPR-Cas9-based genome editing as a potential therapy to treat DMD.

In vivo gene editing in dystrophic mouse muscle and muscle stem cells.

Frame-disrupting mutations in the DMD gene, encoding dystrophin, compromise myofiber integrity and drive muscle deterioration in Duchenne muscular dystrophy (DMD). Removing one or more exons from the mutated transcript can produce an in-frame mRNA and a truncated, but still functional, protein. In this study, we developed and tested a direct gene-editing approach to induce exon deletion and recover dystrophin expression in the mdx mouse model of DMD. Delivery by adeno-associated virus (AAV) of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonucleases coupled with paired guide RNAs flanking the mutated Dmd exon23 resulted in excision of intervening DNA and restored the Dmd reading frame in myofibers, cardiomyocytes, and muscle stem cells after local or systemic delivery. AAV-Dmd CRISPR treatment partially recovered muscle functional deficiencies and generated a pool of endogenously corrected myogenic precursors in mdx mouse muscle.

Rationally engineered Cas9 nucleases with improved specificity.

The RNA-guided endonuclease Cas9 is a versatile genome-editing tool with a broad range of applications from therapeutics to functional annotation of genes. Cas9 creates double-strand breaks (DSBs) at targeted genomic loci complementary to a short RNA guide. However, Cas9 can cleave off-target sites that are not fully complementary to the guide, which poses a major challenge for genome editing. Here, we use structure-guided protein engineering to improve the specificity of Streptococcus pyogenes Cas9 (SpCas9). Using targeted deep sequencing and unbiased whole-genome off-target analysis to assess Cas9-mediated DNA cleavage in human cells, we demonstrate that "enhanced specificity" SpCas9 (eSpCas9) variants reduce off-target effects and maintain robust on-target cleavage. Thus, eSpCas9 could be broadly useful for genome-editing applications requiring a high level of specificity.

Crystal Structure of Staphylococcus aureus Cas9.

The RNA-guided DNA endonuclease Cas9 cleaves double-stranded DNA targets with a protospacer adjacent motif (PAM) and complementarity to the guide RNA. Recently, we harnessed Staphylococcus aureus Cas9 (SaCas9), which is significantly smaller than Streptococcus pyogenes Cas9 (SpCas9), to facilitate efficient in vivo genome editing. Here, we report the crystal structures of SaCas9 in complex with a single guide RNA (sgRNA) and its double-stranded DNA targets, containing the 5'-TTGAAT-3' PAM and the 5'-TTGGGT-3' PAM, at 2.6 and 2.7 Å resolutions, respectively. The structures revealed the mechanism of the relaxed recognition of the 5'-NNGRRT-3' PAM by SaCas9. A structural comparison of SaCas9 with SpCas9 highlighted both structural conservation and divergence, explaining their distinct PAM specificities and orthologous sgRNA recognition. Finally, we applied the structural information about this minimal Cas9 to rationally design compact transcriptional activators and inducible nucleases, to further expand the CRISPR-Cas9 genome editing toolbox.

In vivo genome editing using Staphylococcus aureus Cas9.

The RNA-guided endonuclease Cas9 has emerged as a versatile genome-editing platform. However, the size of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for basic research and therapeutic applications that use the highly versatile adeno-associated virus (AAV) delivery vehicle. Here, we characterize six smaller Cas9 orthologues and show that Cas9 from Staphylococcus aureus (SaCas9) can edit the genome with efficiencies similar to those of SpCas9, while being more than 1 kilobase shorter. We packaged SaCas9 and its single guide RNA expression cassette into a single AAV vector and targeted the cholesterol regulatory gene Pcsk9 in the mouse liver. Within one week of injection, we observed >40% gene modification, accompanied by significant reductions in serum Pcsk9 and total cholesterol levels. We further assess the genome-wide targeting specificity of SaCas9 and SpCas9 using BLESS, and demonstrate that SaCas9-mediated in vivo genome editing has the potential to be efficient and specific.

Reducing the gradient artefact in simultaneous EEG-fMRI by adjusting the subject's axial position.

Large artefacts that compromise EEG data quality are generated when electroencephalography (EEG) and functional magnetic resonance imaging (fMRI) are carried out concurrently. The gradient artefact produced by the time-varying magnetic field gradients is the largest of these artefacts. Although average artefact correction (AAS) and related techniques can remove the majority of this artefact, the need to avoid amplifier saturation necessitates the use of a large dynamic range and strong low-pass filtering in EEG recording. Any intrinsic reduction in the gradient artefact amplitude would allow data with a higher bandwidth to be acquired without amplifier saturation, thus increasing the frequency range of neuronal activity that can be investigated using combined EEG-fMRI. Furthermore, gradient artefact correction methods assume a constant artefact morphology over time, so their performance is compromised by subject movement. Since the resulting, residual gradient artefacts can easily swamp signals from brain activity, any reduction in their amplitude would be highly advantageous for simultaneous EEG-fMRI studies. The aim of this work was to investigate whether adjustment of the subject's axial position in the MRI scanner can reduce the amplitude of the induced gradient artefact, before and after artefact correction using AAS. The variation in gradient artefact amplitude as a function of the subject's axial position was first investigated in six subjects by applying gradient pulses along the three Cartesian axes. The results of this study showed that a significant reduction in the gradient artefact magnitude can be achieved by shifting the subject axially by 4 cm towards the feet relative to the standard subject position (nasion at iso-centre). In a further study, the 4-cm shift was shown to produce a 40% reduction in the RMS amplitude (and a 31% reduction in the range) of the gradient artefact generated during the execution of a standard multi-slice, EPI sequence. By picking out signals occurring at harmonics of the slice acquisition frequency, it was also shown that the 4-cm shift led to a 36% reduction in the residual gradient artefact after AAS. Functional and anatomical MR data quality is not affected by the 4-cm shift, as the head remains in the homogeneous region of the static magnet field and gradients.

Physical modeling of pulse artefact sources in simultaneous EEG/fMRI.

The collection of electroencephalography (EEG) data during simultaneous functional magnetic resonance imaging (fMRI) is impeded by large artefacts in the EEG recordings, with the pulse artefact (PA) being particularly challenging because of its persistence even after application of artefact correction algorithms. Despite several possible causes of the PA having been hypothesized, few studies have rigorously quantified the contributions from the different putative sources. This article presents analytic expressions and simulations describing two possible sources of the PA corresponding to different movements in the strong static field of the MR scanner: cardiac-pulse-driven head rotation and blood-flow-induced Hall voltages. Models of head rotation about a left-right axis and flow in a deep artery running in the anterior-posterior direction reproduced properties of the PA including the left/right spatial variation of polarity. Of these two sources, head rotation was shown to be the most likely source of the PA with simulated magnitudes of >200 muV being generated at 3 T, similar to the in vivo PA magnitudes, for an angular velocity of just 0.5 degrees /s. Smaller artefact voltages of less than 10 muV were calculated for flow in a model artery with physical characteristics similar to the internal carotid artery. A deeper physical understanding of the PA is a key step in working toward production of higher fidelity EEG/fMRI data: analytic expressions for the artefact voltages can guide a redesign of the wiring layout on EEG caps to minimize intrinsic artefact pickup, while simulated artefact maps could be incorporated into selective filters.