Treatment of a Mouse Model of ALS by In Vivo Base Editing.

Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal disorder that can be caused by mutations in the superoxide dismutase 1 (SOD1) gene. Although ALS is currently incurable, CRISPR base editors hold the potential to treat the disease through their ability to create nonsense mutations that can permanently disable the expression of the mutant SOD1 gene. However, the restrictive carrying capacity of adeno-associated virus (AAV) vectors has limited their therapeutic application. In this study, we establish an intein-mediated trans-splicing system that enables in vivo delivery of cytidine base editors (CBEs) consisting of the widely used Cas9 protein from Streptococcus pyogenes. We show that intrathecal injection of dual AAV particles encoding a split-intein CBE engineered to trans-splice and introduce a nonsense-coding substitution into a mutant SOD1 gene prolonged survival and markedly slowed the progression of disease in the G93A-SOD1 mouse model of ALS. Adult animals treated by this split-intein CRISPR base editor had a reduced rate of muscle atrophy, decreased muscle denervation, improved neuromuscular function, and up to 40% fewer SOD1 immunoreactive inclusions at end-stage mice compared to control mice. This work expands the capabilities of single-base editors and demonstrates their potential for gene therapy.

Multiplexed and tunable transcriptional activation by promoter insertion using nuclease-assisted vector integration.

The ability to selectively regulate expression of any target gene within a genome provides a means to address a variety of diseases and disorders. While artificial transcription factors are emerging as powerful tools for gene activation within a natural chromosomal context, current generations often exhibit relatively weak, variable, or unpredictable activity across targets. To address these limitations, we developed a novel system for gene activation, which bypasses native promoters to achieve unprecedented levels of transcriptional upregulation by integrating synthetic promoters at target sites. This gene activation system is multiplexable and easily tuned for precise control of expression levels. Importantly, since promoter vector integration requires just one variable sgRNA to target each gene of interest, this procedure can be implemented with minimal cloning. Collectively, these results demonstrate a novel system for gene activation with wide adaptability for studies of transcriptional regulation and cell line engineering.

SPLICER: A Highly Efficient Base Editing Toolbox That Enables In Vivo Therapeutic Exon Skipping.

Exon skipping technologies enable exclusion of targeted exons from mature mRNA transcripts, which has broad applications in molecular biology, medicine, and biotechnology. Existing exon skipping techniques include antisense oligonucleotides, targetable nucleases, and base editors, which, while effective for specific applications at some target exons, remain hindered by shortcomings, including transient effects for oligonucleotides, genotoxicity for nucleases and inconsistent exon skipping for base editors. To overcome these limitations, we created SPLICER, a toolbox of next-generation base editors consisting of near-PAMless Cas9 nickase variants fused to adenosine or cytosine deaminases for the simultaneous editing of splice acceptor (SA) and splice donor (SD) sequences. Synchronized SA and SD editing with SPLICER improves exon skipping, reduces aberrant outcomes, including cryptic splicing and intron retention, and enables skipping of exons refractory to single splice-site editing. To demonstrate the therapeutic potential of SPLICER, we targeted APP exon 17, which encodes the amino acid residues that are cleaved to form the Aß plaques in Alzheimer's disease. SPLICER reduced the formation of Aß42 peptides in vitro and enabled efficient exon skipping in a mouse model of Alzheimer's disease. Overall, SPLICER is a widely applicable and efficient toolbox for exon skipping with broad therapeutic applications.

In vivo CRISPR base editing for treatment of Huntington's disease.

Huntington's disease (HD) is an inherited and ultimately fatal neurodegenerative disorder caused by an expanded polyglutamine-encoding CAG repeat within exon 1 of the huntingtin (HTT) gene, which produces a mutant protein that destroys striatal and cortical neurons. Importantly, a critical event in the pathogenesis of HD is the proteolytic cleavage of the mutant HTT protein by caspase-6, which generates fragments of the N-terminal domain of the protein that form highly toxic aggregates. Given the role that proteolysis of the mutant HTT protein plays in HD, strategies for preventing this process hold potential for treating the disorder. By screening 141 CRISPR base editor variants targeting splice elements in the HTT gene, we identified platforms capable of producing HTT protein isoforms resistant to caspase-6-mediated proteolysis via editing of the splice acceptor sequence for exon 13. When delivered to the striatum of a rodent HD model, these base editors induced efficient exon skipping and decreased the formation of the N-terminal fragments, which in turn reduced HTT protein aggregation and attenuated striatal and cortical atrophy. Collectively, these results illustrate the potential for CRISPR base editing to decrease the toxicity of the mutant HTT protein for HD.

Protein Engineering Technologies for Development of Next-Generation Genome Editors.

Base editors and prime editors have emerged as promising tools for the modeling and treatment of genetic diseases due to their ability to introduce targeted modifications in the genomic DNA of living cells. Several engineering approaches have been applied to improve their performance, ranging from simple protein design approaches to complex directed evolution schemes that can probe a vast landscape of mutational variants with minimal user intervention. These extensive efforts have led to new generations of editors with enhanced properties such as increased editing activity, tailored editing windows, increased targetability, smaller construct size for viral delivery, and decreased off-target effects. In this manuscript we review protein engineering technologies that have been recently utilized to create an ever-evolving landscape of high-performance gene editing tools specifically designed for genetic targets of interest and that have redefined what is possible in the field of precision medicine.

Targeting Duchenne muscular dystrophy by skipping DMD exon 45 with base editors.

Duchenne muscular dystrophy is an X-linked monogenic disease caused by mutations in the dystrophin gene (DMD) characterized by progressive muscle weakness, leading to loss of ambulation and decreased life expectancy. Since the current standard of care for Duchenne muscular dystrophy is to merely treat symptoms, there is a dire need for treatment modalities that can correct the underlying genetic mutations. While several gene replacement therapies are being explored in clinical trials, one emerging approach that can directly correct mutations in genomic DNA is base editing. We have recently developed CRISPR-SKIP, a base editing strategy to induce permanent exon skipping by introducing C > T or A > G mutations at splice acceptors in genomic DNA, which can be used therapeutically to recover dystrophin expression when a genomic deletion leads to an out-of-frame DMD transcript. We now demonstrate that CRISPR-SKIP can be adapted to correct some forms of Duchenne muscular dystrophy by disrupting the splice acceptor in human DMD exon 45 with high efficiency, which enables open reading frame recovery and restoration of dystrophin expression. We also demonstrate that AAV-delivered split-intein base editors edit the splice acceptor of DMD exon 45 in cultured human cells and in vivo, highlighting the therapeutic potential of this strategy.

Optimizing Quantum Dot Probe Size for Single-Receptor Imaging.

Quantum dots (QDs) are nanocrystals with bright fluorescence and long-term photostability, attributes particularly beneficial for single-molecule imaging and molecular counting in the life sciences. The size of a QD nanocrystal determines its physicochemical and photophysical properties, both of which dictate the success of imaging applications. Larger nanocrystals typically have better optical properties, with higher brightness, red-shifted emission, reduced blinking, and greater stability. However, larger nanocrystals introduce molecular-labeling biases due to steric hindrance and nonspecific binding. Here, we systematically analyze the impact of nanocrystal size on receptor labeling in live and fixed cells. We designed three (core)shell QDs with red emission (600-700 nm) and crystalline sizes of 3.2, 5.5, and 8.3 nm. After coating with the same multidentate polymer, hydrodynamic sizes were 9.2 nm (QD9.2), 13.3 nm (QD13.3), and 17.4 nm (QD17.4), respectively. The QDs were conjugated to streptavidin and applied as probes for biotinylated neurotransmitter receptors. QD9.2 exhibited the highest labeling specificity for receptors in the narrow synaptic cleft (∼20-30 nm) in living neurons. However, for dense receptor labeling for molecular counting in live and fixed HeLa cells, QD13.3 yielded the highest counts. Nonspecific binding rose sharply for hydrodynamic sizes larger than 13.3 nm, with QD17.4 exhibiting particularly diminished specificity. Our comparisons further highlight needs to continue engineering the smallest QDs to increase single-molecule intensity, suppress blinking frequency, and inhibit nonspecific labeling in fixed and permeabilized cells. These results lay a foundation for designing QD probes with further reduced sizes to achieve unbiased labeling for quantitative and single-molecule imaging.

Directed Evolution of CRISPR-Cas9 Base Editors.

A recent publication by Thuronyi et al. described a directed evolution system called phage-assisted continuous evolution (PACE) that was used to generate improved variants of CRISPR-Cas9 base editors. These evolved base editors overcome some of the inherent limitations of the technology such as sequence context preferences, restricted editing windows, and large construct sizes.

Targeted exon skipping with AAV-mediated split adenine base editors.

Techniques for exclusion of exons from mature transcripts have been applied as gene therapies for treating many different diseases. Since exon skipping has been traditionally accomplished using technologies that have a transient effect, it is particularly important to develop new techniques that enable permanent exon skipping. We have recently shown that this can be accomplished using cytidine base editors for permanently disabling the splice acceptor of target exons. We now demonstrate the application of CRISPR-Cas9 adenine deaminase base editors to disrupt the conserved adenine within splice acceptor sites for programmable exon skipping. We also demonstrate that by altering the amino acid sequence of the linker between the adenosine deaminase domain and the Cas9-nickase or by coupling the adenine base editor with a uracil glycosylase inhibitor, the DNA editing efficiency and exon-skipping rates improve significantly. Finally, we developed a split base editor architecture compatible with adeno-associated viral packaging. Collectively, these results represent significant progress toward permanent in vivo exon skipping through base editing and, ultimately, a new modality of gene therapy for the treatment of genetic diseases.

Erratum: Author Correction: Targeted exon skipping with AAV-mediated split adenine base editors.

[This corrects the article DOI: 10.1038/s41421-019-0109-7.].

Publisher Correction: Combining Biomarkers with EMR Data to Identify Patients in Different Phases of Sepsis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Targeted Gene Knock Out Using Nuclease-Assisted Vector Integration: Hemi- and Homozygous Deletion of JAG1.

Gene editing technologies are revolutionizing fields such as biomedicine and biotechnology by providing a simple means to manipulate the genetic makeup of essentially any organism. Gene editing tools function by introducing double-stranded breaks at targeted sites within the genome, which the host cells repair preferentially by Non-Homologous End Joining. While the technologies to introduce double-stranded breaks have been extensively optimized, this progress has not been matched by the development of methods to integrate heterologous DNA at the target sites or techniques to detect and isolate cells that harbor the desired modification. We present here a technique for rapid introduction of vectors at target sites in the genome that enables efficient isolation of successfully edited cells.

CRISPR-SKIP: programmable gene splicing with single base editors.

CRISPR gene editing has revolutionized biomedicine and biotechnology by providing a simple means to engineer genes through targeted double-strand breaks in the genomic DNA of living cells. However, given the stochasticity of cellular DNA repair mechanisms and the potential for off-target mutations, technologies capable of introducing targeted changes with increased precision, such as single-base editors, are preferred. We present a versatile method termed CRISPR-SKIP that utilizes cytidine deaminase single-base editors to program exon skipping by mutating target DNA bases within splice acceptor sites. Given its simplicity and precision, CRISPR-SKIP will be broadly applicable in gene therapy and synthetic biology.

A microfluidic biochip platform for electrical quantification of proteins.

Sepsis, an adverse auto-immune response to an infection often causing life-threatening complications, results in the highest mortality and treatment cost of any illness in US hospitals. Several immune biomarker levels, including Interleukin 6 (IL-6), have shown a high correlation to the onset and progression of sepsis. Currently, no technology diagnoses and stratifies sepsis progression using biomarker levels. This paper reports a microfluidic biochip platform to detect proteins in undiluted human plasma samples. The device uses a differential enumeration platform that integrates Coulter counting principles, antigen specific capture chambers, and micro size bead based immunodetection to quantify cytokines. This microfluidic biochip was validated as a potential point of care technology by quantifying IL-6 from plasma samples (n = 29) with good correlation (R2 = 0.81) and agreement (Bland-Altman) compared to controls. In combination with previous applications, this point of care platform can potentially detect cell and protein biomarkers simultaneously for sepsis stratification.

Combining Biomarkers with EMR Data to Identify Patients in Different Phases of Sepsis.

Sepsis is a leading cause of death and is the most expensive condition to treat in U.S. hospitals. Despite targeted efforts to automate earlier detection of sepsis, current techniques rely exclusively on using either standard clinical data or novel biomarker measurements. In this study, we apply machine learning techniques to assess the predictive power of combining multiple biomarker measurements from a single blood sample with electronic medical record data (EMR) for the identification of patients in the early to peak phase of sepsis in a large community hospital setting. Combining biomarkers and EMR data achieved an area under the receiver operating characteristic (ROC) curve (AUC) of 0.81, while EMR data alone achieved an AUC of 0.75. Furthermore, a single measurement of six biomarkers (IL-6, nCD64, IL-1ra, PCT, MCP1, and G-CSF) yielded the same predictive power as collecting an additional 16 hours of EMR data(AUC of 0.80), suggesting that the biomarkers may be useful for identifying these patients earlier. Ultimately, supervised learning using a subset of biomarker and EMR data as features may be capable of identifying patients in the early to peak phase of sepsis in a diverse population and may provide a tool for more timely identification and intervention.