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.

Corrigendum: Chromatin structure and context-dependent sequence features control prime editing efficiency.

[This corrects the article DOI: 10.3389/fgene.2023.1222112.].

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.

Chromatin structure and context-dependent sequence features control prime editing efficiency.

Prime editing (PE) is a highly versatile CRISPR-Cas9 genome editing technique. The current constructs, however, have variable efficiency and may require laborious experimental optimization. This study presents statistical models for learning the salient epigenomic and sequence features of target sites modulating the editing efficiency and provides guidelines for designing optimal PEs. We found that both regional constitutive heterochromatin and local nucleosome occlusion of target sites impede editing, while position-specific G/C nucleotides in the primer-binding site (PBS) and reverse transcription (RT) template regions of PE guide RNA (pegRNA) yield high editing efficiency, especially for short PBS designs. The presence of G/C nucleotides was most critical immediately 5' to the protospacer adjacent motif (PAM) site for all designs. The effects of different last templated nucleotides were quantified and observed to depend on the length of both PBS and RT templates. Our models found AGG to be the preferred PAM and detected a guanine nucleotide four bases downstream of the PAM to facilitate editing, suggesting a hitherto-unrecognized interaction with Cas9. A neural network interpretation method based on nonextensive statistical mechanics further revealed multi-nucleotide preferences, indicating dependency among several bases across pegRNA. Our work clarifies previous conflicting observations and uncovers context-dependent features important for optimizing PE designs.

Chromatin structure and context-dependent sequence features control prime editing efficiency.

Prime editor (PE) is a highly versatile CRISPR-Cas9 genome editing technique. The current constructs, however, have variable efficiency and may require laborious experimental optimization. This study presents statistical models for learning the salient epigenomic and sequence features of target sites modulating the editing efficiency and provides guidelines for designing optimal PEs. We found that both regional constitutive heterochromatin and local nucleosome occlusion of target sites impede editing, while position-specific G/C nucleotides in the primer binding site (PBS) and reverse transcription (RT) template regions of PE guide-RNA (pegRNA) yield high editing efficiency, especially for short PBS designs. The presence of G/C nucleotides was most critical immediately 5' to the protospacer adjacent motif (PAM) site for all designs. The effects of different last templated nucleotides were quantified and seen to depend on both PBS and RT template lengths. Our models found AGG to be the preferred PAM and detected a guanine nucleotide four bases downstream of PAM to facilitate editing, suggesting a hitherto-unrecognized interaction with Cas9. A neural network interpretation method based on nonextensive statistical mechanics further revealed multi-nucleotide preferences, indicating dependency among several bases across pegRNA. Our work clarifies previous conflicting observations and uncovers context-dependent features important for optimizing PE designs.

Delivering Base Editors In Vivo by Adeno-Associated Virus Vectors.

CRISPR base editors are genome-modifying proteins capable of creating single-base substitutions in DNA but without the requirement for a DNA double-strand break. Given their ability to precisely edit DNA, they hold tremendous therapeutic potential. Here, we describe procedures for delivering base editors in vivo via adeno-associated virus (AAV) vectors, a promising engineered gene delivery vehicle capable of transducing a range of cell types and tissues. We provide step by step protocols for (i) designing and validating base editing systems, (ii) packaging base editors into recombinant AAV vector particles, (iii) delivering AAV to the central nervous system via intrathecal injection, and (iv) quantifying base editing frequencies by next-generation sequencing.

Delineating cooperative effects of Notch and biomechanical signals on patterned liver differentiation.

Controlled in vitro multicellular culture systems with defined biophysical microenvironment have been used to elucidate the role of Notch signaling in the spatiotemporal regulation of stem and progenitor cell differentiation. In addition, computational models incorporating features of Notch ligand-receptor interactions have provided important insights into Notch pathway signaling dynamics. However, the mechanistic relationship between Notch-mediated intercellular signaling and cooperative microenvironmental cues is less clear. Here, liver progenitor cell differentiation patterning was used as a model to systematically evaluate the complex interplay of cellular mechanics and Notch signaling along with identifying combinatorial mechanisms guiding progenitor fate. We present an integrated approach that pairs a computational intercellular signaling model with defined microscale culture configurations provided within a cell microarray platform. Specifically, the cell microarray-based experiments were used to validate and optimize parameters of the intercellular Notch signaling model. This model incorporated the experimentally established multicellular dimensions of the cellular microarray domains, mechanical stress-related activation parameters, and distinct Notch receptor-ligand interactions based on the roles of the Notch ligands Jagged-1 and Delta-like-1. Overall, these studies demonstrate the spatial control of mechanotransduction-associated components, key growth factor and Notch signaling interactions, and point towards a possible role of E-Cadherin in translating intercellular mechanical gradients to downstream Notch signaling.

CRISPR base editing of cis-regulatory elements enables the perturbation of neurodegeneration-linked genes.

CRISPR technology has demonstrated broad utility for controlling target gene expression; however, there remains a need for strategies capable of modulating expression via the precise editing of non-coding regulatory elements. Here, we demonstrate that CRISPR base editors, a class of gene-modifying proteins capable of creating single-base substitutions in DNA, can be used to perturb gene expression via their targeted mutagenesis of cis-acting sequences. Using the promoter region of the human huntingtin (HTT) gene as an initial target, we show that editing of the binding site for the transcription factor NF-κB led to a marked reduction in HTT gene expression in base-edited cell populations. We found that these gene perturbations were persistent and specific, as a transcriptome-wide RNA analysis revealed minimal off-target effects resulting from the action of the base editor protein. We further demonstrate that this base-editing platform could influence gene expression in vivo as its delivery to a mouse model of Huntington's disease led to a potent decrease in HTT mRNA in striatal neurons. Finally, to illustrate the applicability of this concept, we target the amyloid precursor protein, showing that multiplex editing of its promoter region significantly perturbed its expression. These findings demonstrate the potential for base editors to regulate target gene expression.

Development and Implementation of a Remote Enzyme Kinetics Laboratory Exercise.

The COVID-19 pandemic imposed many new challenges on educational systems and increased the demand for novel strategies for effectively teaching laboratory skills without in-person instruction and without access to laboratory space, including critical specialized equipment. While this novel remote instruction modality is compatible with teaching the theory behind experimental techniques, the lack of lab activities that enable learning of laboratory skills severely limits the outcome of instruction. In order to overcome this problem and effectively supplement lectures with hands-on laboratory exercises, we developed an at-home enzyme kinetics lab that provides a safe alternative to traditional enzyme kinetics instructional labs typically performed in a laboratory setting. The combination of a simple design of the activity, accessibility of equipment used, and relatively low overall cost yields an effective exercise for teaching experimental design and basic laboratory skills remotely while providing a unique opportunity for students to learn about enzyme kinetics.

Remote Laboratory Exercise to Develop Micropipetting Skills.

The rapid spread of COVID-19 has fundamentally transformed our educational system. The need to protect both students and instructors from exposure to viral infection has required the implementation of remote instructional models. Although this alternative delivery approach can be successfully implemented to teach the theoretical foundations of multiple disciplines, teaching technical skills poses a major challenge, particularly in various biology fields, where observation of biological safety guidelines and the high cost of analytical equipment represent major impediments for remote instruction. To overcome this problem, we have developed a laboratory exercise to teach students how to use micropipettes that can be completed remotely using materials that can be purchased at a fraction of the cost of the instructional equipment normally reserved for in-person instruction. Our evaluation of the effectiveness of this remote lab indicated that the majority of students who participated in a survey believed they attained the learning objectives and felt confident in their lab technique after completing the exercises. The simplicity, relatively low cost, and effectiveness of this approach makes it highly adaptable for other classrooms and educational settings.

ABC-GWAS: Functional Annotation of Estrogen Receptor-Positive Breast Cancer Genetic Variants.

Over the past decade, hundreds of genome-wide association studies (GWAS) have implicated genetic variants in various diseases, including cancer. However, only a few of these variants have been functionally characterized to date, mainly because the majority of the variants reside in non-coding regions of the human genome with unknown function. A comprehensive functional annotation of the candidate variants is thus necessary to fill the gap between the correlative findings of GWAS and the development of therapeutic strategies. By integrating large-scale multi-omics datasets such as the Cancer Genome Atlas (TCGA) and the Encyclopedia of DNA Elements (ENCODE), we performed multivariate linear regression analysis of expression quantitative trait loci, sequence permutation test of transcription factor binding perturbation, and modeling of three-dimensional chromatin interactions to analyze the potential molecular functions of 2,813 single nucleotide variants in 93 genomic loci associated with estrogen receptor-positive breast cancer. To facilitate rapid progress in functional genomics of breast cancer, we have created "Analysis of Breast Cancer GWAS" (ABC-GWAS), an interactive database of functional annotation of estrogen receptor-positive breast cancer GWAS variants. Our resource includes expression quantitative trait loci, long-range chromatin interaction predictions, and transcription factor binding motif analyses to prioritize putative target genes, causal variants, and transcription factors. An embedded genome browser also facilitates convenient visualization of the GWAS loci in genomic and epigenomic context. ABC-GWAS provides an interactive visual summary of comprehensive functional characterization of estrogen receptor-positive breast cancer variants. The web resource will be useful to both computational and experimental biologists who wish to generate and test their hypotheses regarding the genetic susceptibility, etiology, and carcinogenesis of breast cancer. ABC-GWAS can also be used as a user-friendly educational resource for teaching functional genomics. ABC-GWAS is available at http://education.knoweng.org/abc-gwas/.

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.

Epigenetic engineering of yeast reveals dynamic molecular adaptation to methylation stress and genetic modulators of specific DNMT3 family members.

Cytosine methylation is a ubiquitous modification in mammalian DNA generated and maintained by several DNA methyltransferases (DNMTs) with partially overlapping functions and genomic targets. To systematically dissect the factors specifying each DNMT's activity, we engineered combinatorial knock-in of human DNMT genes in Komagataella phaffii, a yeast species lacking endogenous DNA methylation. Time-course expression measurements captured dynamic network-level adaptation of cells to DNMT3B1-induced DNA methylation stress and showed that coordinately modulating the availability of S-adenosyl methionine (SAM), the essential metabolite for DNMT-catalyzed methylation, is an evolutionarily conserved epigenetic stress response, also implicated in several human diseases. Convolutional neural networks trained on genome-wide CpG-methylation data learned distinct sequence preferences of DNMT3 family members. A simulated annealing interpretation method resolved these preferences into individual flanking nucleotides and periodic poly(A) tracts that rotationally position highly methylated cytosines relative to phased nucleosomes. Furthermore, the nucleosome repeat length defined the spatial unit of methylation spreading. Gene methylation patterns were similar to those in mammals, and hypo- and hypermethylation were predictive of increased and decreased transcription relative to control, respectively, in the absence of mammalian readers of DNA methylation. Introducing controlled epigenetic perturbations in yeast thus enabled characterization of fundamental genomic features directing specific DNMT3 proteins.

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.

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.

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.

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

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

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.

Disruption of the ß1L Isoform of GABP Reverses Glioblastoma Replicative Immortality in a TERT Promoter Mutation-Dependent Manner.

TERT promoter mutations reactivate telomerase, allowing for indefinite telomere maintenance and enabling cellular immortalization. These mutations specifically recruit the multimeric ETS factor GABP, which can form two functionally independent transcription factor species: a dimer or a tetramer. We show that genetic disruption of GABPß1L (ß1L), a tetramer-forming isoform of GABP that is dispensable for normal development, results in TERT silencing in a TERT promoter mutation-dependent manner. Reducing TERT expression by disrupting ß1L culminates in telomere loss and cell death exclusively in TERT promoter mutant cells. Orthotopic xenografting of ß1L-reduced, TERT promoter mutant glioblastoma cells rendered lower tumor burden and longer overall survival in mice. These results highlight the critical role of GABPß1L in enabling immortality in TERT promoter mutant glioblastoma.