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VARSCOT: variant-aware detection and scoring enables sensitive and personalized off-target detection for CRISPR-Cas9.
BACKGROUND: Natural variations in a genome can drastically alter the CRISPR-Cas9 off-target landscape by creating or removing sites. Despite the resulting potential side-effects from such unaccounted for sites, current off-target detection pipelines are not equipped to include variant information. To address this, we developed VARiant-aware detection and SCoring of Off-Targets (VARSCOT). RESULTS: VARSCOT identifies only 0.6% of off-targets to be common between 4 individual genomes and the reference, with an average of 82% of off-targets unique to an individual. VARSCOT is the most sensitive detection method for off-targets, finding 40 to 70% more experimentally verified off-targets compared to other popular software tools and its machine learning model allows for CRISPR-Cas9 concentration aware off-target activity scoring. CONCLUSIONS: VARSCOT allows researchers to take genomic variation into account when designing individual or population-wide targeting strategies. VARSCOT is available from https://github.com/BauerLab/VARSCOT .
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Application of CRISPR-Cas9 technology in diabetes research.
Diabetes is a chronic disorder with rapidly increasing prevalence that is a major global issue of our current era. There are two major types of diabetes. Polygenic forms of diabetes include type 1 diabetes (T1D) and type 2 diabetes (T2D) and its monogenic forms are maturity-onset diabetes of the young (MODY) and neonatal diabetes mellitus (NDM). There are no permanent therapeutic approaches for diabetes and current therapies rely on regular administration of various drugs or insulin injection. Recently, gene editing strategies have offered new promise for treating genetic disorders. Targeted genome editing is a fast-growing technology, recruiting programmable nucleases to specifically modify target genomic sequences. These targeted nucleases generate double-strand breaks at target regions in the genome, which induce cellular repair pathways including non-homologous end joining (NHEJ) and homology-directed repair (HDR). Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a novel gene-editing system, permitting precise genome modification. CRISPR/Cas9 has great potential for various applications in diabetic research such as gene screening, generation of diabetic animal models and treatment. In this article, gene-editing strategies are summarized with a focus on the CRISPR/Cas9 approach in diabetes research.
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Detection of CRISPR/Cas9-Generated Off-Target Effect by Integration-Defective Lentiviral Vector.
Clustered regularly interspaced short palindromic repeat (CRISPR) and other gene editing technologies such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) show great promises for research and therapeutic applications. One major concern is the off-target effects generated by these nucleases at unintended genomic sequences. In silico methods are usually used for off-target site prediction. However, based on currently available algorithms, the predicted cleavage activity at these potential off-target sites does not always reflect the true cleavage in vivo. Here we present an unbiased screening protocol using integration-defective lentiviral vector (IDLV) and deep sequencing to map the off-target sites generated by gene editing tools.
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Protocol to rapidly screen CRISPR-Cas9 gene editing outcomes in a cell population by mutating eGFP to a blue or non-fluorescent phenotype.
When designing genome editing therapy, it is crucial to measure outcomes of DNA damage repair. Here, we present a protocol to distinguish the outcome of targeted DNA damage repair from the bottom up, through a previously established readout of enhanced green fluorescent protein (eGFP) to blue fluorescent protein (BFP) mutations. We describe steps for producing eGFP-positive cells and differentiating between non-homologous end joining-induced gene knockout and homology-directed repair-induced-directed mutation in these cells. This protocol has potential for high-throughput and scalable assessment of gene editing techniques. For complete details on the use and execution of this protocol, please refer to Walther et al.1 and Wilbie et al.2.
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Prime Editing of Mouse Primary Neurons.
Prime editing is a hybrid genome editing technology that introduces small edits on the genome with high precision. It combines nickase Cas9 with reverse transcriptase to prime and synthesizes edited DNA from RNA, reducing unintended insertions and deletions on the genome. This protocol describes the design of prime editing guide RNAs (pegRNAs), cloning of plasmids, nucleofection of mouse primary neurons, and preparation for next-generation sequencing. Directions are given for pegRNA and PE3b gRNA design and construction using PegAssist, a publicly available webtool and plasmid set. Prime editing in neurons allows genome manipulation while maintaining endogenous gene expression, making it ideal for studying protein structure/function relationships and pathogenic variants in a native neuronal context.
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Photoactivable CRISPR for Biosensing and Cancer Therapy.
Photoactivable CRISPR technology represents a transformative approach in the field of genome editing, offering unprecedented control over gene editing with high spatial and temporal precision. By harnessing the power of light to modulate the activity of CRISPR components, this innovative strategy enables precise regulation of Cas proteins, guide RNAs, and ribonucleoprotein complexes. Recent advancements in optical control methodologies, including the development of photoactivable nanocarriers, have significantly expanded the potential applications of CRISPR in biomedical fields. This Concept highlights the latest developments in designing photoactivable CRISPR systems and their promising applications in biosensing and cancer therapy. Additionally, the remaining challenges and future trends are also discussed. It is expected that the photoactivable CRISPR would facilitate translating more precise gene therapies into clinical use.
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Transposons and CRISPR: Rewiring Gene Editing.
CRISPR-Cas is driving a gene editing revolution because of its simple reprogramming. However, off-target effects and dependence on the double-strand break repair pathways impose important limitations. Because homology-directed repair acts primarily in actively dividing cells, many of the current gene correction/replacement approaches are restricted to a minority of cell types. Furthermore, current approaches display low efficiency upon insertion of large DNA cargos (e.g., sequences containing multiple gene circuits with tunable functionalities). Recent research has revealed new links between CRISPR-Cas systems and transposons providing new scaffolds that might overcome some of these limitations. Here, we comment on two new transposon-associated RNA-guided mechanisms considering their potential as new gene editing solutions. Initially, we focus on a group of small RNA-guided endonucleases of the IS200/IS605 family of transposons, which likely evolved into class 2 CRISPR effector nucleases (Cas9s and Cas12s). We explore the diversity of these nucleases (named OMEGA, obligate mobile element-guided activity) and analyze their similarities with class 2 gene editors. OMEGA nucleases can perform gene editing in human cells and constitute promising candidates for the design of new compact RNA-guided platforms. Then, we address the co-option of the RNA-guided activity of different CRISPR effector nucleases by a specialized group of Tn7-like transposons to target transposon integration. We describe the various mechanisms used by these RNA-guided transposons for target site selection and integration. Finally, we assess the potential of these new systems to circumvent some of the current gene editing challenges.
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Efficient Genome Editing Achieved via Plug-and-Play Adenovirus Piggyback Transport of Cas9/gRNA Complex on Viral Capsid Surface.
The capacity to efficiently deliver the gene-editing enzyme complex to target cells is favored over other forms of gene delivery as it offers one-time hit-and-run gene editing, thus improving precision and safety and reducing potential immunogenicity against edited cells in clinical applications. Here we performed a proof-of-mechanism study and demonstrated that a simian adenoviral vector for DNA delivery can be repurposed as a robust intracellular delivery platform for a functional Cas9/guide RNA (gRNA) complex to recipient cells. In this system, the clinically relevant adenovirus was genetically engineered with a plug-and-display technology based on SpyTag003/SpyCatcher003 coupling chemistry. Under physiological conditions, an off-the-shelf mixture of viral vector with SpyTag003 incorporated into surface capsid proteins and Cas9 fused with SpyCatcher003 led to a rapid titration reaction yielding adenovirus carrying Cas9SpyCatcher003 on the virus surface. The Cas9 fusion protein-conjugated viruses in the presence of a reporter gRNA delivered gene-editing functions to cells with an efficiency comparable to that of a commercial CRISPR/Cas9 transfection reagent. Our data fully validate the adenoviral "piggyback" approach to deliver an intracellularly acting enzyme cargo and, thus, warrant the prospect of engineering tissue-targeted adenovirus carrying Cas9/gRNA for in vivo gene editing.
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Editing Candida: Origins and Advances of CRISPR Tools.
Pathogens causing candidiasis encompass a diverse group of ascomycetous yeasts that have become essential models for studying fungal adaptability, pathogenicity, and host-pathogen interactions. Although many candidiasis-promoting species exist as commensals within host microbiota, several have acquired virulence traits that enable opportunistic infections, positioning them as a leading cause of invasive fungal disease in humans. Deciphering the molecular and genetic determinants that underpin the biology of organisms responsible for candidiasis has long been a central objective in medical and molecular mycology. However, research progress has been constrained by intrinsic biological challenges, including noncanonical codon usage and the absence of a complete sexual cycle in diploid species, which have complicated traditional genetic manipulation. CRISPR-Cas9 genome editing has overcome many of these limitations, providing a precise, efficient, and versatile framework for targeted genomic modification. This system has facilitated functional genomic studies ranging from single-gene deletions to high-throughput mutagenesis, yielding new insights into the mechanisms governing virulence, antifungal resistance, and stress adaptation. Since its initial application in Candida albicans, CRISPR-Cas9 technology has been refined and adapted for other clinically and industrially relevant species, including Nakaseomyces glabratus (formerly referred to as Candida glabrata), Candida parapsilosis, and Candida auris. The present work provides an overview of the evolution of genetic approaches employed in research directed against candidiasis-associated species, with a particular focus on the development and optimization of CRISPR-based systems. It highlights how recent advancements have improved the genetic tractability of these pathogens and outlines emerging opportunities for both fundamental and applied studies in fungal biology.
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Bar-seq strategies for the LeishGEdit toolbox.
The number of fully sequenced genomes increases steadily but the function of many genes remains unstudied. To accelerate dissection of gene function in Leishmania spp. and other kinetoplastids we previously developed a streamlined pipeline for CRISPR-Cas9 gene editing, which we termed LeishGEdit. To facilitate high-throughput mutant screens we have adapted this pipeline by barcoding mutants with unique 17-nucleotide barcodes, allowing loss-of-function screens in mixed populations. Here we present primer design and analysis tools that facilitate these bar-seq strategies. We have developed a standalone easy-to-use pipeline to design CRISPR primers suitable for the LeishGEdit toolbox for any given genome and have generated a list of 14,995 barcodes. Barcodes and oligo sequences are now accessible through our website www.leishgedit.net allowing researchers to pursue bar-seq experiments in all currently available TriTrypDB genomes (release 41). This will streamline CRISPR bar-seq assays in kinetoplastids, enabling pooled mutant screens across the community.
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