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CRISPR-Cas9-mediated genome editing in vancomycin-resistant Enterococcus faecium.
The Gram-positive bacterium Enterococcus faecium is becoming increasingly prevalent as a cause of hospital-acquired, antibiotic-resistant infections. A fundamental part of research into E. faecium biology relies on the ability to generate targeted mutants but this process is currently labour-intensive and time-consuming, taking 4 to 5 weeks per mutant. In this report, we describe a method relying on the high recombination rates of E. faecium and the application of the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas9 genome editing tool to more efficiently generate targeted mutants in the E. faecium chromosome. Using this tool and the multi-drug resistant clinical E. faecium strain E745, we generated a deletion mutant in the lacL gene, which encodes the large subunit of the E. faeciumβ-galactosidase. Blue/white screening using 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) could be used to distinguish between the wild-type and lacL deletion mutant. We also inserted two copies of gfp into the intrinsic E. faecium macrolide resistance gene msrC to generate stable green fluorescent cells. We conclude that CRISPR-Cas9 can be used to generate targeted genome modifications in E. faecium in 3 weeks, with limited hands-on time. This method can potentially be implemented in other Gram-positive bacteria with high intrinsic recombination rates.
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Refined sgRNA efficacy prediction improves large- and small-scale CRISPR-Cas9 applications.
Genome editing with the CRISPR-Cas9 system has enabled unprecedented efficacy for reverse genetics and gene correction approaches. While off-target effects have been successfully tackled, the effort to eliminate variability in sgRNA efficacies-which affect experimental sensitivity-is in its infancy. To address this issue, studies have analyzed the molecular features of highly active sgRNAs, but independent cross-validation is lacking. Utilizing fluorescent reporter knock-out assays with verification at selected endogenous loci, we experimentally quantified the target efficacies of 430 sgRNAs. Based on this dataset we tested the predictive value of five recently-established prediction algorithms. Our analysis revealed a moderate correlation (r = 0.04 to r = 0.20) between the predicted and measured activity of the sgRNAs, and modest concordance between the different algorithms. We uncovered a strong PAM-distal GC-content-dependent activity, which enabled the exclusion of inactive sgRNAs. By deriving nine additional predictive features we generated a linear model-based discrete system for the efficient selection (r = 0.4) of effective sgRNAs (CRISPRater). We proved our algorithms' efficacy on small and large external datasets, and provide a versatile combined on- and off-target sgRNA scanning platform. Altogether, our study highlights current issues and efforts in sgRNA efficacy prediction, and provides an easily-applicable discrete system for selecting efficient sgRNAs.
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CRISPR in Neurodegenerative Diseases Treatment: An Alternative Approach to Current Therapies.
Neurodegenerative diseases (NDs) pose a major challenge to global healthcare systems owing to their devastating effects and limited treatment options. These disorders are characterized by progressive loss of neuronal structure and function, resulting in cognitive and motor impairments. Current therapies primarily focus on symptom management rather than on targeting the underlying causes. However, clustered regularly interspaced short palindromic repeat (CRISPR) technology offers a promising alternative by enabling precise genetic modifications that could halt or even reverse ND progression. CRISPR-Cas9, the most widely used CRISPR system, acts as a molecular scissor targeting specific DNA sequences for editing. By designing guide RNAs (gRNAs) to match sequences in genes associated with NDs, researchers can leverage CRISPR to knockout harmful genes, correct mutations, or insert protective genes. This review explores the potential of CRISPR-based therapies in comparison with traditional treatments for NDs. As research advances, CRISPR has the potential to revolutionize ND treatment by addressing its genetic underpinnings. Ongoing clinical trials and preclinical studies continue to expand our understanding and application of this powerful tool to fight debilitating conditions.
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RNA-based gene targeting therapies for human papillomavirus driven cancers.
While platinum-based chemotherapy, radiation therapy and or surgery are effective in reducing human papillomavirus (HPV) driven cancer tumours, they have some significant drawbacks, including low specificity for tumour, toxicity, and severe adverse effects. Though current therapies for HPV-driven cancers are effective, severe late toxicity associated with current treatments contributes to the deterioration of patient quality of life. This warrants the need for novel therapies for HPV derived cancers. In this short review, we examined RNA-based therapies targeting the major HPV oncogenes, including short-interfering RNAs (siRNAs) and clustered regularly interspaced short palindromic repeats (CRISPR) as putative treatment modalities. We also explore other potential RNA-based targeting approaches such as microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and mRNA vaccines as future treatment modalities for HPV cancers. Some of these technologies have already been approved for clinical use for a range of other human diseases but not for HPV cancers. Here we explore the emerging evidence supporting the effectiveness of some of these gene-based therapies for HPV malignancies. In short, the evidence sheds promising light on the feasibility of translating these technologies into a clinically relevant treatment modality for HPV derived cancers and potentially other virally driven human cancers.
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Deciphering pathogenicity of variants of uncertain significance with CRISPR-edited iPSCs.
Genetic variants play an important role in conferring risk for cardiovascular diseases (CVDs). With the rapid development of next-generation sequencing (NGS), thousands of genetic variants associated with CVDs have been identified by genome-wide association studies (GWAS), but the function of more than 40% of genetic variants is still unknown. This gap of knowledge is a barrier to the clinical application of the genetic information. However, determining the pathogenicity of a variant of uncertain significance (VUS) is challenging due to the lack of suitable model systems and accessible technologies. By combining clustered regularly interspaced short palindromic repeats (CRISPR) and human induced pluripotent stem cells (iPSCs), unprecedented advances are now possible in determining the pathogenicity of VUS in CVDs. Here, we summarize recent progress and new strategies in deciphering pathogenic variants for CVDs using CRISPR-edited human iPSCs.
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FlashFry: a fast and flexible tool for large-scale CRISPR target design.
BACKGROUND: Genome-wide knockout studies, noncoding deletion scans, and other large-scale studies require a simple and lightweight framework that can quickly discover and score thousands of candidate CRISPR guides targeting an arbitrary DNA sequence. While several CRISPR web applications exist, there is a need for a high-throughput tool to rapidly discover and process hundreds of thousands of CRISPR targets. RESULTS: Here, we introduce FlashFry, a fast and flexible command-line tool for characterizing large numbers of CRISPR target sequences. With FlashFry, users can specify an unconstrained number of mismatches to putative off-targets, richly annotate discovered sites, and tag potential guides with commonly used on-target and off-target scoring metrics. FlashFry runs at speeds comparable to commonly used genome-wide sequence aligners, and output is provided as an easy-to-manipulate text file. CONCLUSIONS: FlashFry is a fast and convenient command-line tool to discover and score CRISPR targets within large DNA sequences.
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"Designer babies?!" A CRISPR-based learning module for undergraduates built around the CCR5 gene.
CRISPR-cas technology is being incorporated into undergraduate biology curriculum through lab experiences to immerse students in modern technology that is rapidly changing the landscape of science, medicine and agriculture. We developed and implemented an educational module that introduces students to CRISPR-cas technology in a Genetic course and an Advanced Genetics course. Our primary teaching objective was to immerse students in the design, strategy, conceptual modeling, and application of CRISPR-cas technology using the current research claim of the modification of the CCR5 gene in twin girls. This also allowed us to engage students in an open conversation about the bioethical implications of heritable germline and non-heritable somatic genomic editing. We assessed student-learning outcomes and conclude that this learning module is an effective strategy for teaching undergraduates the fundamentals and application of CRISPR-cas gene editing technology and can be adapted to other genes and diseases that are currently being treated with CRISPR-cas technology.
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CRISPRStudio: A User-Friendly Software for Rapid CRISPR Array Visualization.
The CRISPR-Cas system biologically serves as an adaptive defense mechanism against phages. However, there is growing interest in exploiting the hypervariable nature of the CRISPR locus, often of viral origin, for microbial typing and tracking. Moreover, the spacer content of any given strain provides a phage resistance profile. Large-scale CRISPR typing studies require an efficient method for showcasing CRISPR array similarities across multiple isolates. Historically, CRISPR arrays found in microbes have been represented by colored shapes based on nucleotide sequence identity and, while this approach is now routinely used, only scarce computational resources are available to automate the process, making it very time-consuming for large datasets. To alleviate this tedious task, we introduce CRISPRStudio, a command-line tool developed to accelerate CRISPR analysis and standardize the preparation of CRISPR array figures. It first compares nucleotide spacer sequences present in a dataset and then clusters them based on sequence similarity to assign a meaningful representative color. CRISPRStudio offers versatility to suit different biological contexts by including options such as automatic sorting of CRISPR loci and highlighting of shared spacers, while remaining fast and user-friendly.
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Generation of targeted mutant rice using a CRISPR-Cpf1 system.
CRISPR-Cpf1 is a newly identified CRISPR-Cas system, and Cpf1 was recently engineered as a molecular tool for targeted genome editing in mammalian cells. To test whether the engineered CRISPR-Cpf1 system could induce the production of rice mutants, we selected two genome targets in the OsPDS and OsBEL genes. Our results show that both targets could be efficiently mutated in transgenic rice plants using CRISPR-Cpf1. We found that pre-crRNAs with a full-length direct repeat sequence exhibited considerably increased efficiencies compared with mature crRNAs. In addition, the specificity and transmission of the mutation were investigated, and the behaviours of crRNA-Cpf1-induced plant targeted genome mutagenesis were assessed. Taken together, our results indicate that CRISPR-Cpf1 expression via stable transformation can efficiently generate specific and heritable targeted mutations in rice and thereby constitutes a novel and important approach to specific and precise plant genome editing.
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SpRY: Engineered CRISPR/Cas9 Harnesses New Genome-Editing Power.
Due to protospacer adjacent motif (PAM) requirements, CRISPR/Cas9 cannot access many genetic loci. A recent study by Walton et al. structurally engineered Streptococcus pyogenes Cas9 (SpCas9) to near-PAMless SpRY that can target most DNA sequences with high editing efficiency and flexibility. This newly engineered SpRY will potentially expand genome-editing capabilities for basic and applied research.
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