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CRISPR-DT: designing gRNAs for the CRISPR-Cpf1 system with improved target efficiency and specificity.
MOTIVATION: The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cpf1 system has been successfully applied in genome editing. However, target efficiency of the CRISPR-Cpf1 system varies among different guide RNA (gRNA) sequences. RESULTS: In this study, we reanalyzed the published CRISPR-Cpf1 gRNAs data and found many sequence and structural features related to their target efficiency. With the aid of Random Forest in feature selection, a support vector machine model was created to predict target efficiency for any given gRNAs. We have developed the first CRISPR-Cpf1 web service application, CRISPR-DT (CRISPR DNA Targeting), to help users design optimal gRNAs for the CRISPR-Cpf1 system by considering both target efficiency and specificity. CRISPR-DT will empower researchers in genome editing. AVAILABILITY AND IMPLEMENTATION: CRISPR-DT, mainly implemented in Perl, PHP and JavaScript, is freely available at http://bioinfolab.miamioh.edu/CRISPR-DT. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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What are the current bottlenecks in developing and applying CRISPR technologies?
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Editing the Mitochondrial Genome: No CRISPR Required.
Precise gene editing of mitochondrial DNA (mtDNA) is essential for the generation of model systems to study rare mitochondrial diseases but was long deemed impossible - until now. A recent publication by Mok et al. describes a gene editing tool capable of installing point mutations in mtDNA, and it does not involve CRISPR.
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CRISPR: fundamental principles and implications for anaesthesia.
Clustered regularly interspaced short palindromic repeats (CRISPR)-based medical therapies are increasingly gaining regulatory approval worldwide. Consequently, patients receiving CRISPR therapy will come under the care of anaesthesiologists. An understanding of CRISPR, its technological implementations, and the characteristics of patients likely to receive this therapy will be essential to caring for this patient population. However, the role of CRISPR in anaesthesiology extends beyond simply caring for patients with prior CRISPR therapy. CRISPR has multiple direct potential applications in anaesthesia, particularly for managing chronic pain and critical illness. Additionally, given the unique skills anaesthesiologists possess, CRISPR potentially allows new roles for anaesthesiologists in the field of oncology. Consequently, CRISPR technology could enable new domains of anaesthetic practice. This review provides a primer on CRISPR for anaesthesiologists and an overview on how the technology could impact the field.
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Metagenome-Derived CRISPR-Cas12a Mining and Characterization.
The advent of clustered regularly interspaced short palindromic repeats (CRISPR)-based technologies has revolutionized genome editing, with continued interest in expanding the CRISPR-associated proteins (Cas) toolbox with diverse, efficient, and specific effectors. CRISPR-Cas12a is a potent, programmable RNA-guided dual nickase, broadly used for genome editing. Here, we mined dairy cow microbial metagenomes for CRISPR-Cas systems, unraveling novel Cas12a enzymes. Using in silico pipelines, we characterized and predicted key drivers of CRISPR-Cas12a activity, encompassing guides and protospacer adjacent motifs for five systems. We next assessed their functional potential in cell-free transcription-translation assays with GFP-based fluorescence readouts. Lastly, we determined their genome editing potential in vivo in Escherichia coli by generating 1 kb knockouts. Unexpectedly, we observed natural sequence variation in the bridge-helix domain of the best-performing candidate and used mutagenesis to alter the activity of Cas12a orthologs, resulting in increased gene editing capabilities of a relatively inefficient candidate. This study illustrates the potential of underexplored metagenomic sequence diversity for the development and refinement of genome editing effectors.
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The CRISPR-Cas Mechanism for Adaptive Immunity and Alternate Bacterial Functions Fuels Diverse Biotechnologies.
Bacterial and archaeal CRISPR-Cas systems offer adaptive immune protection against foreign mobile genetic elements (MGEs). This function is regulated by sequence specific binding of CRISPR RNA (crRNA) to target DNA/RNA, with an additional requirement of a flanking DNA motif called the protospacer adjacent motif (PAM) in certain CRISPR systems. In this review, we discuss how the same fundamental mechanism of RNA-DNA and/or RNA-RNA complementarity is utilized by bacteria to regulate two distinct functions: to ward off intruding genetic materials and to modulate diverse physiological functions. The best documented examples of alternate functions are bacterial virulence, biofilm formation, adherence, programmed cell death, and quorum sensing. While extensive complementarity between the crRNA and the targeted DNA and/or RNA seems to constitute an efficient phage protection system, partial complementarity seems to be the key for several of the characterized alternate functions. Cas proteins are also involved in sequence-specific and non-specific RNA cleavage and control of transcriptional regulator expression, the mechanisms of which are still elusive. Over the past decade, the mechanisms of RNA-guided targeting and auxiliary functions of several Cas proteins have been transformed into powerful gene editing and biotechnological tools. We provide a synopsis of CRISPR technologies in this review. Even with the abundant mechanistic insights and biotechnology tools that are currently available, the discovery of new and diverse CRISPR types holds promise for future technological innovations, which will pave the way for precision genome medicine.
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CRISPR Editing Takes Aim at Ischemia/Reperfusion Injury.
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The Right Tools for the Right Job: CRISPR-pass Could Offer Safe Gene Correction for Many Disorders.
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Deconvolution of Complex DNA Repair (DECODR): Establishing a Novel Deconvolution Algorithm for Comprehensive Analysis of CRISPR-Edited Sanger Sequencing Data.
During CRISPR-directed gene editing, multiple gene repair mechanisms interact to produce a wide and largely unpredictable variety of sequence changes across an edited population of cells. Shortcomings inherent to previously available proposal-based insertion and deletion (indel) analysis software necessitated the development of a more comprehensive tool that could detect a larger range and variety of indels while maintaining the ease of use of tools currently available. To that end, we developed Deconvolution of Complex DNA Repair (DECODR). DECODR can detect indels formed from single or multi-guide CRISPR experiments without a limit on indel size. The software is accurate in determining the identities and positions of inserted and deleted bases in DNA extracts from both clonally expanded and bulk cell populations. The accurate identification and output of any potential indel allows for DECODR analysis to be executed in experiments utilizing potentially any configuration of donor DNA sequences, CRISPR-Cas, and endogenous DNA repair pathways.
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Optimizing Prime Editing in Zebrafish.
Prime editing is a clustered regularly interspaced short palindromic repeats-based approach that enables the introduction of precise genetic modifications, including missense mutations, making it valuable for generating disease models. The comparative performance of novel prime editor (PE) variants in zebrafish remains largely unexplored. Here, we systematically evaluated the efficiency of five PEs-PE2, PE6b, PE6c, PEmax, and PE7-in zebrafish. We tested mRNA encoding for each of these PEs with prime editing guide RNAs (pegRNAs) designed to install five missense mutations. Efficient editing was achieved at four of the five sites with multiple PEs. Among these, PEmax emerged as the most efficient editor for introducing pure prime edits, with rates reaching 15.34%. We found that strategies proposed to block 3' degradation of pegRNAs (epegRNAs and addition of a La RNA binding motif to the PE) did not improve performance in our assays. Together, these findings establish PEmax as a robust tool to introduce missense mutations into zebrafish.
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