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CRISPR/Cpf1 enables fast and simple genome editing of Saccharomyces cerevisiae.
Cpf1 represents a novel single RNA-guided CRISPR/Cas endonuclease system suitable for genome editing with distinct features compared with Cas9. We demonstrate the functionality of three Cpf1 orthologues - Acidaminococcus spp. BV3L6 (AsCpf1), Lachnospiraceae bacterium ND2006 (LbCpf1) and Francisella novicida U112 (FnCpf1) - for genome editing of Saccharomyces cerevisiae. These Cpf1-based systems enable fast and reliable introduction of donor DNA on the genome using a two-plasmid-based editing approach together with linear donor DNA. LbCpf1 and FnCpf1 displayed editing efficiencies comparable with the CRISPR/Cas9 system, whereas AsCpf1 editing efficiency was lower. Further characterization showed that AsCpf1 and LbCpf1 displayed a preference for their cognate crRNA, while FnCpf1-mediated editing with similar efficiencies was observed using non-cognate crRNAs of AsCpf1 and LbCpf1. In addition, multiplex genome editing using a single LbCpf1 crRNA array is shown to be functional in yeast. This work demonstrates that Cpf1 broadens the genome editing toolbox available for Saccharomyces cerevisiae. © 2017 The Authors. Yeast published by John Wiley & Sons, Ltd.
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Genome editing in archaeal viruses and endogenous viral protein purification.
Archaea-infecting viruses are morphologically and genomically among the most diverse entities. Unfortunately, they are also fairly understudied due to a lack of efficient genetic tools. Here, we present a detailed protocol for the CRISPR/Cas-based genome editing of the virus SIRV2 infecting the genus Sulfolobus, which could easily be adapted to other archaeal viruses. This protocol also includes the procedure for endogenous viral protein purification and identification, allowing for assessing the molecular mechanisms behind virus life cycle and virus-host interactions. For complete details on the use and execution of this protocol, please refer to Mayo-Muñoz et al. (2018) and Bhoobalan-Chitty et al. (2019).
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Knockout of secondary alcohol dehydrogenase in Nocardia cholesterolicum NRRL 5767 by CRISPR/Cas9 genome editing technology.
Nocardia cholesterolicum NRRL 5767 is well-known for its ability to convert oleic acid to 10-hydroxystearic acid (~88%, w/w) and 10-ketostearic acid (~11%, w/w). Conversion of oleic acid to 10-hydroxystearic acid and then to 10-ketostearic acid has been proposed to be catalyzed by oleate hydratase and secondary alcohol dehydrogenase, respectively. Hydroxy fatty acids are value-added with many industrial applications. The objective of this study was to improve the Nocardia cholesterolicum NRRL5767 strain by CRISPR/Cas9 genome editing technology to knockout the secondary alcohol dehydrogenase gene, thus blocking the conversion of 10-hydroxystearic acid to 10-ketostearic acid. The improved strain would produce 10-hydroxystearic acid solely from oleic acid. Such improvement would enhance the production of 10-hydroxystearic acid by eliminating downstream separation of 10-hydroxystearic acid from 10-ketostearic acid. Here, we report: (1) Molecular cloning and characterization of two functional recombinant oleate hydratase isozymes and a functional recombinant secondary alcohol dehydrogenase from Nocardia cholesterolicum NRRL5767. Existence of two oleate hydratase isozymes may explain the high conversion yield of 10-hydroxystearic acid from oleic acid. (2) Construction of a CRISPR/Cas9/sgRNA chimeric plasmid that specifically targeted the secondary alcohol dehydrogenase gene by Golden Gate Assembly. (3) Transformation of the chimeric plasmid into Nocardia cholesterolicum NRRL 5767 by electroporation and screening of secondary alcohol dehydrogenase knockout mutants. Two mutants were validated by their lack of secondary alcohol dehydrogenase activity at the protein level and mutation at the targeted 5' coding region and the 5' upstream at the DNA level. The knockout mutants offer improvements by converting added oleic acid to solely 10-hydroxystearic acid, thus eliminating downstream separation of 10-hydroxystearic acid from 10-ketostearic acid. To the best of our knowledge, we report the first successful knockout of a target gene in the Nocardia species using CRISPR/Cas9/sgRNA-mediated genome editing technology.
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Discovering CRISPR-Cas off-target breaks.
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Novel gene-based therapeutic approaches for the management of hepatic complications in diabetes: Reviewing recent advances.
Diabetes mellitus is a chronic metabolic disorder marked by hyperglycemia and systemic complications, including hepatic dysfunction, significantly contributing to disease progression and morbidity. This article reviews recent advances in gene-based therapeutic strategies targeting hepatic complications in diabetes, offering a promising approach for precision medicine by addressing underlying molecular mechanisms. Traditional treatments for hepatic complications in diabetes often manage symptoms rather than molecular causes, showing limited efficacy. Gene-based therapies are poised to correct dysfunctional pathways and restore hepatic function. Fundamental gene therapy approaches include gene silencing via small interfering RNAs (siRNAs) to target hepatic glucose production, lipid metabolism, and inflammation. Viral vectors can restore insulin sensitivity and reduce oxidative stress in diabetic livers. Genome editing, especially CRISPR-Cas9, allows the precise modification of disease-associated genes, offering immense potential for hepatic complication treatment. Strategies using CRISPR-Cas9 to enhance insulin receptor expression and modulate aberrant lipid regulatory genes are explored. Safety challenges in gene-based therapies, such as off-target effects and immune responses, are discussed. Advances in nanoparticle-based delivery systems and targeted gene editing techniques offer solutions to enhance specificity and minimize adverse effects. In conclusion, gene-based therapeutic approaches are a transformative direction in managing hepatic complications in diabetes. Further research is needed to optimize efficacy, safety, and long-term outcomes. Nevertheless, these innovative strategies promise to improve the lives of individuals with diabetes by addressing hepatic dysfunction's genetic root causes.
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The Geneva Statement on Heritable Human Genome Editing: A Criticism.
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Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors.
CRISPR/Cas9-based genome editing can easily generate knockout mouse models by disrupting the gene sequence, but its efficiency for creating models that require either insertion of exogenous DNA (knock-in) or replacement of genomic segments is very poor. The majority of mouse models used in research involve knock-in (reporters or recombinases) or gene replacement (e.g., conditional knockout alleles containing exons flanked by LoxP sites). A few methods for creating such models have been reported that use double-stranded DNA as donors, but their efficiency is typically 1-10% and therefore not suitable for routine use. We recently demonstrated that long single-stranded DNAs (ssDNAs) serve as very efficient donors, both for insertion and for gene replacement. We call this method efficient additions with ssDNA inserts-CRISPR (Easi-CRISPR) because it is a highly efficient technology (efficiency is typically 30-60% and reaches as high as 100% in some cases). The protocol takes ∼2 months to generate the founder mice.
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Fanzors: Mysterious TnpB-Like Bacterial Transposon-Related RNA-Guided DNA Nucleases of Eukaryotes.
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Gene Targeting in Mouse Embryonic Stem Cells via CRISPR/Cas9 Ribonucleoprotein (RNP)-Mediated Genome Editing.
The CRISPR/Cas9-mediated genome-editing system enables the development of gene-modified mice using fertilized eggs. However, while the efficiency in developing gene knockout mice by inducing small indel mutations would be good enough, the successful ratio to create large side DNA knock-in (KI) by embryonic genome editing is still low. In contrast to the direct embryo KI method, gene targeting using embryonic stem cells (ESC) followed by chimeric mouse development by blastocyst injection still has several advantages, e.g., high-throughput in vitro targeting/screening or large-size DNA KI such as Cre, CreERT, TetON, and reporter fluorescent protein, or their fusion proteins can be carried out without serving animal lives. The ESC targeting can also be applied to strains such as BALB/c, of which embryos are known to be difficult to handle in vitro. This text describes the optimized method for either short- or large-size DNA KI in ESC by applying CRISPR/Cas9-mediated genome editing followed by chimera mice production to develop gene-manipulated mouse models.
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Genetic Editing of Long Noncoding RNA Using CRISPR/Cas9 Technology.
Long noncoding RNAs (lncRNAs) are a class of RNA transcripts greater than 200 nucleotides in length and makeup a considerable part of the human genome. LncRNAs are well established as crucial players in a myriad of physiological and pathological processes; however, despite their abundance and versatility, the functional characteristics of lncRNAs remain largely unknown predominantly due to the lack of suitable genetic editing strategies. The complexity of their genetic structure and regulation combined with their unique functionality poses several limitations in the application of classic genetic manipulation methods in lncRNA functional studies. Several reports have demonstrated the successful implementation of CRISPR/Cas9 technology in screening and identifying the function of specific lncRNAs. Here, we describe a detailed protocol utilizing CRISPR/Cas9 genetic editing technology for knocking down lncRNAs in vitro.
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