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LeishGEdit: A Method for Rapid Gene Knockout and Tagging Using CRISPR-Cas9.
Postgenomic analyses of Leishmania biology benefit from rapid and precise methods for gene manipulation. Traditional methods of gene knockout or tagging by homologous recombination have limitations: they tend to be slow and require successive transfection and selection rounds to knock out multiple alleles of a gene. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems overcome these limitations. We describe here in detail a simple, rapid, and scalable method for CRISPR-Cas9-mediated gene knockout and tagging in Leishmania. This method details how to use simple PCR to generate (1) templates for single guide RNA (sgRNA) transcription in cells expressing Cas9 and T7 RNA polymerase and (2) drug-selectable editing cassettes, using a modular set of plasmids as templates. pT plasmids allow for amplification of drug resistance genes for knockouts and pPLOT plasmids provide a choice of different tags to generate N- or C-terminally tagged proteins. We describe how to use an online platform ( LeishGEdit.net ) for automated primer design and how to perform PCRs and transfections in small batches or on 96-well plates for large-scale knockout or tagging screens. This method allows generation of knockout mutants or tagged cell lines within 1 week.
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Precision Therapy for Inherited Retinal Disease: At the Forefront of Genomic Medicine.
Inherited retinal diseases (IRDs) represent a diverse array of conditions characterized by dysfunction or loss of 1 or more retinal cell types. Next-generation sequencing has enabled rapid and relatively inexpensive genotyping, with more than 250 genes identified as responsible for IRDs. This expansion in molecular diagnostic accuracy, in combination with the retina's relative accessibility and immune privilege, has fostered the development of precision therapies to treat these myriad conditions. Novel techniques are being used in early trials. Precision molecular therapies for IRDs hold great promise as diagnostic and treatment strategies continue to expand.
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Genome Editing Using the Endogenous Type I-E CRISPR-Cas System in Lactobacillus paracasei ATCC334.
Lactobacillus paracasei ATCC334 is a well-known beneficial strain that plays a crucial role in food industry and promotion of human health. However, despite its significance, our understanding of its gene functions remains limited due to obstacles in gene editing techniques. This gap hinders the full utilization and development of this beneficial bacterium. In this study, we targeted L. paracasei ATCC334 as editing chassis. Initially, bioinformatics tools were used to explore a type I-E endogenous clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system within L. paracasei ATCC334. We further analyzed its repeat sequences, spacer sequences, and leader sequence predicted the protospacer adjacent motif (PAM) recognized by this system. To validate our findings, we assessed the accuracy of potential PAM, evaluated the cutting activity of the endogenous CRISPR-Cas system, and studied the impact of the artificial mini-CRISPR array through plasmid interference and genome interference experiments. These results helped us to achieve successful gene knockout and gene integration. Finally, we engineered a strain capable of nicotine degradation. Our study provides valuable insights for the broader development and application of lactobacilli.
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Delivery of CRISPR-Cas12a Ribonucleoprotein Complex for Genome Editing in an Embryogenic Citrus Cell Line.
Clustered regularly interspaced short palindromic repeats (CRISPR) technology is a powerful genome editing tool. Recently developed CRISPR-Cas12a system confers several advantages over CRISPR-Cas9, making it ideal for use in plant genome editing and crop improvement. While traditional transformation methods based on plasmid delivery pose concerns associated with transgene integration and off-target effects, CRISPR-Cas12a delivered as ribonucleoproteins (RNPs) can effectively alleviate these potential issues. Here we present a detailed protocol for LbCas12a-mediated genome editing using RNP delivery in Citrus protoplasts. This protocol provides a comprehensive guideline for RNP component preparation, RNP complex assembly and delivery, and editing efficiency assessment.
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Discovery and Characterization of Novel Type V Cas12f Nucleases with Diverse Protospacer Adjacent Motif Preferences.
Small Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) effectors are key to developing gene editing therapies due to the packaging constraints of viral vectors. While Cas9 and Cas12a CRISPR-Cas effectors have advanced into select clinical applications, their size is prohibitive for efficient delivery of both nuclease and guide RNA in a single viral vector. Type V Cas12f effectors present a solution given their small size. In this study, we describe a novel set of miniature (<490AA) Cas12f nucleases that cleave double-stranded DNA in human cells. We determined their optimal trans-activating RNA empirically through rational modifications, which resulted in an optimal single guide RNA. We show that these nucleases have broad protospacer adjacent motif (PAM) preferences, allowing for expanded genome targeting. The unique characteristics of these novel nucleases add to the diversity of the miniature CRISPR-Cas toolbox while the expanded PAM allows for the editing of genomic locations that could not be accessed with existing Cas12f nucleases.
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Current applications and future perspective of CRISPR/Cas9 gene editing in cancer.
Clustered regularly interspaced short palindromic repeats (CRISPR) system provides adaptive immunity against plasmids and phages in prokaryotes. This system inspires the development of a powerful genome engineering tool, the CRISPR/CRISPR-associated nuclease 9 (CRISPR/Cas9) genome editing system. Due to its high efficiency and precision, the CRISPR/Cas9 technique has been employed to explore the functions of cancer-related genes, establish tumor-bearing animal models and probe drug targets, vastly increasing our understanding of cancer genomics. Here, we review current status of CRISPR/Cas9 gene editing technology in oncological research. We first explain the basic principles of CRISPR/Cas9 gene editing and introduce several new CRISPR-based gene editing modes. We next detail the rapid progress of CRISPR screening in revealing tumorigenesis, metastasis, and drug resistance mechanisms. In addition, we introduce CRISPR/Cas9 system delivery vectors and finally demonstrate the potential of CRISPR/Cas9 engineering to enhance the effect of adoptive T cell therapy (ACT) and reduce adverse reactions.
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Localized delivery of CRISPR/dCas9 via layer-by-layer self-assembling peptide coating on nanofibers for neural tissue engineering.
The clustered regularly interspaced short palindromic repeat (CRISPR) systems have a wide variety of applications besides precise genome editing. In particular, the CRISPR/dCas9 system can be used to control specific gene expression by CRISPR activation (CRISPRa) or interference (CRISPRi). However, the safety concerns associated with viral vectors and the possible off-target issues of systemic administration remain huge concerns to be safe delivery methods for CRISPR/Cas9 systems. In this study, a layer-by-layer (LbL) self-assembling peptide (SAP) coating on nanofibers is developed to mediate localized delivery of CRISPR/dCas9 systems. Specifically, an amphiphilic negatively charged SAP- is first coated onto PCL nanofibers through strong hydrophobic interactions, and the pDNA complexes and positively charged SAP+-RGD are then absorbed via electrostatic interactions. The SAPcoated scaffolds facilitate efficient loading and sustained release of the pDNA complexes, while enhancing cell adhesion and proliferation. As a proof of concept, the scaffolds are used to activate GDNF expression in mammalian cells, and the secreted GDNF subsequently promotes neurite outgrowth of rat neurons. These promising results suggest that the LbL self-assembling peptide coated nanofibers can be a new route to establish a bioactive interface, which provides a simple and efficient platform for the delivery of CRISPR/dCas9 systems for regenerative medicine.
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Recognizing CRISPR as the new age disease-modifying drug: Strategies to bioengineer CRISPR/Cas for direct in vivo delivery.
Clustered regularly interspaced short palindromic repeats (CRISPR) have established itself as a frontier technology in genetic engineering. Researchers have successfully used the CRISPR/Cas system as precise gene editing tools and have further expanded their scope beyond both imaging and diagnostic applications. The most prominent utility of CRISPR is its capacity for gene therapy, serving as the contemporary, disease-modifying drug at the genetic level of human medical disorders. Correcting these diseases using CRISPR-based gene editing has developed to the extent of preclinical trials and possible patient treatments. A major impediment in actualizing this is the complications associated with in vivo delivery of the CRISPR/Cas complex. Currently, only the viral vectors (e.g., lentivirus) and non-viral encapsulation (e.g., lipid particles, polymer-based, and gold nanoparticles) techniques have been extensively reviewed, neglecting the efficiency of direct delivery. However, the direct delivery of CRISPR/Cas for in vivo gene editing therapies is an intricate process with numerous drawbacks. Hence, this paper discusses in detail both the need and the strategies that can potentially improve the direct delivery aspects of CRISPR/Cas biomolecules for gene therapy of human diseases. Here, we focus on enhancing the molecular and functional features of the CRISPR/Cas system for targeted in vivo delivery such as on-site localization, internalization, reduced immunogenicity, and better in vivo stability. We additionally emphasize the CRISPR/Cas complex as a multifaceted, biomolecular vehicle for co-delivery with therapeutic agents in targeted disease treatments. The delivery formats of efficient CRISPR/Cas systems for human gene editing are also briefly elaborated.
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The evolution of CRISPR/Cas9 and their cousins: hope or hype?
Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system allows biologists to edit genomic DNA of any cell in precise and specific way, entailing great potential for crop improvement, drug development and gene therapy. The system involves a nuclease (Cas9) and a designed guide RNA that are involved in wide range of applications such as genome modification, transcriptional modulation, genomic loci marking and RNA tracking. The limitation of the technique, in view of resistance of thymidine-rich genome to Cas9 cleavage, has now been overcome by the use of Cpf1 nuclease. In this review, we present an overview of CRISPR nucleases (Cas9 or Cpf1) with particular emphasis on human genome modification and compare their advantages and limitations. Furthermore, we summarize some of the pros and cons of CRISPR technology particularly in human therapeutics.
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Shortened CRISPR-Cas9 arrays enable multiplexed gene targeting in bacteria from a smaller DNA footprint.
CRISPR technologies comprising a Cas nuclease and a guide RNA (gRNA) can utilize multiple gRNAs to enact multi-site editing or regulation in the same cell. Nature devised a highly compact means of encoding gRNAs in the form of CRISPR arrays composed of conserved repeats separated by targeting spacers. However, the capacity to acquire new spacers keeps the arrays longer than necessary for CRISPR technologies. Here, we show that CRISPR arrays utilized by the Cas9 nuclease can be shortened without compromising and sometimes even enhancing targeting activity. Using multiplexed gene repression in E. coli, we found that each region could be systematically shortened to varying degrees before severely compromising targeting activity. Surprisingly, shortening some spacers yielded enhanced targeting activity, which was linked to folding of the transcribed array prior to processing. Overall, shortened CRISPR-Cas9 arrays can facilitate multiplexed editing and gene regulation from a smaller DNA footprint across many bacterial applications of CRISPR technologies.
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