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CRISPR Comparison Toolkit: Rapid Identification, Visualization, and Analysis of CRISPR Array Diversity.
CRISPR-Cas systems provide immunity against mobile genetic elements (MGEs) through sequence-specific targeting by spacer sequences encoded in CRISPR arrays. Spacers are highly variable between microbial strains and can be acquired rapidly, making them well suited for use in strain typing of closely related organisms. However, no tools are currently available to automate the process of reconstructing strain histories using CRISPR spacers. We therefore developed the CRISPR Comparison Toolkit (CCTK) to enable analyses of array relationships. The CCTK includes tools to identify arrays, analyze relationships between arrays using CRISPRdiff and CRISPRtree, and predict targets of spacers. CRISPRdiff visualizes arrays and highlights the similarities between them. CRISPRtree infers a phylogenetic tree from array relationships and presents a hypothesis of the evolutionary history of the arrays. The CCTK unifies several CRISPR analysis tools into a single command line application, including the first tool to infer phylogenies from array relationships.
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Efficient and Scalable Precision Genome Editing in Staphylococcus aureus through Conditional Recombineering and CRISPR/Cas9-Mediated Counterselection.
Staphylococcus aureus is an important human pathogen, but studies of the organism have suffered from the lack of a robust tool set for its genetic and genomic manipulation. Here we report the development of a system for the facile and high-throughput genomic engineering of S. aureus using single-stranded DNA (ssDNA) oligonucleotide recombineering coupled with clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated counterselection. We identify recombinase EF2132, derived from Enterococcus faecalis, as being capable of integrating single-stranded DNA oligonucleotides into the S. aureus genome. We found that EF2132 can readily mediate recombineering across multiple characterized strains (3 of 3 tested) and primary clinical isolates (6 of 6 tested), typically yielding thousands of recombinants per transformation. Surprisingly, we also found that some S. aureus strains are naturally recombinogenic at measurable frequencies when oligonucleotides are introduced by electroporation, even without exogenous recombinase expression. We construct a temperature-sensitive, two-vector system which enables conditional recombineering and CRISPR/Cas9-mediated counterselection in S. aureus without permanently introducing exogenous genetic material or unintended genetic lesions. We demonstrate the ability of this system to efficiently and precisely engineer point mutations and large single-gene deletions in the S. aureus genome and to yield highly enriched populations of engineered recombinants even in the absence of an externally selectable phenotype. By virtue of utilizing inexpensive, commercially synthesized synthetic DNA oligonucleotides as substrates for recombineering and counterselection, this system provides a scalable, versatile, precise, inexpensive, and generally useful tool for producing isogenic strains in S. aureus which will enable the high-throughput functional assessment of genome variation and gene function across multiple strain backgrounds.IMPORTANCE Engineering genetic changes in bacteria is critical to understanding the function of particular genes or mutations but is currently a laborious and technically challenging process to perform for the important human pathogen Staphylococcus aureus In an effort to develop methods which are rapid, easy, scalable, versatile, and inexpensive, here we describe a system for incorporating synthetic, mutagenic DNA molecules into the S. aureus genome and for eliminating cells that lack the engineered mutation. This method allows efficient, precise, and high-throughput genetic engineering of S. aureus strains and will facilitate studies seeking to address a variety of issues about the function of particular genes and specific mutations.
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Design and assessment of engineered CRISPR-Cpf1 and its use for genome editing.
Cpf1, a CRISPR endonuclease discovered in Prevotella and Francisella 1 bacteria, offers an alternative platform for CRISPR-based genome editing beyond the commonly used CRISPR-Cas9 system originally discovered in Streptococcus pyogenes. This protocol enables the design of engineered CRISPR-Cpf1 components, both CRISPR RNAs (crRNAs) to guide the endonuclease and Cpf1 mRNAs to express the endonuclease protein, and provides experimental procedures for effective genome editing using this system. We also describe quantification of genome-editing activity and off-target effects of the engineered CRISPR-Cpf1 in human cell lines using both T7 endonuclease I (T7E1) assay and targeted deep sequencing. This protocol enables rapid construction and identification of engineered crRNAs and Cpf1 mRNAs to enhance genome-editing efficiency using the CRISPR-Cpf1 system, as well as assessment of target specificity within 2 months. This protocol may also be appropriate for fine-tuning other types of CRISPR systems.
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Electroporation-based CRISPR gene editing in adult buffalo fibroblast cells.
Electroporation is a widely used method for delivering CRISPR components into cells; however, it presents challenges when applied to difficult-to-transfect cells like adult buffalo fibroblasts. In this study, the ITGB2 gene (encoding the CD18 protein), plays vital for cellular adhesion and immune responses, was selected for editing experiments. To optimize electroporation conditions, we investigated parameters such as electric field strength, pulse duration, plasmid DNA amount, cuvette type, and cell type. The best transfection rates were obtained in a 4 mm gap cuvette with a single 20-millisecond pulse of 300 V using a 10 μg of all-in-one CRISPR plasmid for 106 cells in 100 μL of electroporation buffer. Increasing DNA quantity enhanced transfection rates but compromised cell viability. The 4 mm cuvette gap had high transfection rates than the 2 mm gap, and newborn cells exhibited higher transfection rates than adult cells. We achieved transfection rates of 10-12% with a cell viability of 25-30% for adult fibroblast cells. Subsequently, successfully edited the ITGB2 gene with a 30% editing efficiency, confirmed through various analysis methods, including T7E1 assay, TIDE and ICE analysis, and TA cloning. In conclusion, electroporation conditions reported here can edit buffalo gene(s) for various biotechnological research applications.
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Analyzing CRISPR genome-editing experiments with CRISPResso.
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Non-viral strategies for delivering genome editing enzymes.
Genome-editing tools such as Cre recombinase (Cre), zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and most recently the clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein system have revolutionized biomedical research, agriculture, microbial engineering, and therapeutic development. Direct delivery of genome editing enzymes, as opposed to their corresponding DNA and mRNA precursors, is advantageous since they do not require transcription and/or translation. In addition, prolonged overexpression is a problem when delivering viral vector or plasmid DNA which is bypassed when delivering whole proteins. This lowers the risk of insertional mutagenesis and makes for relatively easier manufacturing. However, a major limitation of utilizing genome editing proteins in vivo is their low delivery efficiency, and currently the most successful strategy involves using potentially immunogenic viral vectors. This lack of safe and effective non-viral delivery systems is still a big hurdle for the clinical translation of such enzymes. This review discusses the challenges of non-viral delivery strategies of widely used genome editing enzymes, including Cre recombinase, ZFNs and TALENs, CRISPR/Cas9, and Cas12a (Cpf1) in their protein format and highlights recent innovations of non-viral delivery strategies which have the potential to overcome current delivery limitations and advance the clinical translation of genome editing.
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Delivery of genome editing tools: A promising strategy for HPV-related cervical malignancy therapy.
INTRODUCTION: Persistent high-risk human papillomavirus infection is the main cause of various types of cancer especially cervical cancer. The E6 and E7 oncoproteins of HPV play critical roles in promoting carcinogenesis and cancer cell growth. As a result, E6 and E7 oncogenes are considered as promising therapeutic targets for cervical cancer. Recently, the development of genome-editing technologies including transcription activator-like effector nucleases (TALEN), meganucleases (MNs), zinc finger nucleases (ZFN), and more importantly clustered regularly interspaced short palindromic repeat-CRISPR-associated protein (CRISPR-Cas) has sparked a revolution in the cervical cancer-targeted therapy. However, due to immunogenicity, off-target effect, renal clearance, guide RNA (gRNA) nuclease degradation, and difficult direct transportation into the cytoplasm and nucleus, the safe and effective delivery is considered as the Achilles' heel of this robust strategy. AREAS COVERED: In this review, we discuss cutting-edge available strategies for in vivo delivery of genome-editing technologies for HPV-induced cervical cancer therapy. Moreover, the combination of genome-editing tools and other therapies has been fully discussed. EXPERT OPINION: The combination of nanoparticle-based delivery systems and genome-editing tools is a promising powerful strategy for cervical cancer therapy. The most significant limitations of this strategy that need to be focused on are low efficiency and off-target events.
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Expansion of YALIcloneHR toolkit for Yarrowia lipolytica combined with Golden Gate and CRISPR technology.
Metabolic Engineering of yeast is a critical approach to improving the production capacity of cell factories. To obtain genetically stable recombinant strains, the exogenous DNA is preferred to be integrated into the genome. Previously, we developed a Golden Gate toolkit YALIcloneNHEJ, which could be used as an efficient modular cloning toolkit for the random integration of multigene pathways through the innate non-homologous end-joining repair mechanisms of Yarrowia lipolytica. We expanded the toolkit by designing additional building blocks of homologous arms and using CRISPR technology. The reconstructed toolkit was thus entitled YALIcloneHR and designed for gene-specific knockout and integration. To verify the effectiveness of the system, the gene PEX10 was selected as the target for the knockout. This system was subsequently applied for the arachidonic acid production, and the reconstructed strain can accumulate 4.8% of arachidonic acid. The toolkit will expand gene editing technology in Y. lipolytica, which would help produce other chemicals derived from acetyl-CoA in the future.
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(Broken) Promises of Sustainable Food and Agriculture through New Biotechnologies: The CRISPR Case.
In recent years, the development of diverse CRISPR-based technologies has revolutionized genome manipulation and enabled a broad scientific community in industry, academia, and beyond to redefine research and development for biotechnology products encompassing food, agriculture, and medicine. CRISPR-based genome editing affords tremendous opportunities in agriculture for the breeding of crops and livestock across the food supply chain that could benefit larger portions of the population compared to CRISPR applications in medicine, for example by helping to feed a growing global population, reach sustainability goals, and possibly mitigate the effects of climate change. These promises come alongside concerns of risks and adverse impacts associated with CRISPR-based genome editing and concerns that governance systems that are ill equipped or not well suited to evaluate these risks. The international community will continue to gather, in multiple venues, in the coming years to discuss these concerns. At the same time, responsible research and innovation paradigms also promise to evaluate the risks and benefits better while incorporating broad stakeholder engagement across the research and development process. The CRISPR community therefore must actively engage with these international deliberations, society, and national governance systems that have promised to build better agricultural systems and provide better food products to achieve equitable outcomes while protecting the environment. Without this active engagement, the promises discussed in this paper are sure to be broken.
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Nature's 10: Ten people who mattered in science in 2018.
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