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Enhanced scale and scope of genome engineering and regulation using CRISPR/Cas in Saccharomyces cerevisiae.
Although only 6 years old, the CRISPR system has blossomed into a tool for rapid, on-demand genome engineering and gene regulation in Saccharomyces cerevisiae. In this minireview, we discuss fundamental CRISPR technologies, tools to improve the efficiency and capabilities of gene targeting, and cutting-edge techniques to explore gene editing and transcriptional regulation at genome scale using pooled approaches. The focus is on applications to metabolic engineering with topics including development of techniques to edit the genome in multiplex, tools to enable large numbers of genetic modifications using pooled single-guide RNA libraries and efforts to enable programmable transcriptional regulation using endonuclease-null Cas enzymes.
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Generation of Genome-Edited Mice by Cytoplasmic Injection of CRISPR-Cas9 RNA.
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR-Cas9) gene editing enables rapid production of genome-edited animals. The Cas9/guide RNA (gRNA) component can be introduced into zygotes in several ways. Here, we provide an instructional guide for the generation of knockout mice using cytoplasmic injection of in vitro transcribed Cas9 RNA and gRNA.
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Large fragment deletion using a CRISPR/Cas9 system in Saccharomyces cerevisiae.
Large chromosomal modifications have been performed in natural and laboratory evolution studies and hold tremendous potential for use in foundational research, medicine, and biotechnology applications. Recently, the type II bacterial Clustered Regularly Interspaced Short Palindromic Repeat and CRISPR-associated (CRISPR/Cas9) system has emerged as a powerful tool for genome editing in various organisms. In this study, we applied the CRISPR/Cas9 system to preform large fragment deletions in Saccharomyces cerevisiae and compared the performance activity to that of a traditional method that uses the Latour system. Here we report in S. Cerevisiae the CRIPR/Cas9 system has been used to delete fragments exceeding 30 kb. The use of the CRISPR/Cas9 system for generating chromosomal segment excision showed some potential advantages over the Latour system. All the results indicated that CRISPR/Cas9 system was a rapid, efficient, low-cost, and versatile method for genome editing and that it can be applied in further studies in the fields of biology, agriculture, and medicine.
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Production of Human CRISPR-Engineered CAR-T Cells.
Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with hematological malignancies and are currently being investigated for various solid tumors. CAR-T cells are generated by removing T cells from a patient's blood and engineering them to express a synthetic immune receptor that redirects the T-cells to recognize and eliminate target tumor cells. Gene editing of CAR-T cells has the potential to improve safety of current CAR-T cell therapies and further increase the efficacy of CAR-T cells. Here, we describe methods for the activation, expansion, and characterization of human CRISPR-engineered CD19 directed CAR-T cells. This comprises transduction of the CAR lentiviral vector and use of single guide RNA (sgRNA) and Cas9 endonuclease to target genes of interest in T cells. The methods described in this protocol can be universally applied to other CAR constructs and target genes beyond the ones used for this study. Furthermore, this protocol discusses strategies for gRNA design, lead gRNA selection and target gene knockout validation to reproducibly achieve high-efficiency, multiplex CRISPR-Cas9 engineering of clinical grade human T cells.
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CRISPR/Cas9 and TALEN-mediated knock-in approaches in zebrafish.
The targeted introduction of mutations utilizing sequence specific transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system (RNA-guided nucleases, RGNs) has revolutionized reverse genetic approaches in numerous model organisms. In zebrafish, both systems were successfully applied to generate loss-of-function alleles by targeting open reading frames or deletion and inversion of whole chromosomal regions. In addition to the production of these loss-of-function alleles, genomic engineering by insertion of short sequences utilizing single stranded DNA oligonucleotides as templates for homology based repair was made possible, enabling effective insertion of loxP sites or tags for protein coding genes. Recent studies based on homologous recombination and non-homologous end joining have also broadened the repertoire for genome editing. These approaches allow the targeted insertion of open reading frames or even whole donor vectors. In this review we summarize the use of TALENs and RNA-guided nucleases in the field of zebrafish genetics with a special focus on knock-in approaches.
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Enhanced Bioreduction of Radionuclides by Driving Microbial Extracellular Electron Pumping with an Engineered CRISPR Platform.
Dissimilatory metal-reducing bacteria (DMRB) with extracellular electron transfer (EET) capability show great potential in bioremediating the subsurface environments contaminated by uranium through bioreduction and precipitation of hexavalent uranium [U(VI)]. However, the low EET efficiency of DMRB remains a bottleneck for their applications. Herein, we develop an engineered CRISPR platform to drive the extracellular electron pumping of Shewanella oneidensis, a representative DMRB species widely present in aquatic environments. The CRISPR platform allows for highly efficient and multiplex genome editing and rapid platform elimination post-editing in S. oneidensis. Enabled by such a platform, a genomic promoter engineering strategy (GPS) for genome-widely engineering the EET-encoding gene network was established. The production of electron conductive Mtr complex, synthesis of electron shuttle flavin, and generation of NADH as intracellular electron carrier are globally optimized and promoted, leading to a significantly enhanced EET ability. Applied to U(VI) bioreduction, the edited strains achieve up to 3.62-fold higher reduction capacity over the control. Our work endows DMRB with an enhanced ability to remediate the radionuclides-contaminated environments and provides a gene editing approach to handle the growing environmental challenges of radionuclide contaminations.
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Evaluation of the effects of sequence length and microsatellite instability on single-guide RNA activity and specificity.
Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology is effective for genome editing and now widely used in life science research. However, the key factors determining its editing efficiency and off-target cleavage activity for single-guide RNA (sgRNA) are poorly documented. Here, we systematically evaluated the effects of sgRNA length on genome editing efficiency and specificity. Results showed that sgRNA 5'-end lengths can alter genome editing activity. Although the number of predicted off-target sites significantly increased after sgRNA length truncation, sgRNAs with different lengths were highly specific. Because only a few predicted off-targets had detectable cleavage activity as determined by Target capture sequencing (TargetSeq). Interestingly, > 20% of the predicted off-targets contained microsatellites for selected sgRNAs targeting the dystrophin gene, which can produce genomic instability and interfere with accurate assessment of off-target cleavage activity. We found that sgRNA activity and specificity can be sensitively detected by TargetSeq in combination with in silico prediction. Checking whether the on- and off-targets contain microsatellites is necessary to improve the accuracy of analyzing the efficiency of genome editing. Our research provides new features and novel strategies for the accurate assessment of CRISPR sgRNA activity and specificity.
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Simple and efficient CRISPR/Cas9-mediated targeted mutagenesis in Xenopus tropicalis.
We have assessed the efficacy of the recently developed CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system for genome modification in the amphibian Xenopus tropicalis. As a model experiment, targeted mutations of the tyrosinase gene were verified, showing the expected albinism phenotype in injected embryos. We further tested this technology by interrupting the six3 gene, which is required for proper eye and brain formation. Expected eye and brain phenotypes were observed when inducing mutations in the six3 coding regions, as well as when deleting the gene promoter by dual targeting. We describe here a standardized protocol for genome editing using this system. This simple and fast method to edit the genome provides a powerful new reverse genetics tool for Xenopus researchers.
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CRISPR Correction of the GBA Mutation in Human-Induced Pluripotent Stem Cells Restores Normal Function to Gaucher Macrophages and Increases Their Susceptibility to Mycobacterium tuberculosis.
Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the β-glucocerebrosidase (GCase) GBA gene, which result in macrophage dysfunction. CRISPR (clustered regularly interspaced short palindromic repeats) editing of the homozygous L444P (1448T→C) GBA mutation in type 2 GD (GBA-/-) human-induced pluripotent stem cells (hiPSCs) yielded both heterozygous (GBA+/-) and homozygous (GBA+/+) isogenic lines. Macrophages derived from GBA-/-, GBA+/- and GBA+/+ hiPSCs showed that GBA mutation correction restores normal macrophage functions: GCase activity, motility, and phagocytosis. Furthermore, infection of GBA-/-, GBA+/- and GBA+/+ macrophages with the Mycobacterium tuberculosis H37Rv strain showed that impaired mobility and phagocytic activity were correlated with reduced levels of bacterial engulfment and replication suggesting that GD may be protective against tuberculosis.
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[Progress of new-generation genome editing mediated by engineered endonucleases].
Genome editing refers to the experimental methods to targeted modify specific loci in the genomic DNA sequence. In recent years, engineered endonucleases, including ZFN, TALEN and CRISPR/Cas, have been developed as a new-generation genome editing technique, and greatly improved the feasibility of gene function analyses, gene therapy, etc. Here, we briefly summarize the basic principle, developmental process and applications of this technology.
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