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WheatCRISPR: a web-based guide RNA design tool for CRISPR/Cas9-mediated genome editing in wheat.
BACKGROUND: CRISPR/Cas9 gene editing has become a revolutionary technique for crop improvement as it can facilitate fast and efficient genetic changes without the retention of transgene components in the final plant line. Lack of robust bioinformatics tools to facilitate the design of highly specific functional guide RNAs (gRNAs) and prediction of off-target sites in wheat is currently an obstacle to effective application of CRISPR technology to wheat improvement. DESCRIPTION: We have developed a web-based bioinformatics tool to design specific gRNAs for genome editing and transcriptional regulation of gene expression in wheat. A collaborative study between the Broad Institute and Microsoft Research used large-scale empirical evidence to devise algorithms (Doech et al., 2016, Nature Biotechnology 34, 184-191) for predicting the on-target activity and off-target potential of CRISPR/SpCas9 (Streptococcus pyogenes Cas9). We applied these prediction models to determine on-target specificity and potential off-target activity for individual gRNAs targeting specific loci in the wheat genome. The genome-wide gRNA mappings and the corresponding Doench scores predictive of the on-target and off-target activities were used to create a gRNA database which was used as a data source for the web application termed WheatCRISPR. CONCLUSION: The WheatCRISPR tool allows researchers to browse all possible gRNAs targeting a gene or sequence of interest and select effective gRNAs based on their predicted high on-target and low off-target activity scores, as well as other characteristics such as position within the targeted gene. It is publicly available at https://crispr.bioinfo.nrc.ca/WheatCrispr/ .
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Predicted multispecies unintended effects from outdoor genome editing.
CRISPR/Cas9, a potent genetic engineering tool widely adopted in agriculture, is capable of introducing new characteristics into plants on a large scale and without conventional breeding methods. Despite its remarkable efficiency, concerns have arisen regarding unintended consequences in uncontrolled environments. Our aim was to assess potential activity in organisms that could be exposed to genome editing in uncontrolled environments. We developed three scenarios, using irrigation, fumigation and fertilization as delivery methods, based on outdoor uses in agriculture, namely pest and disease control. Using publicly available software (Cas-OFFinder, NCBI Genome Data Viewer and STRING), off-target effects were predicted in multiple species commonly found in the agroecosystem, including humans (16 of 38 (42 %) sampled). Metabolic enrichment analysis (gene IDs), by connecting off-target genes into a physiological network, predicted effects on the development of nervous and respiratory systems. Our findings emphasize the importance of exercising caution when considering the use of this genome editing in uncontrolled environments. Unintended genomic alterations may occur in unintended organisms, underscoring the significance of understanding potential hazards and implementing safety measures to protect human health and the environment.
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Genome Editing in Human Pluripotent Stem Cells.
Genome editing in human pluripotent stem cells (hPSCs) enables the generation of reporter lines and knockout cell lines. Zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and CRISPR/Cas9 technology have recently increased the efficiency of proper gene editing by creating double strand breaks (DSB) at defined sequences in the human genome. These systems typically use plasmids to transiently transcribe nucleases within the cell. Here, we describe the process for preparing hPSCs for transient expression of nucleases via electroporation and subsequent analysis to create genetically modified stem cell lines.
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Generation of genetically modified rat models via the CRISPR/Cas9 technology.
The RNA-guided CRISPR/Cas9 genomic editing system consists of a single guide RNA (sgRNA) and a Cas9 nuclease. The two components form a complex in cells and target the genomic loci complementary to the sgRNA. The Cas9 nuclease cleaves the target site creating a double stranded DNA break (DSB). In mammalian cells, DSBs are often repaired via error prone non-homologous end joining (NHEJ) or via homology directed repair (HDR) with the presence of donor DNA templates. Micro-injection of the CRISPR/Cas9 system into the rat embryos enables generation of genetically modified rat models. Here, we describe a detailed protocol for creating gene knockout or knockin rat models via the CRISPR/Cas9 technology.
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Using Prime Editing Guide Generator (PEGG) for High-Throughput Generation of Prime Editing Sensor Libraries.
Prime editing enables the generation of nearly any small genetic variant. However, the process of prime editing guide RNA (pegRNA) design is challenging and requires automated computational design tools. We developed Prime Editing Guide Generator (PEGG), a fast, flexible, and user-friendly Python package that enables the rapid generation of pegRNA and pegRNA-sensor libraries. Here, we describe the installation and use of PEGG ( https://pegg.readthedocs.io ) to rapidly generate custom pegRNA-sensor libraries for use in high-throughput prime editing screens.
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Engineering Cas9 for human genome editing.
Since the initial reports describing CRISPR-Cas9, labs across the globe have leveraged this valuable gene editing tool to alter the genomes of living cells. With the goal of generating more precise and efficient genome changes, scientists and engineers have mutated, evolved, and covalently altered Cas9 in order to predictably edit the genetic code. Here, we highlight recent advancements and contributions to the growing field of Cas9 engineering. We present key aspects of Cas9 engineering efforts focused on sgRNA manipulation, PAM-recognition, specificity, deaminase fusions, reverse-transcriptase fusions, and structural rearrangements of this important gene-modifying tool.
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A CRISPR-based system for temporal, combinatorial gene knockout in mice.
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A Versatile Nonviral Delivery System for Multiplex Gene-Editing in the Liver.
Recent advances in CRISPR present attractive genome-editing toolsets for therapeutic strategies at the genetic level. Here, a liposome-coated mesoporous silica nanoparticle (lipoMSN) is reported as an effective CRISPR delivery system for multiplex gene-editing in the liver. The MSN provides efficient loading of Cas9 plasmid as well as Cas9 protein/guide RNA ribonucleoprotein complex (RNP), while liposome-coating offers improved serum stability and enhanced cell uptake. Hypothesizing that loss-of-function mutation in the lipid-metabolism-related genes pcsk9, apoc3, and angptl3 would improve cardiovascular health by lowering blood cholesterol and triglycerides, the lipoMSN is used to deliver a combination of RNPs targeting these genes. When targeting a single gene, the lipoMSN achieved a 54% gene-editing efficiency, besting the state-of-art Lipofectamine CRISPRMax. For multiplexing, lipoMSN maintained significant gene-editing at each gene target despite reduced dosage of target-specific RNP. By delivering combinations of targeting RNPs in the same nanoparticle, synergistic effects on lipid metabolism are observed in vitro and vivo. These effects, such as a 50% decrease in serum cholesterol after 4 weeks of post-treatment with lipoMSN carrying both pcsk9 and angptl3-targeted RNPs, could not be reached with a single gene-editing approach. Taken together, this lipoMSN represents a versatile platform for the development of efficient, combinatorial gene-editing therapeutics.
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CRISPR/Cas9-based efficient genome editing in Staphylococcus aureus.
Staphylococcus aureus is an important pathogenic bacterium prevalent in nosocomial infections and associated with high morbidity and mortality rates, which arise from the significant pathogenicity and multi-drug resistance. However, the typical genetic manipulation tools used to explore the relevant molecular mechanisms of S. aureus have multiple limitations: leaving a scar in the genome, comparatively low gene-editing efficiency, and prolonged experimental period. Here, we present a single-plasmid based on the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system which allows rapid and efficient chromosomal manipulation in S. aureus. The plasmid carries the cas9 gene under the control of the constitutive promoter Pxyl/tet, a single guide RNA-encoding sequence transcribed via a strong promoter Pspac, and donor DNA used to repair the double strand breaks. The function of the CRISPR/Cas9 vector was demonstrated by deleting the tgt gene and the rocA gene, and by inserting the erm R cassette in S. aureus. This research establishes a CRISPR/Cas9 genome editing tool in S. aureus, which enables marker-free, scarless and rapid genetic manipulation, thus accelerating the study of gene function in S. aureus.
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Spider Eye Development Editing and Silk Fiber Engineering Using CRISPR-Cas.
CRISPR-Cas9 gene editing represents an effective and precise technology to induce mutations in the genome, and it has been applied to a wide range of organisms for diverse purposes. However, CRISPR-based gene editing in spiders has not been reported to date. In this study, we demonstrate CRISPR-mediated microinjection in parental spiders leading to both knock-out (KO) and knock-in (KI) mutations within the spider's offspring. The KO of the gene sine oculis causes total eye loss, confirming the role of the gene in the development of all spider eyes. The KI of a monomeric red fluorescent protein (mRFP-KI) within a spider silk gene encoding one compound of the major ampullate silk of the spider Parasteatoda tepidariorum yields red fluorescent silk fibers. This finding demonstrates the feasibility of functionalizing silk proteins in spiders using CRISPR-based gene editing without influencing silk assembly. Our study expands the application of CRISPR to spiders and provides insights in the fields of developmental genetics as well as material sciences.
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