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Found: 26 119
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Structural Basis for the Altered PAM Recognition by Engineered CRISPR-Cpf1.
The RNA-guided Cpf1 nuclease cleaves double-stranded DNA targets complementary to the CRISPR RNA (crRNA), and it has been harnessed for genome editing technologies. Recently, Acidaminococcus sp. BV3L6 (AsCpf1) was engineered to recognize altered DNA sequences as the protospacer adjacent motif (PAM), thereby expanding the target range of Cpf1-mediated genome editing. Whereas wild-type AsCpf1 recognizes the TTTV PAM, the RVR (S542R/K548V/N552R) and RR (S542R/K607R) variants can efficiently recognize the TATV and TYCV PAMs, respectively. However, their PAM recognition mechanisms remained unknown. Here we present the 2.0 Å resolution crystal structures of the RVR and RR variants bound to a crRNA and its target DNA. The structures revealed that the RVR and RR variants primarily recognize the PAM-complementary nucleotides via the substituted residues. Our high-resolution structures delineated the altered PAM recognition mechanisms of the AsCpf1 variants, providing a basis for the further engineering of CRISPR-Cpf1.
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Coordinated regulation of IFITM1, 2 and 3 genes by an IFN-responsive enhancer through long-range chromatin interactions.
Interferon-induced transmembrane protein (IFITM) 1, 2 and 3 genes encode a family of interferon (IFN)-induced transmembrane proteins that block entry of a broad spectrum of pathogens. However, the transcriptional regulation of these genes, especially whether there exist any enhancers and their roles during the IFN induction process remain elusive. Here, through public data mining, episomal luciferase reporter assay and in vivo CRISPR-Cas9 genome editing, we identified an IFN-responsive enhancer located 35kb upstream of IFITM3 gene promoter upregulating the IFN-induced expression of IFITM1, 2 and 3 genes. Chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA) and luciferase reporter assay demonstrated that signal transducers and activators of transcription (STAT) 1 bound to the enhancer with the treatment of IFN and was indispensable for the enhancer activity. Furthermore, using chromosome conformation capture technique, we revealed that the IFITM1, 2 and 3 genes physically clustered together and constitutively looped to the distal enhancer through long-range interactions in both HEK293 and A549 cells, providing structural basis for coordinated regulation of IFITM1, 2 and 3 by the enhancer. Finally, we showed that in vivo truncation of the enhancer impaired IFN-induced resistance to influenza A virus (IAV) infection. These findings expand our understanding of the mechanisms underlying the transcriptional regulation of IFITM1, 2 and 3 expression and its ability to mediate IFN signaling.
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Versatile and precise gene-targeting strategies for functional studies in mammalian cell lines.
The advent of programmable nucleases such as ZFNs, TALENs and CRISPR/Cas9 has brought the power of genetic manipulation to widely used model systems. In mammalian cells, nuclease-mediated DNA double strand break is mainly repaired through the error-prone non-homologous end-joining (NHEJ) repair pathway, eventually leading to accumulation of small deletions or insertions (indels) that can inactivate gene function. However, due to the variable size of the indels and the polyploid status of many cell lines (e.g., cancer-derived cells), obtaining a knockout usually requires lengthy screening and characterization procedures. Given the more precise type of modifications that can be introduced upon homology-directed repair (HDR), we have developed HDR-based gene-targeting strategies that greatly facilitate the process of knockout generation in cell lines. To generate reversible knockouts (R-KO), a selectable promoter-less STOP cassette is inserted in an intron, interrupting transcription. Loss-of-function can be validated by RT-qPCR and is removable, enabling subsequent restoration of gene function. A variant of the R-KO procedure can be used to introduce point mutations. To generate constitutive knockouts (C-KO), an exon is targeted, which makes use of HDR-based gene disruption together with NHEJ-induced indels on non-HDR targeted allele(s). Hence the C-KO procedure greatly facilitates simultaneous inactivation of multiple alleles. Overall these genome-editing tools offer superior precision and efficiency for functional genetic approaches. We provide detailed protocols guiding in the design of targeting vectors and in the analysis and validation of gene targeting experiments.
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Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a.
The CRISPR-associated protein Cas12a (Cpf1), which has been repurposed for genome editing, possesses two distinct nuclease activities: endoribonuclease activity for processing its own guide RNAs and RNA-guided DNase activity for target DNA cleavage. To elucidate the molecular basis of both activities, we determined crystal structures of Francisella novicida Cas12a bound to guide RNA and in complex with an R-loop formed by a non-cleavable guide RNA precursor and a full-length target DNA. Corroborated by biochemical experiments, these structures reveal the mechanisms of guide RNA processing and pre-ordering of the seed sequence in the guide RNA that primes Cas12a for target DNA binding. Furthermore, the R-loop complex structure reveals the strand displacement mechanism that facilitates guide-target hybridization and suggests a mechanism for double-stranded DNA cleavage involving a single active site. Together, these insights advance our mechanistic understanding of Cas12a enzymes and may contribute to further development of genome editing technologies.
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Using the Endogenous CRISPR-Cas System of Heliobacterium modesticaldum To Delete the Photochemical Reaction Center Core Subunit Gene.
In Heliobacterium modesticaldum, as in many Firmicutes, deleting genes by homologous recombination using standard techniques has been extremely difficult. The cells tend to integrate the introduced plasmid into the chromosome by a single recombination event rather than perform the double recombination required to replace the targeted locus. Transformation with a vector containing only a homologous recombination template for replacement of the photochemical reaction center gene pshA produced colonies with multiple genotypes, rather than a clean gene replacement. To address this issue, we required an additional means of selection to force a clean gene replacement. In this study, we report the genetic structure of the type I-A and I-E CRISPR-Cas systems from H. modesticaldum, as well as methods to leverage the type I-A system for genome editing. In silico analysis of the CRISPR spacers revealed a potential consensus protospacer adjacent motif (PAM) required for Cas3 recognition, which was then tested using an in vivo interference assay. Introduction of a homologous recombination plasmid that carried a miniature CRISPR array targeting sequences in pshA (downstream of a naturally occurring PAM sequence) produced nonphototrophic transformants with clean replacements of the pshA gene with ∼80% efficiency. Mutants were confirmed by PCR, sequencing, optical spectroscopy, and growth characteristics. This methodology should be applicable to any genetic locus in the H. modesticaldum genome.IMPORTANCE The heliobacteria are the only phototrophic members of the largely Gram-positive phylum Firmicutes, which contains medically and industrially important members, such as Clostridium difficile and Clostridium acetobutylicum Heliobacteria are of interest in the study of photosynthesis because their photosynthetic system is unique and the simplest known. Since their discovery in the early 1980s, work on the heliobacteria has been hindered by the lack of a genetic transformation system. The problem of introducing foreign DNA into these bacteria has been recently rectified by our group; however, issues still remained for efficient genome editing. The significance of this work is that we have characterized the endogenous type I CRISPR-Cas system in the heliobacteria and leveraged it to assist in genome editing. Using the CRISPR-Cas system allowed us to isolate transformants with precise replacement of the pshA gene encoding the main subunit of the photochemical reaction center.
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Gene knockout in highly purified mouse hematopoietic stem cells by CRISPR/Cas9 technology.
The CRISPR/Cas9 system has been used for genome editing of human and mouse cells. In this study, we established a protocol for gene knockout (KO) in mouse hematopoietic stem cells (HSCs). HSCs were highly purified from the bone marrow of tamoxifen-treated Cas9-EGFP/Cre-ER transgenic mice, maintained in serum-free polyvinyl alcohol culture with cytokines, lentivirally transduced with sgRNA-Crimson, and transplanted into lethally irradiated mice with competitor cells. Previous studies of Pax5 KO mice have shown B cell differentiation block. To verify our KO HSC strategy, we deleted Pax5 gene in 600 CD201+CD150+CD48-c-Kit+Sca-1+Lin- cells (HSC1 cells), highly enriched in myeloid-biased HSCs, and CD201+CD150-CD48- c-Kit+Sca-1+Lin- cells (HSC2 cells), highly enriched in lymphoid-biased HSCs. As predicted, both Pax5 KO HSC1 and HSC2 cells showed few B cells in the peripheral blood and the accumulation of pro-B cells in the bone marrow of recipient mice. Our data suggesetd that myeloid-biased and lymphoid-biased HSCs share a common B cell differentiation pathway. This population-specific KO strategy will find its applications for gene editing in a varity of somatic cells, particuarly rare stem and progenitor cells from different tissues.
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Generation of beta-lactoglobulin knock-out goats using CRISPR/Cas9.
Goat's milk, considered a substitute for cow's milk, has a high nutritional value. However, goat's milk contains various allergens, predominantly β-lactoglobulin (BLG). In this study, we employed the CRISPR/Cas9 system to target the BLG locus in goat fibroblasts for sgRNA optimization and generate BLG knock-out goats through co-injection of Cas9 mRNA and small guide RNAs (sgRNAs) into goat embryos at the one-cell stage. We firstly tested sgRNA editing efficiencies in goat fibroblast cells, and approximately 8.00%-9.09% of the cells were modified in single sgRNA-guided targeting experiment. Among the kids, the genome-targeting efficiencies of single sgRNA were 12.5% (10 ng/μL sg1) and 0% (10 ng/μL sg2) and efficiencies of dual sgRNAs were 25.0% (25 ng/μL sg2+sg3 group) and 28.6% (50 ng/μL sg2+sg3 group). Relative expression of BLG in BLG knock-out goat mammary glands significantly (p < 0.01) decreased as well as other milk protein coding genes, such as CSN1S1, CSN1S2, CSN2, CSN3 and LALBA (p < 0.05). As expected, BLG protein had been abolished in the milk of the BLG knock-out goat. In addition, most of the targeted kids were chimeric (3/4), and their various body tissues were edited simultaneously. Our study thus provides a basis for optimizing the quality of goat milk, which can be applied to biomedical and agricultural research.
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Nanofountain Probe Electroporation Enables Versatile Single-Cell Intracellular Delivery and Investigation of Postpulse Electropore Dynamics.
Introducing exogenous molecules into cells with high efficiency and dosage control is a crucial step in basic research as well as clinical applications. Here, the capability of the nanofountain probe electroporation (NFP-E) system to deliver proteins and plasmids in a variety of continuous and primary cell types with appropriate dosage control is reported. It is shown that the NFP-E can achieve fine control over the relative expression of two cotransfected plasmids. Finally, the dynamics of electropore closure after the pulsing ends with the NFP-E is investigated. Localized electroporation has recently been utilized to demonstrate the converse process of delivery (sampling), in which a small volume of the cytosol is retrieved during electroporation without causing cell lysis. Single-cell temporal sampling confers the benefit of monitoring the same cell over time and can provide valuable insights into the mechanisms underlying processes such as stem cell differentiation and disease progression. NFP-E parameters that maximize the membrane resealing time, which is essential for increasing the sampled volume and in meeting the challenge of monitoring low copy number biomarkers, are identified. Its application in CRISPR/Cas9 gene editing, stem cell reprogramming, and single-cell sampling studies is envisioned.
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CRISPR-Cas9-Mediated Targeting of Multidrug Resistance Genes in Methicillin-Resistant Staphylococcus aureus.
Antibiotic resistance poses a global health crisis limiting the efficacy of available therapeutic agents. We explored CRISPR-Cas-based antimicrobials to combat multidrug resistance in methicillin-resistant Staphylococcus aureus (MRSA), targeting methicillin (mecA), gentamicin (aacA), and ciprofloxacin (grlA, grlB) resistance genes. Engineered CRISPR plasmids with specific single-guide RNAs were electroporated into MRSA strains. Real-time polymerase chain reaction assessed gene expression changes, while antibiotic susceptibility tests (ASTs) evaluated resistance status. Results showed a 1.5-fold decrease in mecA, a 5.5-fold decrease in grlA, a 6-fold decrease in grlB, and a 4-fold decrease in aacA expression. ASTs demonstrated the reversal of resistance to beta-lactam, quinolone, and aminoglycoside antibiotics. Western blot analysis revealed a 70% decrease in penicillin-binding protein 2a expression. Sanger sequencing confirmed point mutations in the grlB and aacA genes. Our findings highlight the potential of CRISPR-Cas9 technology to restore antibiotic efficacy against multidrug-resistant pathogens.
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Large-scale reconstruction of cell lineages using single-cell readout of transcriptomes and CRISPR-Cas9 barcodes by scGESTALT.
Lineage relationships among the large number of heterogeneous cell types generated during development are difficult to reconstruct in a high-throughput manner. We recently established a method, scGESTALT, that combines cumulative editing of a lineage barcode array by CRISPR-Cas9 with large-scale transcriptional profiling using droplet-based single-cell RNA sequencing (scRNA-seq). The technique generates edits in the barcode array over multiple timepoints using Cas9 and pools of single-guide RNAs (sgRNAs) introduced during early and late zebrafish embryonic development, which distinguishes it from similar Cas9 lineage-tracing methods. The recorded lineages are captured, along with thousands of cellular transcriptomes, to build lineage trees with hundreds of branches representing relationships among profiled cell types. Here, we provide details for (i) generating transgenic zebrafish; (ii) performing multi-timepoint barcode editing; (iii) building scRNA-seq libraries from brain tissue; and (iv) concurrently amplifying lineage barcodes from captured single cells. Generating transgenic lines takes 6 months, and performing barcode editing and generating single-cell libraries involve 7 d of hands-on time. scGESTALT provides a scalable platform to map lineage relationships between cell types in any system that permits genome editing during development, regeneration, or disease.
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