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A G-Rich Motif in the lncRNA Braveheart Interacts with a Zinc-Finger Transcription Factor to Specify the Cardiovascular Lineage.
Long non-coding RNAs (lncRNAs) are an emerging class of transcripts that can modulate gene expression; however, their mechanisms of action remain poorly understood. Here, we experimentally determine the secondary structure of Braveheart (Bvht) using chemical probing methods and show that this ∼590 nt transcript has a modular fold. Using CRISPR/Cas9-mediated editing of mouse embryonic stem cells, we find that deletion of 11 nt in a 5' asymmetric G-rich internal loop (AGIL) of Bvht (bvhtdAGIL) dramatically impairs cardiomyocyte differentiation. We demonstrate a specific interaction between AGIL and cellular nucleic acid binding protein (CNBP/ZNF9), a zinc-finger protein known to bind single-stranded G-rich sequences. We further show that CNBP deletion partially rescues the bvhtdAGIL mutant phenotype by restoring differentiation capacity. Together, our work shows that Bvht functions with CNBP through a well-defined RNA motif to regulate cardiovascular lineage commitment, opening the door for exploring broader roles of RNA structure in development and disease.
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More precise, more universal and more specific - the next generation of RNA-guided endonucleases for genome editing.
CRISPR is a prokaryotic defence system that was adapted as a tool for genome editing and has become one of the most important discoveries of this century. CRISPR-associated endonucleases cleave DNA at precise sites, which are marked by complementary short-guided RNA. The recently developed versions of endonucleases are compatible with a broad range of PAM motifs, have a higher specificity and enable a specific nucleotide to be replaced.
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Ultrasound-activated particles as CRISPR/Cas9 delivery system for androgenic alopecia therapy.
Compared to a plasmid, viral, and other delivery systems, direct Cas9/sgRNA protein delivery has several advantages such as low off-targeting effects and non-integration, but it still has limitations due to low transfer efficiency. As such, the CRISPR/Cas9 system is being developed in combination with nano-carrier technology to enhance delivery efficiency and biocompatibility. We designed a microbubble-nanoliposomal particle as a Cas9/sgRNA riboprotein complex carrier, which effectively facilitates local delivery to a specific site when agitated by ultrasound activation. In practice, we successfully transferred the protein constructs into dermal papilla cells in the hair follicle of androgenic alopecia animals by microbubble cavitation induced sonoporation of our particle. The delivered Cas9/sgRNA recognized and edited specifically the target gene with high efficiency in vitro and in vivo, thus recovering hair growth. We demonstrated the topical application of ultrasound-activated nanoparticles for androgenic alopecia therapy through the suppression of SRD5A2 protein production by CRISPR-based genomic editing.
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CRISPR/Cas9-loaded stealth liposomes effectively cleared established HPV16-driven tumours in syngeneic mice.
Gene-editing has raised the possibility of being able to treat or cure cancers, but key challenges remain, including efficient delivery, in vivo efficacy, and its safety profile. Ideal targets for cancer therapy are oncogenes, that when edited, cause cell death. Here, we show, using the human papillomavirus (HPV) type 16 cancer cell line TC1, that CRISPR/Cas9 targeting the E7 oncogene and packaged in PEGylated liposomes cleared established tumours in immunocompetent mice. Treatment caused no significant toxicity in the spleen or liver. An ideal therapeutic outcome would be the induction of an immunogenic cell death (ICD), such that recurrent tumours would be eliminated by the host immune system. We show here for the first time that CRISPR/Cas9-mediated cell death via targeting E7 did not result in ICD. Overall, our data show that in vivo CRISPR/Cas targeting of oncogenes is an effective treatment approach for cancer.
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CRISPR-Csy4-Mediated Editing of Rotavirus Double-Stranded RNA Genome.
CRISPR-nucleases have been widely applied for editing cellular and viral genomes, but nuclease-mediated genome editing of double-stranded RNA (dsRNA) viruses has not yet been reported. Here, by engineering CRISPR-Csy4 nuclease to localize to rotavirus viral factories, we achieve the nuclease-mediated genome editing of rotavirus, an important human and livestock pathogen with a multisegmented dsRNA genome. Rotavirus replication intermediates cleaved by Csy4 is edited through the formation of precise deletions in the targeted genome segments in a single replication cycle. Using CRISPR-Csy4-mediated editing of rotavirus genome, we label the products of rotavirus secondary transcription made by newly assembled viral particles during rotavirus replication, demonstrating that this step largely contributes to the overall production of viral proteins. We anticipate that the nuclease-mediated cleavage of dsRNA virus genomes will promote an advanced level of understanding of viral replication and host-pathogen interactions, also offering opportunities to develop therapeutics.
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Multiplexed Genome Engineering with Cas12a.
Genome engineering technologies based on CRISPR-Cas systems are fueling efforts to study genotype-phenotype relationships in a high-throughput and multiplexed fashion. While many genome engineering technologies exist and provide a means to efficiently manipulate one or a few genes in a singular context-knockout, inhibition, or activation in a constitutive, conditional, or inducible manner-progress towards engineering complex cellular programs has been hampered by the lack of technologies that can integrate these functions within a unified framework. To address this challenge, our lab created single transcript CRISPR-Cas12a (SiT-Cas12a), which enables conditional, inducible, orthogonal, and massively multiplexed genome engineering of dozens, to potentially hundreds, of genomic targets in eukaryotic cells simultaneously-providing a novel way to interrogate and engineer complex genetic programs. In this chapter, we outline the utility of SiT-Cas12a in human cells and describe experimental procedures for executing massively multiplexed genome engineering experiments-including strategies for designing and assembling customized multiplexed CRISPR guide RNA arrays as well as validating and analyzing CRISPR guide RNA array processing and genome engineering outcomes.
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Application of genetic modification technologies in molecular design breeding of sheep.
Genetic modification technologies can be used for modifying animal genome to express exogenous genes or affect the function of endogenous genes. In animal breeding, genetic modification technologies allow the rapid generation of germplasms with beneficial traits. It includes traditional genetic modification, virus or sperm carrier-mediated genetic modification and nuclease-mediated genome editing, especially the CRISPR/Cas9, one of the artificial nuclease genome editing technologies, have been applied in genome editing in many domestic animals including sheep (Ovis aries). Compared with conventional strategies used for animal breeding, there is great value for sheep breeding improvement by using genome editing technology, which is more effective and timesaving. In this review, we summarize the approaches of genetic modification in sheep and discuss the possibility of molecular design and breeding of sheep by genome editing technologies. We also identify the potential bottlenecks and challenges of these technologies in sheep breeding.
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Generation of knockout mice by Cpf1-mediated gene targeting.
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Targeted mutagenesis in wheat microspores using CRISPR/Cas9.
CRISPR/Cas9 genome editing is a transformative technology that will facilitate the development of crops to meet future demands. However, application of gene editing is hindered by the long life cycle of many crop species and because desired genotypes generally require multiple generations to achieve. Single-celled microspores are haploid cells that can develop into double haploid plants and have been widely used as a breeding tool to generate homozygous plants within a generation. In this study, we combined the CRISPR/Cas9 system with microspore technology and developed an optimized haploid mutagenesis system to induce genetic modifications in the wheat genome. We investigated a number of factors that may affect the delivery of CRISPR/Cas9 reagents into microspores and found that electroporation of a minimum of 75,000 cells using 10-20 µg DNA and a pulsing voltage of 500 V is optimal for microspore transfection using the Neon transfection system. Using multiple Cas9 and sgRNA constructs, we present evidence for the seamless introduction of targeted modifications in an exogenous DsRed gene and two endogenous wheat genes, including TaLox2 and TaUbiL1. This study demonstrates the value and feasibility of combining microspore technology and CRISPR/Cas9-based gene editing for trait discovery and improvement in plants.
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Breasi-CRISPR: an efficient genome-editing method to interrogate protein localization and protein-protein interactions in the embryonic mouse cortex.
In developing tissues, knowing the localization and interactors of proteins of interest is key to understanding their function. Here, we describe the Breasi-CRISPR approach (Brain Easi-CRISPR), combining Easi-CRISPR with in utero electroporation to tag endogenous proteins within embryonic mouse brains. Breasi-CRISPR enables knock-in of both short and long epitope tag sequences with high efficiency. We visualized epitope-tagged proteins with varied expression levels, such as ACTB, LMNB1, EMD, FMRP, NOTCH1 and RPL22. Detection was possible by immunohistochemistry as soon as 1 day after electroporation and we observed efficient gene editing in up to 50% of electroporated cells. Moreover, tagged proteins could be detected by immunoblotting in lysates from individual cortices. Next, we demonstrated that Breasi-CRISPR enables the tagging of proteins with fluorophores, allowing visualization of endogenous proteins by live imaging in organotypic brain slices. Finally, we used Breasi-CRISPR to perform co-immunoprecipitation mass-spectrometry analyses of the autism-related protein FMRP to discover its interactome in the embryonic cortex. Together, these data demonstrate that Breasi-CRISPR is a powerful tool with diverse applications that will propel the understanding of protein function in neurodevelopment.
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