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A Guanidinobenzol-Rich Polymer Overcoming Cascade Delivery Barriers for CRISPR-Cas9 Genome Editing.
The efficient cytosolic delivery of the CRISPR-Cas9 machinery remains a challenge for genome editing. Herein, we performed ligand screening and identified a guanidinobenzol-rich polymer to overcome the cascade delivery barriers of CRISPR-Cas9 ribonucleoproteins (RNPs) for genome editing. RNPs were stably loaded into the polymeric nanoparticles (PGBA NPs) by their inherent affinity. The polymer facilitated rapid endosomal escape of RNPs via a dynamic multiple-step cascade process. Importantly, the incorporation of fluorescence in the polymer helps to identify the correlation between cellular uptake and editing efficiency, increasing the efficiency up to 70% from the initial 30% for the enrichment of edited cells. The PGBA NPs efficiently deliver RNPs for in vivo gene editing via both local and systemic injections and dramatically reduce PCSK9 level. These results indicate that PGBA NPs enable the cascade delivery of RNPs for genome editing, showing great promise in broadening the therapeutic potential of the CRISPR-Cas9 technique.
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A Cas12a-based gene editing system for Phytophthora infestans reveals monoallelic expression of an elicitor.
Phytophthora infestans is a destructive pathogen of potato and a model for investigations of oomycete biology. The successful application of a CRISPR gene editing system to P. infestans is so far unreported. We discovered that it is difficult to express CRISPR/Cas9 but not a catalytically inactive form in transformants, suggesting that the active nuclease is toxic. We were able to achieve editing with CRISPR/Cas12a using vectors in which the nuclease and its guide RNA were expressed from a single transcript. Using the elicitor gene Inf1 as a target, we observed editing of one or both alleles in up to 13% of transformants. Editing was more efficient when guide RNA processing relied on the Cas12a direct repeat instead of ribozyme sequences. INF1 protein was not made when both alleles were edited in the same transformant, but surprisingly also when only one allele was altered. We discovered that the isolate used for editing, 1306, exhibited monoallelic expression of Inf1 due to insertion of a copia-like element in the promoter of one allele. The element exhibits features of active retrotransposons, including a target site duplication, long terminal repeats, and an intact polyprotein reading frame. Editing occurred more often on the transcribed allele, presumably due to differences in chromatin structure. The Cas12a system not only provides a tool for modifying genes in P. infestans, but also for other members of the genus by expanding the number of editable sites. Our work also highlights a natural mechanism that remodels oomycete genomes.
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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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Application of Protoplast Regeneration to CRISPR/Cas9 Mutagenesis in Nicotiana tabacum.
Protoplast transfection is widely used in plant research to rapidly evaluate RNA degradation, reporter assay, gene expression, subcellular localization, and protein-protein interactions. In order to successfully use protoplast transfection with the newly emerging clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) protein editing platform, high yield of protoplasts, stable transfection efficiency, and reliable regeneration protocols are necessary. The Nicotiana tabacum transient protoplast transfection and regeneration system can effectively obtain target gene mutations in regenerated plants without transgenes and is thus a very attractive technique for evaluating gene editing reagents using CRISPR/Cas-based systems. Here, we describe in detail sterilized seed germination, culture conditions, isolation of Nicotiana tabacum protoplasts from tissue culture explants, construction of a vector containing the Cas protein and sgRNA cassette, highly efficient polyethylene glycol-calcium transient transfection of plasmids delivered into protoplasts, evaluation of mutagenesis efficiency and genotype analysis from protoplasts and regenerated plants, and the regeneration conditions to obtain CRISPR-edited plants from single protoplasts.
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Multiplexed Targeted Genome Engineering Using a Universal Nuclease-Assisted Vector Integration System.
Engineered nucleases are capable of efficiently modifying complex genomes through introduction of targeted double-strand breaks. However, mammalian genome engineering remains limited by low efficiency of heterologous DNA integration at target sites, which is typically performed through homologous recombination, a complex, ineffective and costly process. In this study, we developed a multiplexable and universal nuclease-assisted vector integration system for rapid generation of gene knock outs using selection that does not require customized targeting vectors, thereby minimizing the cost and time frame needed for gene editing. Importantly, this system is capable of remodeling native mammalian genomes through integration of DNA, up to 50 kb, enabling rapid generation and screening of multigene knockouts from a single transfection. These results support that nuclease assisted vector integration is a robust tool for genome-scale gene editing that will facilitate diverse applications in synthetic biology and gene therapy.
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Delivery of genetic load during ex situ liver machine perfusion with potential for CRISPR-Cas9 gene editing: An innovative strategy for graft treatment.
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Generation of knock-in cynomolgus monkey via CRISPR/Cas9 editing.
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Application of CRISPR/CasΦ2 System for Genome Editing in Plants.
CRISPR/Cas system has developed a new technology to modify target genes. In this study, CasΦ2 is a newly Cas protein that we used for genome modification in Arabidopsis and tobacco. PDS and BRI1 of marker genes were chosen for targeting. CasΦ2 has the function to cleave pre-crRNA. In the presence of 10 mM Mg2+ irons concentration, sgRNA3 type guided CasΦ2 to edit target gene and generate mutation, and a mutant seedling of AtBRI1 gene with an expected male sterile phenotype was obtained. In the process of tobacco transformation, the gene editing activity of CasΦ2 can be activated by 100 nM Mg2+ irons concentration, and sgRNA1 type guided CasΦ2 to edit target gene. Mutant seedlings of NtPDS gene with an expected albino were obtained. The results indicate that CasΦ2 can effectively edit target genes under the guidance of different sgRNA type in the presence of Mg2+ ions. Together, our results verify that the CRISPR/CasΦ2 system is an effective and precise tool for genome editing in plants.
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Effects of Electroporation Timing and Cumulus Cell Attachment on In Vitro Development and Genome Editing of Porcine Embryos.
Pig genome editing using the oviductal nucleic acid delivery (GONAD) method with electroporation would allow the efficient obtention of genetically modified pigs. However, oocytes and zygotes at various stages after ovulation must be targeted, and cumulus cell attachment and mosaic mutations are major obstacles. Therefore, we investigated whether two parameters (electroporation timing and the cumulus cell attachment) influence the effectiveness of multiplex genome editing by electroporation in porcine oocytes or zygotes. Three gRNAs targeting either GGTA1, CMAH or B4GALNT2 were introduced individually into oocytes and zygotes with and without cumulus cells at three different time points, 0 h before in vitro fertilisation (IVF) and 5 h and 10 h after IVF initiation. The introduction of gRNAs into oocytes and zygotes did not significantly affect the rates of blastocyst formation and total mutation of the resulting blastocysts irrespective of cumulus cell attachment and electroporation timing. In conclusion, the electroporation timing and the cumulus cell attachment did not interfere with the efficient delivery of the CRISPR/Cas9 system to the oocytes/zygotes, indicating that porcine genome editing in the oviduct using GONAD method may be possible.
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Application of CRISPR/Cas9 for Rapid Genome Editing of Pseudorabies Virus and Bovine Herpesvirus-1.
The CRISPR/Cas9 system is widely used to manipulate viral genomes. Although Alphaherpesvirinae genomes are large and complicated to edit, in recent years several Pseudorabies virus (PRV) mutants have been successfully generated using the CRISPR/Cas9 system. However, the application of CRISPR/Cas9 editing on another member of alpha herpesviruses, bovine herpesvirus-1 (BHV-1), is rarely reported. This paper reports a rapid and straightforward approach to manipulating herpesviruses genome using CRISPR/Cas9. The recombinant plasmids contained the left and right arm of the thymidine kinase (TK) gene of PRV or of the glycoprotein I (gI) and glycoprotein E (gE) of BHV-1. Upon the cleavage of the TK or gIgE gene by Cas9 protein, this was replaced by the enhanced green fluorescence protein (eGFP) by homologous recombination. With this approach, we generated recombinant TK-/eGFP+ PRV and gIgE-/eGFP+ BHV-1 mutants and then proceeded to characterize their biological activities in vitro and in vivo. In conclusion, we showed that alpha herpesvirus, including PRV and BHV-1, can be rapidly edited using the CRISPR/Cas9 approach paving the way to the development of animal herpesvirus vaccines.
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