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Prospects and challenges of CRISPR/Cas genome editing for the study and control of neglected vector-borne nematode diseases.
Neglected tropical diseases caused by parasitic nematodes inflict an immense health and socioeconomic burden throughout much of the developing world. Current estimates indicate that more than two billion people are infected with nematodes, resulting in the loss of 14 million disability-adjusted life years per annum. Although these parasites cause significant mortality, they primarily cause chronic morbidity through a wide range of severe clinical ailments. Treatment options for nematode infections are restricted to a small number of anthelmintic drugs, and the rapid expansion of anthelmintic mass drug administration raises concerns of drug resistance. Preservation of existing drugs is necessary, as well as the development of new treatment options and methods of control. We focus this review on how the democratization of CRISPR/Cas9 genome editing technology can be enlisted to improve our understanding of the biology of nematode parasites and our ability to treat the infections they cause. We will first explore how this robust method of genome manipulation can be used to newly exploit the powerful model nematode Caenorhabditis elegans for parasitology research. We will then discuss potential avenues to develop CRISPR/Cas9 editing protocols in filarial nematodes. Lastly, we will propose potential ways in which CRISPR/Cas9 can be used to engineer gene drives that target the transmission of mosquito-borne filarial nematodes.
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An improved CRISPR-Cas9 protein-based method for knocking out insect Sf9 cell genes.
Insect cells are one of the uprising expression systems in the biopharmaceutical industry to produce vaccines and gene therapy vectors, but cell line development has been limited by the lack of established genetic engineering tools and genomic characterization. CRISPR-Cas9 has arisen as a powerful tool for gene editing but has seen little application in insect cells. In this work, a gene editing pipeline for the delivery of a ribonucleoprotein (RNP) complex comprised of a guide RNA and the enzyme Cas9 to insect Sf9 cells was implemented and then applied to knockout caspase initiator Sf-Dronc, aiming at alleviating cell apoptosis during an infection process. The resulting engineered cell lines were characterized as per their phenotype and production of three different product modalities. Utilizing the established workflow, a knockout rate of 68% was achieved with the implemented protocol (vs. the 12% presumed efficiency of a previously reported system) when targeting the fdl gene. When applied to Sf-Dronc, mutants containing deletions in several alleles of the host genome were identified and confirmed by next-generation sequencing. Generated clones exhibited higher apoptosis resistance and delayed onset of cell viability drop following infection with baculovirus. While Sf-Dronc deletion was shown to have negligible impact on the production of rAAV and PfRipr5, production of iVLPS showed an > twofold increase over wild-type Sf9. Overall, this study showcases the successful implementation of an efficient CRISPR-Cas9 pipeline, further leveraging the usage of genetic engineering in insect Sf9 cells towards the development of enhanced cell hosts for biopharmaceutical production. KEY POINTS: • Implementation of an efficient CRISPR-Cas9 RNP complex delivery strategy to insect cells. • Establishment of the genome editing pipeline demonstrated through Sf-Dronc knockout, resulting in increased apoptosis resistance and delayed loss of viability upon baculovirus infection. • Sf-Dronc deletion led to over a twofold increase in the production of influenza VLPs compared to wild-type Sf9 cells.
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Genome Editing: Promoting Responsible Research.
For more than 40 years, scientists have been developing tools and technologies for genome modification; however, initial progress was slow and few outside of the molecular biology community took an interest in the field. Everything has dramatically changed with the recent appearance of the so-called precision approaches, and especially with the 'CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) revolution'. With great powers come great responsibilities. CRISPR-derived technologies have been proven efficient, cheap, rather easy and fast, and provided universal access to genome modification techniques beyond the leading research centers and reference laboratories. The popularization of techniques to manipulate the human genome and that of all other living beings consequently raises many essential questions, on the ethical and legal sides, both for the scientific community and the lay public. In order to mitigate excessive hype and concern among citizens, a call for the mobilization of the various stakeholders is now urgent through a global governance of genome editing.
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Inhibition of HSV-1 Replication by Gene Editing Strategy.
HSV-1 induced illness affects greater than 85% of adults worldwide with no permanent curative therapy. We used RNA-guided CRISPR/Cas9 gene editing to specifically target for deletion of DNA sequences of the HSV-1 genome that span the region directing expression of ICP0, a key viral protein that stimulates HSV-1 gene expression and replication. We found that CRISPR/Cas9 introduced InDel mutations into exon 2 of the ICP0 gene profoundly reduced HSV-1 infectivity in permissive human cell culture models and protected permissive cells against HSV-1 infection. CRISPR/Cas9 mediated targeting ICP0 prevented HSV-1-induced disintegration of promonocytic leukemia (PML) nuclear bodies, an intracellular event critical to productive HSV-1 infection that is initiated by interaction of the ICP0 N-terminus with PML. Combined treatment of cells with CRISPR targeting ICP0 plus the immediate early viral proteins, ICP4 or ICP27, completely abrogated HSV-1 infection. We conclude that RNA-guided CRISPR/Cas9 can be used to develop a novel, specific and efficacious therapeutic and prophylactic platform for targeted viral genomic ablation to treat HSV-1 diseases.
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CRISPR/Cas9-Mediated Genome Editing of the Komagataella phaffii to Obtain a Phytase-Producer Markerless Strain.
Using CRISPR/Cas9 system, the recipient strains K. phaffii VKPM Y-5013 (His- phenotype) and K. phaffii VKPM Y-5014 (Leu- phenotype) were derived from the K. phaffii VKPM Y-4287 strain, which has a high expression potential. Based on the developed recipient strains, markerless producers of heterologous proteins could be obtained. Efficiency of the gene inactivation with different variants of sgRNA ranged from 65 to 98% and from 15 to 72% for the HIS4 and LEU2 genes, respectively. The recipient strains retained growth characteristics of the parent strain and exhibited high expression potential, as estimated by the production of heterologous phytase from Citrobacter gillenii. Average productivity of the transformants based on the K. phaffii VKPM Y-5013 and K. phaffii VKPM Y-5014 strains was 2.1 and 2.0 times higher than productivity of the transformants of the commercial K. phaffii GS115 strain. Method for sequential integration of genetic material into genome of the K. phaffii VKPM Y-5013 strain was proposed. A highly effective multicopy markerless strain producing C. gillenii phytase was obtained.
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DeepCRISTL: deep transfer learning to predict CRISPR/Cas9 on-target editing efficiency in specific cellular contexts.
MOTIVATION: CRISPR/Cas9 technology has been revolutionizing the field of gene editing. Guide RNAs (gRNAs) enable Cas9 proteins to target specific genomic loci for editing. However, editing efficiency varies between gRNAs and so computational methods were developed to predict editing efficiency for any gRNA of interest. High-throughput datasets of Cas9 editing efficiencies were produced to train machine-learning models to predict editing efficiency. However, these high-throughput datasets have a low correlation with functional and endogenous datasets, which are too small to train accurate machine-learning models on. RESULTS: We developed DeepCRISTL, a deep-learning model to predict the editing efficiency in a specific cellular context. DeepCRISTL takes advantage of high-throughput datasets to learn general patterns of gRNA editing efficiency and then fine-tunes the model on functional or endogenous data to fit a specific cellular context. We tested two state-of-the-art models trained on high-throughput datasets for editing efficiency prediction, our newly improved DeepHF and CRISPRon, combined with various transfer-learning approaches. The combination of CRISPRon and fine-tuning all model weights was the overall best performer. DeepCRISTL outperformed state-of-the-art methods in predicting editing efficiency in a specific cellular context on functional and endogenous datasets. Using saliency maps, we identified and compared the important features learned by DeepCRISTL across cellular contexts. We believe DeepCRISTL will improve prediction performance in many other CRISPR/Cas9 editing contexts by leveraging transfer learning to utilize both high-throughput datasets and smaller and more biologically relevant datasets. AVAILABILITY AND IMPLEMENTATION: DeepCRISTL is available via https://github.com/OrensteinLab/DeepCRISTL.
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Recent Advances in Genome-Engineering Strategies.
In October 2020, the chemistry Nobel Prize was awarded to Emmanuelle Charpentier and Jennifer A. Doudna for the discovery of a new promising genome-editing tool: the genetic scissors of CRISPR-Cas9. The identification of CRISPR arrays and the subsequent identification of cas genes, which together represent an adaptive immunological system that exists not only in bacteria but also in archaea, led to the development of diverse strategies used for precise DNA editing, providing new insights in basic research and in clinical practice. Due to their advantageous features, the CRISPR-Cas systems are already employed in several biological and medical research fields as the most suitable technique for genome engineering. In this review, we aim to describe the CRISPR-Cas systems that have been identified among prokaryotic organisms and engineered for genome manipulation studies. Furthermore, a comprehensive comparison between the innovative CRISPR-Cas methodology and the previously utilized ZFN and TALEN editing nucleases is also discussed. Ultimately, we highlight the contribution of CRISPR-Cas methodology in modern biomedicine and the current plethora of available applications for gene KO, repression and/or overexpression, as well as their potential implementation in therapeutical strategies that aim to improve patients' quality of life.
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Plenary Symposia.
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Selector adeno-associated viral vectors facilitate on-target precise genome editing and purge off-target chromosomal insertions.
Adeno-associated viral (AAV) vectors are commonly used for genome editing owing to the proclivity with which their single-stranded genomes serve as homologous recombination (donor) substrates during programmable nuclease-assisted gene targeting. However, the highly recombinogenic nature of AAV genomes also facilitates their nonhomologous end joining at off-target chromosomal breaks ('capture') created by said nucleases, mutagens, or DNA metabolic processes. Moreover, AAV donor constructs can equally yield imprecise on-target edits resulting from end-joining recombination pathways. Here, we demonstrate that endowing AAV vectors with exogenous marker-free selectable sequences permits enrichment for cells precisely coedited at endogenous target and ATP1A1 alleles. These selector AAV vectors install ATP1A1 polymorphisms conferring resistance to the small molecule ouabain, yielding high frequencies of on-target and precisely edited cell populations. Crucially, we further report that selector AAV vectors achieve a thorough removal of heterogeneous off-target DNA species resulting from conventional AAV-based genome editing procedures.
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Transcriptional regulation by CRISPR/dCas9 in common wheat.
The application of CRISPR/Cas9 system for gene editing, as a technical coup for biotechnology, is worldwide and encompasses multiple of species. The inactivation of catalytical site in Cas9 (dCas9) has been reprogrammed as an effective approach to regulate the transcriptional level of target genes, especially for the functionally essential genes and redundant genes. Here, we exploited the CRISPR/dCas9 system to manipulate the transcriptional level of target genes in common wheat. To improve target gene's expression, we generated transcriptional activator by fusing 6×TAL-VP128 activation domain to the C-terminus of dCas9 in frame. For target gene's repressing expression transcriptionally, 3×SRDX repression domain was conjugated to the C-terminus of dCas9 in frame. Our results showed that dCas9 fused activation or repression domain could increase or decrease the transcriptional level of target gene effectively in stable transgenic lines of wheat. The study on the tRNA-processing system in CRISPR/dCas9 based transcriptional regulation system demonstrated that this robust multiplex targeted tool can be incorporated to the CRISPR/dCas9 system to facilitate the target regulation of several genes' transcriptional level. Our data broaden the application of CRISPR/dCas9 based transcriptional regulation and provide great opportunities to investigate the function of essential genes in common wheat.
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