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Mos1 Element-Mediated CRISPR Integration of Transgenes in Caenorhabditis elegans.
The introduction of exogenous genes in single-copy at precise genomic locations is a powerful tool that has been widely used in the model organism Caenorhabditis elegans Here, we have streamlined the process by creating a rapid, cloning-free method of single-copy transgene insertion we call Mos1 element-mediated CRISPR integration (mmCRISPi). The protocol combines the impact of Mos1 mediated single-copy gene insertion (mosSCI) with the ease of CRISPR/Cas9 mediated gene editing, allowing in vivo construction of transgenes from linear DNA fragments integrated at defined loci in the C. elegans genome. This approach was validated by defining its efficiency at different integration sites in the genome and by testing transgene insert size. The mmCRISPi method benefits from in vivo recombination of overlapping PCR fragments, allowing researchers to mix-and-match between promoters, protein-coding sequences, and 3' untranslated regions, all inserted in a single step at a defined Mos1 loci.
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Genome editing with the CRISPR-Cas system: an art, ethics and global regulatory perspective.
Over the last three decades, the development of new genome editing techniques, such as ODM, TALENs, ZFNs and the CRISPR-Cas system, has led to significant progress in the field of plant and animal breeding. The CRISPR-Cas system is the most versatile genome editing tool discovered in the history of molecular biology because it can be used to alter diverse genomes (e.g. genomes from both plants and animals) including human genomes with unprecedented ease, accuracy and high efficiency. The recent development and scope of CRISPR-Cas system have raised new regulatory challenges around the world due to moral, ethical, safety and technical concerns associated with its applications in pre-clinical and clinical research, biomedicine and agriculture. Here, we review the art, applications and potential risks of CRISPR-Cas system in genome editing. We also highlight the patent and ethical issues of this technology along with regulatory frameworks established by various nations to regulate CRISPR-Cas-modified organisms/products.
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CRISPR-Cpf1-Mediated Gene-Editing System Based on a Single Bidirectional Promoter.
Recent advances in gene therapy have highlighted the potential of CRISPR-based gene-editing systems combined with adeno-associated virus (AAV) vectors. However, the limited packaging capacity of AAV remains a significant challenge for the simultaneous expression of Cas effector proteins and guide RNAs within a single vector. To address this limitation, we developed a compact AAV vector that enables the co-expression of Acidaminococcus sp. Cas12a (AsCpf1) and CRISPR RNAs (crRNAs) using a single bidirectional promoter derived from the mouse H1 promoter. Our single bidirectional H1 promoter supported indel formation comparable to that achieved by dual-promoter systems and facilitated scalable genome editing with single-, dual-, and triple-target configurations. Genome editing was successfully accomplished both in vitro and in vivo following AAV delivery. This study shows that our engineered compact AAV vector platform is capable of simultaneously delivering AsCpf1 and multiplexed crRNAs.
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Exon skipping induced by CRISPR-directed gene editing regulates the response to chemotherapy in non-small cell lung carcinoma cells.
We have been developing CRISPR-directed gene editing as an augmentative therapy for the treatment of non-small cell lung carcinoma (NSCLC) by genetic disruption of Nuclear Factor Erythroid 2-Related Factor 2 (NRF2). NRF2 promotes tumor cell survival in response to therapeutic intervention and thus its disablement should restore or enhance effective drug action. Here, we report how NRF2 disruption leads to collateral damage in the form of CRISPR-mediated exon skipping. Heterogeneous populations of transcripts and truncated proteins produce a variable response to chemotherapy, dependent on which functional domain is missing. We identify and characterize predicted and unpredicted transcript populations and discover that several types of transcripts arise through exon skipping; wherein one or two NRF2 exons are missing. In one specific case, the presence or absence of a single nucleotide determines whether an exon is skipped or not by reorganizing Exonic Splicing Enhancers (ESEs). We isolate and characterize the diversity of clones induced by CRISPR activity in a NSCLC tumor cell population, a critical and often overlooked genetic byproduct of this exciting technology. Finally, gRNAs must be designed with care to avoid altering gene expression patterns that can account for variable responses to solid tumor therapy.
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Performing an In Vitro Genome-Wide CRISPR Knockout Screen in Chimeric Antigen Receptor T Cells.
Chimeric antigen receptor T (CART) cell therapy is an innovative form of targeted immunotherapy that has revolutionized the treatment of cancer. However, the durable response remains limited. Recent studies have shown that the epigenetic landscape of preinfusion CART cell products can influence response to therapy, and gene editing has been proposed as a solution. However, more work needs to be done to determine the optimal gene editing strategy. Genome-wide CRISPR screens have become popular tools to both investigate mechanisms of resistance and optimize gene editing strategies. Yet their application to primary cells presents many challenges. Here we describe a method to complete a genome-wide CRISPR knockout screen in CART cells from healthy donors. As a proof-of-concept model, we designed this method to investigate the development of exhaustion in CART cells targeting the CD19 antigen. However, we believe that this approach can be used to address a variety of mechanisms of resistance to therapy in different CAR constructs and tumor models.
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A Method for Rapid Selection of Randomly Induced Mutations in a Gene of Interest Using CRISPR/Cas9 Mediated Activation of Gene Expression.
We have developed a CRISPR/Cas9 based method for isolating randomly induced recessive lethal mutations in a gene of interest (GOI) by selection within the F1 progeny of a single genetic cross. Our method takes advantage of the ability to overexpress a GOI using CRISPR/Cas9 mediated activation of gene expression. In essence, the screening strategy is based upon the idea that if overexpression of a wild type allele can generate a phenotype, then overexpression of a newly induced loss-of-function allele will lack this phenotype. As a proof-of-principle, we used this method to select EMS induced mutations of the Drosophila gene hindsight (hnt). From approximately 45,000 F1 progeny we recovered 8 new EMS induced loss-of-function hnt alleles that we characterized as an allelic series of hypomorphic mutations. This new method can, in theory, be used to recover randomly induced point mutants in a GOI and can be applied to any circumstance where CRISPR/Cas9 mediated activation of gene expression is associated with lethality or a visible phenotype.
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Notes on Functional Modules in the Assembly of CRISPR/Cas9-Mediated Epigenetic Modifiers.
CRISPR/cas9 is a popular tool, widely used today for genome editing. However, the modular organization of this tool allows it to be used not only for DNA modifications but also for introducing epigenetic modifications both in DNA (methylation/demethylation) and in histones (acetylation/deacetylation). In these notes we will concentrate on the ways to adapt the CRISPR/cas9 system for epigenetic DNA modification of specific regions of interest. The modular organization represents a universal principal, that allows to create infinite number of functions with a limited number of tools. CRISPR/cas9, in which each subunit can be adapted for a particular task, is an excellent example of this rule. Made of two main subunits, it can be modified for targeted delivery of foreign activity (effector, an epigenetic enzyme in our case) to a selected part of the genome. In doing this the CRISPR/cas9 system represents a unique method that allows the introduction of both genomic and epigenetic modifications. This chapter gives a detailed review of how to prepare DNA for the fully functional CRISPR/cas9 system, able to introduce required modifications in the region of interest. We will discuss specific requirements for each structural component of the system as well as for auxiliary elements (modules), which are needed to ensure efficient expression of the elements of the system within the cell and the needs of selection and visualization.
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CRISPR/Cas9 gene editing for the creation of an MGAT1-deficient CHO cell line to control HIV-1 vaccine glycosylation.
Over the last decade, multiple broadly neutralizing monoclonal antibodies (bN-mAbs) to the HIV-1 envelope protein (Env) gp120 have been described. Many of these recognize epitopes consisting of both amino acid and glycan residues. Moreover, the glycans required for binding of these bN-mAbs are early intermediates in the N-linked glycosylation pathway. This type of glycosylation substantially alters the mass and net charge of Envs compared to molecules with the same amino acid sequence but possessing mature, complex (sialic acid-containing) carbohydrates. Since cell lines suitable for biopharmaceutical production that limit N-linked glycosylation to mannose-5 (Man5) or earlier intermediates are not readily available, the production of vaccine immunogens displaying these glycan-dependent epitopes has been challenging. Here, we report the development of a stable suspension-adapted Chinese hamster ovary (CHO) cell line that limits glycosylation to Man5 and earlier intermediates. This cell line was created using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing system and contains a mutation that inactivates the gene encoding Mannosyl (Alpha-1,3-)-Glycoprotein Beta-1,2-N-Acetylglucosaminyltransferase (MGAT1). Monomeric gp120s produced in the MGAT1- CHO cell line exhibit improved binding to prototypic glycan-dependent bN-mAbs directed to the V1/V2 domain (e.g., PG9) and the V3 stem (e.g., PGT128 and 10-1074) while preserving the structure of the important glycan-independent epitopes (e.g., VRC01). The ability of the MGAT1- CHO cell line to limit glycosylation to early intermediates in the N-linked glycosylation pathway without impairing the doubling time or ability to grow at high cell densities suggests that it will be a useful substrate for the biopharmaceutical production of HIV-1 vaccine immunogens.
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Otoferlin gene editing in sheep via CRISPR-assisted ssODN-mediated Homology Directed Repair.
Different mutations of the OTOF gene, encoding for otoferlin protein expressed in the cochlear inner hair cells, induces a form of deafness that is the major cause of nonsyndromic recessive auditory neuropathy spectrum disorder in humans. We report the generation of the first large animal model of OTOF mutations using the CRISPR system associated with different Cas9 components (mRNA or protein) assisted by single strand oligodeoxynucleotides (ssODN) to induce homology-directed repair (HDR). Zygote microinjection was performed with two sgRNA targeting exon 5 and 6 associated to Cas9 mRNA or protein (RNP) at different concentrations in a mix with an ssODN template targeting HDR in exon 5 containing two STOP sequences. A total of 73 lambs were born, 13 showing indel mutations (17.8%), 8 of which (61.5%) had knock-in mutations by HDR. Higher concentrations of Cas9-RNP induced targeted mutations more effectively, but negatively affected embryo survival and pregnancy rate. This study reports by the first time the generation of OTOF disrupted sheep, which may allow better understanding and development of new therapies for human deafness related to genetic disorders. These results support the use of CRISPR/Cas system assisted by ssODN as an effective tool for gene editing in livestock.
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CRISPR Tackles Emerging Viral Pathogens.
Understanding the dynamic relationship between viral pathogens and cellular host factors is critical to furthering our knowledge of viral replication, disease mechanisms and development of anti-viral therapeutics. CRISPR genome editing technology has enhanced this understanding, by allowing identification of pro-viral and anti-viral cellular host factors for a wide range of viruses, most recently the cause of the COVID-19 pandemic, SARS-CoV-2. This review will discuss how CRISPR knockout and CRISPR activation genome-wide screening methods are a robust tool to investigate the viral life cycle and how other class 2 CRISPR systems are being repurposed for diagnostics.
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