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The CRISPR Revolution in the Drug Discovery Workflow: An Industry Perspective.
In a relatively short time, the pharmaceutical industry has witnessed a rapid integration of the CRISPR technology in multiple areas of research, development, therapy, and diagnostics. A unique feature to this system compared with other technologies is the exceptional versatility in adapting to the broad range of needs across the drug discovery pipeline, such as target identification, cell engineering, and in vivo modeling. As a consequence, the CRISPR toolbox has been evolving to address key questions around preclinical and clinical drug development. In this review, we provide a high-level perspective of how CRISPR has impacted several aspects of the drug discovery workflow and the future ahead for this exciting technology.
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Delivery of genome-editing biomacromolecules for treatment of lung genetic disorders.
Genome-editing systems based on clustered, regularly interspaced, short palindromic repeat (CRISPR)/associated protein (CRISPR/Cas), are emerging as a revolutionary technology for the treatment of various genetic diseases. To date, the delivery of genome-editing biomacromolecules by viral or non-viral vectors have been proposed as new therapeutic options for lung genetic disorders, such as cystic fibrosis (CF) and α-1 antitrypsin deficiency (AATD), and it has been accepted that these delivery vectors can introduce CRISPR/Cas9 machineries into target cells or tissues in vitro, ex vivo and in vivo. However, the efficient local or systemic delivery of CRISPR/Cas9 elements to the lung, enabled by either viral or by non-viral carriers, still remains elusive. Herein, we first introduce lung genetic disorders and their current treatment options, and then summarize CRISPR/Cas9-based strategies for the therapeutic genome editing of these disorders. We further summarize the pros and cons of different routes of administration for lung genetic disorders. In particular, the potentials of aerosol delivery for therapeutic CRISPR/Cas9 biomacromolecules for lung genome editing are discussed and highlighted. Finally, current challenges and future outlooks in this emerging area are briefly discussed.
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Chimeric DNA-RNA Guide RNA Designs.
CRISPR-associated nuclease (Cas) has been widely applied to modify the genomes of various cell types. As RNA-guided endonucleases, Cas enzymes can target different genomic sequences simply by changing the guide sequence of the CRISPR RNA (crRNA) or single guide RNA (sgRNA). Recent studies have demonstrated that DNA-RNA chimeric crRNA or sgRNA can efficiently guide the Cas9 protein for genome editing with reduced off-target effects. This chapter aims to describe a procedure for using chimeric RNA to modify the genomes of mammalian cells.
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A robust and practical CRISPR/crRNA screening system for soybean cultivar editing using LbCpf1 ribonucleoproteins.
Calli protoplasts isolated from three soybean cultivars are useful tools to evaluate guide RNAs for clustered regularly interspaced short palindromic repeats (CRISPR)-based precise gene editing. A type V CRISPR effector, LbCpf1(Cas12a) from Lachnospiraceae bacterium ND 2006, has been used for precision editing of the plant genome. We report that callus-derived protoplasts from three soybeans, including Glycine Max var. Williams 82 and two Korean cultivars (Kwangan and Daewon) represent efficient systems for the screening of active crRNA for CRISPR/LbCpf1. CRISPR/LbCpf1 ribonucleoproteins (RNPs) were delivered as complexes of purified endonucleases mixed with designed crRNA to simultaneously edit target genes of GlymaFAD2-1A and GlymaFAD2-1B transfected into three soybean protoplasts including genome-sequenced Williams 82 with cultivars, Kwangan and Daewon. Previously, we reported that nine crRNAs designed for LbCpf1 exhibited varying degrees of editing efficacy for two FAD2 genes. Among the nine crRNAs, the LbCpf1-crRNA3 complexes showed the highest efficiency in soybean cotyledon protoplasts. The new screening systems of callus protoplasts from three soybeans have been successfully used to transfect GFP-tagged markers and CRISPR/LbCpf1 RNPs. The callus protoplasts confirm that the LbCpf1-crRNA3 complex is an active crRNA for LbCpf1 to edit two FAD2 genes similar to cotyledon protoplasts. These results demonstrate that soybean callus protoplast-based CRISPR/crRNA selection is a new and practical tool to screen the efficacy of crRNAs and a prerequisite for progressive regeneration of the edited soybean.
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Gene Editing of Mouse Embryonic and Epiblast Stem Cells.
Efficient and reliable methods for gene editing are critical for the generation of loss-of-gene function stem cells and genetically modified mice. Here, we outline the application of CRISPR-Cas9 technology for gene editing in mouse embryonic stem cells (mESCs) to generate knockout ESC chimeras for the fast-tracked analysis of gene function. Furthermore, we describe the application of gene editing directly to mouse epiblast stem cells (mEpiSCs) for modelling germ layer differentiation in vitro.
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Research Status of Clustered Regulary Interspaced Short Palindromic Repeats Technology in the Treatment of Human Papillomavirus (HPV) Infection Related Diseases.
Background: CRISPR/Cas9 technology has rapidly advanced as a pivotal tool in cancer research, particularly in the precision targeting required for both detecting and treating malignancies. Its high specificity and low off-target effects make it exceptionally effective in applications involving Human Papillomavirus (HPV) related diseases, most notably cervical cancer. This approach offers a refined methodology for the rapid detection of viral infections and provides a robust platform for the safe and effective treatment of diseases associated with viral infections through gene therapy.Purpose: Gene therapy, within this context, involves the strategic delivery of genetic material into target cells via a vector. This is followed by the meticulous modulation of gene expression, whether through correction, addition, or suppression, specifically honed to target tumor cells while sparing healthy cells. This dual capacity to diagnose and treat at such a precise level underscores the transformative potential of CRISPR/Cas9 in contemporary medical science, particularly in oncology and virology.Research Design: This article provides an overview of the advancements made in utilizing the CRISPR-Cas9 system as a research tool for HPV-related treatments while summarizing its application status in basic research, diagnosis, and treatment of HPV.Data Collection: Furthermore, it discusses the future prospects for this technology within emerging areas of HPV research and precision medicine in clinical practice, while highlighting technical challenges and potential directions for future development.
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CRISPR-Edited Immune Effectors: The End of the Beginning.
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Efficient genome editing of Brassica campestris based on the CRISPR/Cas9 system.
Conventional methods for gene function study in Brassica campestris have lots of drawbacks, which greatly hinder the identification of important genes' functions and molecular breeding. The clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9) system is a versatile tool for genome editing that has been widely utilized in many plant species and has many advantages over conventional methods for gene function study. However, the application of CRISPR/Cas9 system in B. campestris remains unreported. The pectin-methylesterase genes Bra003491, Bra007665, and Bra014410 were selected as the targets of the CRISPR/Cas9 system. A single-targeting vector and a multitargeting vector were constructed. Different types of mutations were detected in T0 generation through Agrobacterium transformation. The mutation rate of the three designed sgRNA seeds varied from 20 to 56%. Although the majority of T0 mutants were chimeric, four homozygous mutants were identified. Transformation with the multitargeting vector generated one line with a large fragment deletion and one line with mutations in two target genes. Mutations in Bra003491 were stable and inherited by T1 and T2 generations. Nine mutants which did not contain T-DNA insertions were also obtained. No mutations were detected in predicted potential off-target sites. Our work demonstrated that CRISPR/Cas9 system is efficient on single and multiplex genome editing without off-targeting in B. campestris and that the mutations are stable and inheritable. Our results may greatly facilitate gene functional studies and the molecular breeding of B. campestris and other plants.
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First Trial of CRISPR-Edited T cells in Lung Cancer.
The clinical application of CRISPR-Cas9 gene editing has been eagerly awaited since the first description of the technique in 2013. Lu and colleagues now describe the treatment of 12 patients with nonsmall-cell lung cancer (NSCLC) with PD-1 gene-edited bulk autologous T cells, with results supporting both the feasibility and safety of gene editing in cell therapy.
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SNP-CRISPR: A Web Tool for SNP-Specific Genome Editing.
CRISPR-Cas9 is a powerful genome editing technology in which a single guide RNA (sgRNA) confers target site specificity to achieve Cas9-mediated genome editing. Numerous sgRNA design tools have been developed based on reference genomes for humans and model organisms. However, existing resources are not optimal as genetic mutations or single nucleotide polymorphisms (SNPs) within the targeting region affect the efficiency of CRISPR-based approaches by interfering with guide-target complementarity. To facilitate identification of sgRNAs (1) in non-reference genomes, (2) across varying genetic backgrounds, or (3) for specific targeting of SNP-containing alleles, for example, disease relevant mutations, we developed a web tool, SNP-CRISPR (https://www.flyrnai.org/tools/snp_crispr/). SNP-CRISPR can be used to design sgRNAs based on public variant data sets or user-identified variants. In addition, the tool computes efficiency and specificity scores for sgRNA designs targeting both the variant and the reference. Moreover, SNP-CRISPR provides the option to upload multiple SNPs and target single or multiple nearby base changes simultaneously with a single sgRNA design. Given these capabilities, SNP-CRISPR has a wide range of potential research applications in model systems and for design of sgRNAs for disease-associated variant correction.
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