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Guide RNA Design for Genome-Wide CRISPR Screens in Yarrowia lipolytica.
Genome-wide functional genomic screens are essential to determining the genetic underpinning of a biological process. Novel and powerful tools for perturbing gene function, with the help of genetic and epigenetic information, have made it possible to systematically investigate the contribution of every gene to evolved and engineered phenotypes. Functional genomics and screening for enhanced phenotypes become ever more important when dealing with nonconventional hosts. Non-model organisms are valuable to metabolic engineering as they present a range of desirable phenotypes and can help in avoiding complex and intensive engineering of less suitable hosts that do not possess the desired phenotype(s). Domestication of such hosts however requires a suite of synthetic biology tools that allow for targeted genome engineering, regulation of gene expression, and genome-wide mutational screens. The widespread adoption of CRISPR-Cas9 and CRISPR-Cpf1 based systems has allowed for such screens in many organisms. Key considerations in any genome-wide CRISPR screen are the design of a set of unique guide RNAs targeting the required set of genes in the genome and the design of nontargeting guide RNAs that function as appropriate negative controls for the experiment. In this methods chapter, we present protocols for the design of guides for a CRISPR screen, targeting every gene in the genome of the industrially relevant oleaginous yeast Yarrowia lipolytica. The first set of protocols describes the algorithm for the design of genome targeting and nontargeting guides for a genome-wide CRISPR-Cpf1 screen. The second set of protocols describes modifications to the first for the design of guides for a CRISPR-Cas9 screen. The strategies described here should serve as an efficient guide to design a library of gRNAs for most genome-wide CRISPR screens.
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Warrior Spirit: An Interview with Victoria Gray, Sickle Cell Pioneer.
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CRISPR therapies march into clinic, but genotoxicity concerns linger.
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CRISPR/Cas advancements for genome editing, diagnosis, therapeutics, and vaccine development for Plasmodium parasites, and genetic engineering of Anopheles mosquito vector.
Malaria as vector-borne disease remains important health concern with over 200 million cases globally. Novel antimalarial medicines and more effective vaccines must be developed to eliminate and eradicate malaria. Appraisal of preceding genome editing approaches confirmed the CRISPR/Cas nuclease system as a novel proficient genome editing system and a tool for species-specific diagnosis, and drug resistance researches for Plasmodium species, and gene drive to control Anopheles population. CRISPR/Cas technology, as a handy tool for genome editing can be justified for the production of transgenic malaria parasites like Plasmodium transgenic lines expressing Cas9, chimeric Plasmodium transgenic lines, knockdown and knockout transgenic parasites, and transgenic parasites expressing alternative alleles, and also mutant strains of Anopheles such as only male mosquito populations, generation of wingless mosquitoes, and creation of knock-out/ knock-in mutants. Though, the incorporation of traditional methods and novel molecular techniques could noticeably enhance the quality of results. The striking development of a CRISPR/Cas-based diagnostic kit that can specifically diagnose the Plasmodium species or drug resistance markers is highly required in malaria settings with affordable cost and high-speed detection. Furthermore, the advancement of genome modifications by CRISPR/Cas technologies resolves contemporary restrictions to culturing, maintaining, and analyzing these parasites, and the aptitude to investigate parasite genome functions opens up new vistas in the better understanding of pathogenesis.
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CRISPR/Cas9-Mediated Homology-Directed Genome Editing in Pichia pastoris.
State-of-the-art strain engineering techniques for the methylotrophic yeast Pichia pastoris (syn. Komagataella spp.) include overexpression of endogenous and heterologous genes and deletion of host genes. For efficient gene deletion, methods such as the split-marker technique have been established. However, synthetic biology trends move toward building up large and complex reaction networks, which often require endogenous gene knockouts and simultaneous overexpression of individual genes or whole pathways. Realization of such engineering tasks by conventional approaches employing subsequent steps of transformations and marker recycling is very time- and labor-consuming. Other applications require tagging of certain genes/proteins or promoter exchange approaches, which are hard to design and construct with conventional methods. Therefore, efficient systems are required that allow precise manipulations of the P. pastoris genome, including simultaneous overexpression of multiple genes. To meet this challenge, we have developed a CRISPR/Cas9-based kit for gene insertions, deletions, and replacements, which paves the way for precise genomic modifications in P. pastoris. In this chapter, the versatile method for performing these modifications without the integration of a selection marker is described. A ready-to-use plasmid kit for performing CRISPR/Cas9-mediated genome editing in P. pastoris based on the GoldenPiCS modular cloning vectors is available at Addgene as CRISPi kit (#1000000136).
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Perspectives of Gene Editing for the Conservation of Plant Genetic Resources.
The rapid development of genome editing tools, particularly CRISPR-Cas9 and virus-induced genome editing (VIGE), offers transformative opportunities for the conservation of plant genetic resources. These technologies enable precise modifications that can enhance genetic variation, restore endangered populations, and strengthen resilience against climate change and invasive species. This chapter reviews methodological frameworks, including guide design, delivery systems, regeneration protocols, and verification of off-target effects, as well as ecological and ethical considerations. Case studies are highlighted in both wild and cultivated species: American chestnut, ash, elm, Pacific yew, hemlock, and whitebark pine, alongside crops of global significance such as coffee, cacao, wild apple, and banana. Applications include resistance to blight, borers, Dutch elm disease, woolly adelgid, rust, rots, and endogenous viral elements. Finally, the chapter outlines future directions emphasizing biosafety, ecological validation, and equitable governance, underscoring the potential of CRISPR-based strategies to integrate conservation science with sustainable agricultural practices.
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Efficient CRISPR-based genome editing for inducible degron systems to enable temporal control of protein function in large double-stranded DNA virus genomes.
CRISPR-Cas9-based gene editing enables precise genetic modifications. However, its application to human cytomegalovirus (HCMV) remains challenging due to the large size of the viral genome and the essential roles of key regulatory genes. Here, we establish an optimized CRISPR-Cas9 system for precise labeling and functional analysis of HCMV immediate early (IE) genes. By integrating a multifunctional cassette encoding an auxin-inducible degron (AID), a self-cleaving peptide (P2A), and GFP into the viral genome via homology-directed repair (HDR), we achieved efficient knock-ins without reliance on bacterial artificial chromosome (BAC) cloning, a labor-intensive and time-consuming approach. We optimized delivery strategies, donor template designs, and component ratios to enhance HDR efficiency, significantly improving knock-in success rates. This system enables real-time fluorescent tracking and inducible protein degradation, allowing temporal control of essential viral proteins through auxin-mediated depletion. Our approach provides a powerful tool for dissecting the dynamic roles of viral proteins throughout the HCMV life cycle, facilitating a deeper understanding of viral pathogenesis and potential therapeutic targets.
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Gene Modulation with CRISPR-based Tools in Human iPSC-Cardiomyocytes.
Precise control of gene expression (knock-out, knock-in, knockdown or overexpression) is at the heart of functional genomics - an approach to dissect the contribution of a gene/protein to the system's function. The development of a human in vitro system that can be patient-specific, induced pluripotent stem cells, iPSC, and the ability to obtain various cell types of interest, have empowered human disease modeling and therapeutic development. Scalable tools have been deployed for gene modulation in these cells and derivatives, including pharmacological means, DNA-based RNA interference and standard RNA interference (shRNA/siRNA). The CRISPR/Cas9 gene editing system, borrowed from bacteria and adopted for use in mammalian cells a decade ago, offers cell-specific genetic targeting and versatility. Outside genome editing, more subtle, time-resolved gene modulation is possible by using a catalytically "dead" Cas9 enzyme linked to an effector of gene transcription in combination with a guide RNA. The CRISPRi / CRISPRa (interference/activation) system evolved over the last decade as a scalable technology for performing functional genomics with libraries of gRNAs. Here, we review key developments of these approaches and their deployment in cardiovascular research. We discuss specific use with iPSC-cardiomyocytes and the challenges in further translation of these techniques.
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Introduction of Genome Editing Reagents and Genotyping of Derived Edited Alleles in Soybean (Glycine max (L.) Merr.).
Cas9-based genome editing is a powerful genetic tool for loci specifically targeted for genome modification. This chapter describes up-to-date protocols using Cas9-based genome editing technology, including vector construction with GoldenBraid assembly, Agrobacterium-mediated soybean transformation, and identification of editing in the genome.
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Improved Cas9 activity by specific modifications of the tracrRNA.
CRISPR/Cas is a transformative gene editing tool, that offers a simple and effective way to target a catalytic Cas9, the most widely used is derived from Streptococcus pyogenes (SpCas9), with a complementary small guide RNA (sgRNA) to inactivate endogenous genes resulting from insertions and deletions (indels). CRISPR/Cas9 has been rapidly applied to basic research as well as expanded for potential clinical applications. Utilization of spCas9 as an ribonuclearprotein complex (RNP) is considered the most safe and effective method to apply Cas9 technology, and the efficacy of this system is critically dependent on the ability of Cas9 to generate high levels of indels. We find here that novel sequence changes to the tracrRNA significantly improves Cas9 activity when delivered as an RNP. We demonstrate that a dual-guide RNA (dgRNA) with a modified tracrRNA can improve reporter knockdown and indel formation at several targets within the long terminal repeat (LTR) of HIV. Furthermore, the sequence-modified tracrRNAs improved Cas9-mediated reduction of CCR5 surface receptor expression in cell lines, which correlated with higher levels of indel formation. It was demonstrated that a Cas9 RNP with a sequence modified tracrRNA enhanced indel formation at the CCR5 target site in primary CD4+ T-cells. Finally, we show improved activity at two additional targets within the HBB locus and the BCL11A GATA site. Overall, the data presented here suggests that novel facile tracrRNA sequence changes could potentially be integrated with current dgRNA technology, and open up the possibility for the development of sequence modified tracrRNAs to improve Cas9 RNP activity.
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