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CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum.
Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical regions, is caused by infection with Plasmodium parasites. The development of more effective drugs to combat malaria can be accelerated by improving our understanding of the biology of this complex parasite. Genetic manipulation of these parasites is key to understanding their biology; however, historically the genome of P. falciparum has been difficult to manipulate. Recently, CRISPR/Cas9 genome editing has been utilized in malaria parasites, allowing for easier protein tagging, generation of conditional protein knockdowns, and deletion of genes. CRISPR/Cas9 genome editing has proven to be a powerful tool for advancing the field of malaria research. Here, we describe a CRISPR/Cas9 method for generating glmS-based conditional knockdown mutants in P. falciparum. This method is highly adaptable to other types of genetic manipulations, including protein tagging and gene knockouts.
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Prime Editing: A Novel Cas9-Reverse Transcriptase Fusion May Revolutionize Genome Editing.
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Online Databases of Genome Editing in Cardiovascular and Metabolic Diseases.
Metabolic and cardiovascular diseases are world-concerning pathologies that affect an important percentage of the population. Nowadays, advances in the genetic background of these diseases allow new approaches to models and therapies, as well as different gene edition trials. Furthermore, technological improvements in gene editing go along with the development of new online and biocomputational tools that provide us alternative ways to explore pathologies. In this chapter, historical gene editing methods are discussed but focusing on CRISPR-Cas system in detail and also online resources available to perform these types of experiments. Here, the different strategies for gene editing and their online tools are gathered, putting the light on its application in the study and treatment of cardiovascular and metabolic diseases.
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Rapid Generation of Human Genetic Loss-of-Function iPSC Lines by Simultaneous Reprogramming and Gene Editing.
Specifically ablating genes in human induced pluripotent stem cells (iPSCs) allows for studies of gene function as well as disease mechanisms in disorders caused by loss-of-function (LOF) mutations. While techniques exist for engineering such lines, we have developed and rigorously validated a method of simultaneous iPSC reprogramming while generating CRISPR/Cas9-dependent insertions/deletions (indels). This approach allows for the efficient and rapid formation of genetic LOF human disease cell models with isogenic controls. The rate of mutagenized lines was strikingly consistent across experiments targeting four different human epileptic encephalopathy genes and a metabolic enzyme-encoding gene, and was more efficient and consistent than using CRISPR gene editing of established iPSC lines. The ability of our streamlined method to reproducibly generate heterozygous and homozygous LOF iPSC lines with passage-matched isogenic controls in a single step provides for the rapid development of LOF disease models with ideal control lines, even in the absence of patient tissue.
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In situ gene editing of hematopoietic stem cells via AAV-delivered CRISPR guide RNAs.
Hematopoietic stem cells (HSCs) are self-renewing, multipotent, and engraftable precursors of all blood cells. Efficient delivery of therapeutic gene products and gene editing machinery to correct disease-causing gene variants in endogenous HSCs while they remain in the body holds exciting potential to leverage HSC potency for the treatment of monogenic blood disorders. Toward this goal, we used adeno-associated virus (AAV) to deliver CRISPR guide RNAs (gRNAs) to edit HSC genomes in situ in Ai9;SpCas9-EGFP transgenic mice carrying a Cas9-activatable Lox-STOP-Lox-tdTomato reporter cassette together with a constitutive SpCas9-2A-EGFP. Using a variety of conditions and vector designs, we tested whether systemic administration to these mice of AAVs carrying SpCas9-compatible gRNAs designed to cut DNA upstream and downstream of the STOP cassette would induce tdTomato expression in HSCs. Our findings identify self-complementary AAVs (scAAVs) and increased ratio of guide to Cas9 as parameters facilitating higher editing efficiency. Of note, we find preserved multilineage output and engraftability of HSCs upon scAAV-gRNA editing. In an example application of this technology, we explore the potential for in situ HSC gene editing by dual AAV-CRISPR delivery and demonstrate robust gene modification, concurrent with induction of therapeutic fetal hemoglobin, in a sickle cell disease mouse model modified to express SpCas9. In summary, this work offers a sensitive and adaptable platform that allows robust modification of HSC genomes in situ.
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Editing nature: Local roots of global governance.
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Genome Editing Mediated by Primordial Germ Cell in Chicken.
Genome editing technology has facilitated the studies on exploring specific gene functions in diverse living organisms. The technology has also contributed to creating high-value livestock in industry fields in terms of enhancing productivity or acquiring disease resistance. Particularly, applying genome editing technologies in avian species has been emphasized in both academic and industrial fields due to their unique developmental patterns as well as application possibilities. To accomplish genome editing in avian species, gene integration into chicken primordial germ cell (PGC) genome using a virus or transposition systems has been widely used, and recently developed programmable genome editing technologies including clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas9) systems enable to edit the genetic information precisely for maximizing the application potentials of avian species. In these regards, this chapter will cover the methods for producing genome-edited chickens, particularly by CRISPR/Cas9 technologies allowing targeted gene insertion, gene knockout, and gene tagging.
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Protocol for editing fibroblasts with in vitro transcribed Cas9 mRNA and profile off-target editing by optimized GUIDE-seq.
CRISPR-Cas9 gene editing is an efficient technique to modify specific sites/regions of DNA. Delivery of the Cas9 by mRNA is particularly promising in pre-clinical genome editing applications for its transient, nonintegrating feature. However, the off-target of Cas9-gRNA still remains a concern and needs a specific monitor. Here, we present a revised protocol to edit fibroblasts by in vitro transcribed Cas9 mRNA and profile its off-target effect by the optimized GUIDE-seq method. This protocol can also be applied to other cell lines. For complete details on the use and execution of this protocol, please refer to Ganna Reint et al. (2021).1.
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Biomimetic gene editing system for precise tumor cell reprogramming and augmented tumor therapy.
The abnormal level of hypoxia-inducible factor-1 alpha (HIF-1α) is closely related to cancer metastasis and treatment resistance. CRISPR-Cas9-based gene editing technology has sparked profound hope to solve this issue by precise gene disruption, although the in vivo application remains hindered by the lack of a safe and efficient delivery strategy. Herein, we developed a cell membrane biomimetic core-shell system for light-controllable, precise gene editing. The inner core of the system comprises protamine for CRISPR-Cas9/sgRNA plasmid (pCas9) loading and calcium ions for efficient pCas9 transfection. The shell of the system is camouflaged by a cell membrane and modified with AS1411 aptamers for tumor targeting and photosensitizers to induce lysosomal escape and pCas9 release through reactive oxygen species production, thereby producing light-controllable enhanced gene editing. Neoplastic H1299 cells were reprogrammed using the biomimetic gene editing system upon laser irradiation with reduced VEGF and Vimentin expression, leading to enhanced antimetastatic effects. Genetic disruption of HIF-1α augmented the in vivo chemotherapeutic efficacy of paclitaxel. Our approach of using a membrane-camouflaged system combined with light augmentation provides a potential solution for the in vivo delivery of CRISPR-Cas9 as well as a feasible strategy for cancer therapy.
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[In vivo gene editing for gene therapy].
In vivo gene editing has been achieved in a phase I clinical trial and results in a strong reduction of the level of a pathogenic protein. While preliminary, these results open the way for many applications in gene therapy.
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