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[Improving the Efficiency and Safety of Human CCR5 Gene Editing by Selection of Optimal Guide RNAs for SpCAS9 and CAS12A].
Advances in CRISPR/Cas-mediated genome editing have opened up treatment alternatives for many human diseases, including HIV infection. Knockout of the CCR5 gene as a potential way to treat HIV infection has long been studied. Here we analyzed guide RNAs for SpCas9 and AsCas12a nucleases targeting CCR5 gene which had been previously studied and selected the most effective among them. We also designed novel guide RNAs for the same nucleases using bioinformatics resources. We compared the efficiency of target site cleavage for all selected gRNAs using three nucleases: wt SpCas9, SpCas9-HF1-plus, and AsCas12a, as well as their off- target activities. We demonstrated that among the tested guide RNAs two for SpCas9- HF1-plus and three for AsCas12a exhibited high cleavage activity, cutting CCR5 gene in 60-72% of cells, and had off-target activities below the limit of detection. Thus, these guide RNAs may be candidates for future development of gene therapies against HIV infection.
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Advanced editing of the nuclear and plastid genomes in plants.
Genome editing is a powerful suite of technologies utilized in basic and applied plant research. Both nuclear and plastid genomes have been genetically engineered to alter traits in plants. While the most frequent molecular outcome of gene editing has been knockouts resulting in a simple deletion of an endogenous protein of interest from the host's proteome, new genes have been added to plant genomes and, in several instances, the sequence of endogenous genes have been targeted for a few coding changes. Targeted plant characteristics for genome editing range from single gene targets for agronomic input traits to metabolic pathways to endow novel plant function. In this paper, we review the fundamental approaches to editing nuclear and plastid genomes in plants with an emphasis on those utilizing synthetic biology. The differences between the eukaryotic-type nuclear genome and the prokaryotic-type plastid genome (plastome) in plants has profound consequences in the approaches employed to transform, edit, select transformants, and indeed, nearly all aspects of genetic engineering procedures. Thus, we will discuss the two genomes targeted for editing in plants, the toolbox used to make edits, along with strategies for future editing approaches to transform crop production and sustainability. While CRISPR/Cas9 is the current method of choice in editing nuclear genomes, the plastome is typically edited using homologous recombination approaches. A particularly promising synthetic biology approach is to replace the endogenous plastome with a 'synplastome' that is computationally designed, and synthesized and assembled in the lab, then installed into chloroplasts. The editing strategies, transformation methods, characteristics of the novel plant also affect how the genetically engineered plant may be governed and regulated. Each of these components and final products of gene editing affect the future of biotechnology and farming.
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Transposon-Associated CRISPR-Cas System: A Powerful DNA Insertion Tool.
The transposon-associated CRISPR-Cas system is emerging as a powerful tool for targeted genetic manipulation. Using this system, targeted genomic knockins and knockouts can be achieved, independently of the host-cell repair machinery, thereby significantly improving our ability to perform genome editing in a wider variety of cell types.
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CRISPR/Cas genome editing in plants: Dawn of Agrobacterium transformation for recalcitrant and transgene-free plants for future crop breeding.
Genome editing tools based on CRISPR/Cas system have been posed to solve many issues in agriculture and improve food production. Genetic engineering by Agrobacterium-mediated transformation has helped to impart specific traits straightaway in many crops. Many GM crops have also reached the field for commercial cultivation. Genetic engineering requires mostly a transformation protocol often mediated by Agrobacterium to insert a specific gene at a random locus. Genome editing with CRISPR/Cas system is a more precise technique for the targeted modification of genes/bases in the host plant genome. Unlike the conventional transformation system, wherein elimination of marker/foreign gene was possible only post-transformation, CRISPR/Cas system could generate transgene-free plants by delivering CRISPR/Cas reagents such as the Cas protein and guide RNAs gRNA(s) preassembled to form ribonucleoproteins (RNPs) into plant cells. CRISPR reagent delivery might be helpful to overcome issues with plants that are recalcitrant to Agrobacterium transformation and the legal hurdles due to the presence of the foreign gene. More recently, the grafting of wild-type shoots to transgenic donor rootstocks developed by the CRISPR/Cas system has reported transgene-free genome editing. CRISPR/Cas system also requires only a small piece of gRNA besides Cas9 or other effectors to target a specific region in the genome. So this system has been projected to be a key contributor to future crop breeding. In this article, we recap the main events of plant transformation, compare the difference between genetic transformation and CRISPR/Cas-mediated genome editing, and draw insights into the future application of the CRISPR/Cas system.
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Genome Editing in Mouse Zygotes and Embryonic Stem Cells by Introducing SgRNA/Cas9 Expressing Plasmids.
In mammalian cells, genome editing with the single guide RNA (sgRNA)/Cas9 complex allows for high targeting efficiency within a relatively short time frame with the added benefits of being low cost and easy to design. sgRNA/Cas9-mediated editing in mouse zygotes has accelerated the analysis of gene functions and the generation of mouse models of human diseases. Despite the benefits, this method still suffers from several problems, such as mosaicism in the founder generation which complicates genotyping and phenotypical analyses, and the low efficiency of more complicated genome editing. Thus, we recently established the system for genome editing in embryonic stem (ES) cells and its application for chimeric analysis in mice. In this section, we introduce the procedure for genome editing in mouse zygotes and ES cells.
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Speciation and adaptation research meets genome editing.
Understanding the genetic basis of reproductive isolation and adaptive traits in natural populations is one of the fundamental goals in evolutionary biology. Genome editing technologies based on CRISPR-Cas systems and site-specific recombinases have enabled us to modify a targeted genomic region as desired and thus to conduct functional analyses of target loci, genes and mutations even in non-conventional model organisms. Here, we review the technical properties of genome editing techniques by classifying them into the following applications: targeted gene knock-out for investigating causative gene functions, targeted gene knock-in of marker genes for visualizing expression patterns and protein functions, precise gene replacement for identifying causative alleles and mutations, and targeted chromosomal rearrangement for investigating the functional roles of chromosomal structural variations. We describe examples of their application to demonstrate functional analysis of naturally occurring genetic variations and discuss how these technologies can be applied to speciation and adaptation research. This article is part of the theme issue 'Genetic basis of adaptation and speciation: from loci to causative mutations'.
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Harnessing the natural diversity and in vitro evolution of Cas9 to expand the genome editing toolbox.
In the past few years, the Cas9 endonuclease from the type II CRISPR-Cas bacterial antiviral defense system has revolutionized the genome editing field. Guided by an RNA molecule, Cas9 can be reprogrammed to target almost any DNA sequence: the only limitation being the short nucleotide sequence in the vicinity of the target, termed the PAM, which is characteristic for each Cas9 protein. Streptococcus pyogenes Cas9 which recognizes the NGG PAM is currently most widely used for genome manipulation. However, Cas9 orthologues and engineered Cas9 variants offer expanded genome targeting capabilities, improved specificity and biochemical properties.
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Pioneering genome editing in parthenogenetic stick insects: CRISPR/Cas9-mediated gene knockout in Medauroidea extradentata.
The parthenogenetic life cycle of the stick insect Medauroidea extradentata offers unique advantages for the generation of genome-edited strains, as an isogenic and stable mutant line can in principle be achieved already in the first generation (G0). However, genetic tools for the manipulation of their genes had not been developed until now. Here, we successfully implement CRISPR/Cas9 as a technique to modify the genome of the stick insect M. extradentata. As proof-of-concept we targeted two genes involved in the ommochrome pathway of eye pigmentation (cinnabar and white, second and first exon, respectively), to generate knockout (KO) mutants. Microinjections were performed within 24 h after oviposition, to focus on the mononuclear (and haploid) stage of development. The KOs generated resulted in distinct eye and cuticle colour phenotypes for cinnabar and white. Homozygous cinnabar mutants showed pale pigmentation of eyes and cuticle. They develop into adults capable of producing viable eggs. Homozygous white KO resulted in a completely unpigmented phenotype in developing embryos that were unable to hatch. In conclusion, we show that CRISPR/Cas9 can be successfully applied to the genome of M. extradentata by creating phenotypically different and viable insects. This powerful gene editing technique can now be employed to create stable genetically modified lines using a parthenogenetic non-model organism.
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Protocol for CRISPR-Cas12a genome editing of protein tyrosine phosphatases in human pluripotent stem cells and functional β-like cell generation.
Gene editing of human pluripotent stem cells is a promising approach for developing targeted gene therapies for metabolic diseases. Here, we present a protocol for generating a CRISPR-Cas12a gene knockout of protein tyrosine phosphatases in human embryonic stem cells. We describe steps for differentiating the edited clones into pancreatic islet-like spheroids rich in β-like cells. We then detail procedures for implanting these spheroids under the murine kidney capsule for in vivo maturation.
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Testing the genome-editing toolkit in cardiomyopathy.
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