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Synthetic minichromosomes in plants: past, present, and promise.
The status of engineered mini-chromosomes/artificial chromosomes/synthetic chromosomes in plants is summarized. Their promise is that they provide a means to accumulate foreign genes on an independent entity other than the normal chromosomes, which would facilitate stacking of novel traits in a way that would not be linked to endogenous genes and that would facilitate transfer between lines. Centromeres in plants are epigenetic, and therefore the isolation of DNA underlying centromeres and reintroduction into plant cells will not establish a functional kinetochore, which obviates this approach for in vitro assembly of plant artificial chromosomes. This issue was bypassed by using telomere-mediated chromosomal truncation to produce mini-chromosomes with little more than an endogenous centromere that could in turn be used as a foundation to build synthetic chromosomes. Site-specific recombinases and various iterations of CRISPR-Cas9 editing provide many tools for the development and re-engineering of synthetic chromosomes.
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Multiplexed CRISPR technologies for gene editing and transcriptional regulation.
Multiplexed CRISPR technologies, in which numerous gRNAs or Cas enzymes are expressed at once, have facilitated powerful biological engineering applications, vastly enhancing the scope and efficiencies of genetic editing and transcriptional regulation. In this review, we discuss multiplexed CRISPR technologies and describe methods for the assembly, expression and processing of synthetic guide RNA arrays in vivo. Applications that benefit from multiplexed CRISPR technologies, including cellular recorders, genetic circuits, biosensors, combinatorial genetic perturbations, large-scale genome engineering and the rewiring of metabolic pathways, are highlighted. We also offer a glimpse of emerging challenges and emphasize experimental considerations for future studies.
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Efficient genome editing by CRISPR/Cas9 with a tRNA-sgRNA fusion in the methylotrophic yeast Ogataea polymorpha.
The methylotrophic yeast Ogataea polymorpha (syn. Hansenula polymorpha) is an attractive industrial non-conventional yeast showing high thermo-tolerance (up to 50°C) and xylose assimilation. However, genetic manipulation of O. polymorpha is often laborious and time-consuming because it has lower homologous recombination efficiency relative to Saccharomyces cerevisiae. To overcome this disadvantage, we applied the CRISPR/Cas9 system as a powerful genome editing tool in O. polymorpha. In this system, both single guide RNA (sgRNA) and endonuclease Cas9 were expressed by a single autonomously-replicable plasmid and the sgRNA portion could be easily changed by using PCR and In-Fusion cloning techniques. Because the mutation efficiency of the CRISPR/Cas9 system was relatively low when the sgRNA was expressed under the control of the OpSNR6 promoter, the tRNACUG gene was used for sgRNA expression. The editing efficiency of this system ranged from 17% to 71% of transformants in several target genes tested (ADE12, PHO1, PHO11, and PHO84). These findings indicate that genetic manipulation of O. polymorpha will be more convenient and accelerated by using this CRISPR/Cas9 system.
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Cas9-induced immune response: A potential caution for human genome editing.
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CRISPR/Cas9 Genome Engineering in Human Pluripotent Stem Cells for Modeling of Neurological Disorders.
Recent advances in genome editing have brought new hopes for personalized and precision medicine but have also dramatically facilitated disease modeling studies. Combined with reprogramming approaches, stem cells and differentiation toward neural lineages, genome engineering holds great potential for regenerative approaches and to model neurological disorders. The use of patient-specific induced pluripotent stem cells combined with neural differentiation allows studying the effect of specific mutations in different brain cells. New genome editing tools such as CRISPR/Cas9 represent a step further by facilitating the introduction or correction of specific mutations within the same cell line, thus eliminating variability due to differences in the genetic background. Here, we present a step-by-step protocol from design to generation of human pluripotent stem cell lines with specific mutations introduced or corrected with CRISPR/Cas9 gene editing that can be used in combination with transcription factor-based protocols to dissect underlying mechanisms of neurological disorders.
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Highly efficient genome editing in N. gerenzanensis using an inducible CRISPR/Cas9-RecA system.
OBJECTIVE: To develop an inducible CRISPR/Cas9-Recombinase A (RecA) system to manipulate genes in Nonomuraea gerenzanensis effectively. RESULTS: Compared with traditional homologous recombination, the inducible CRISPR/Cas9 system achieved 68.8% editing efficiency, whereas, with both the inducible Cas9 and the overexpressed RecA, the efficiency of the combined genome editing system reached 100%. The dbv23-deleted mutant obtained by the inducible CRISPR/Cas9-RecA system was confirmed to produce more A40926 with an approximate yield of 200 mg L-1 than that of around 150 mg L-1 produced by the wild-type strain. CONCLUSIONS: This inducible CRISPR/Cas9-RecA system was successfully constructed and can be utilized as an efficient genome editing tool for Actinomyces able to shorten editing time simultaneously.
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Genome Editing and Cardiac Regeneration.
Although the field of cardiac regeneration is relatively young, it is progressing rapidly with technological advancements. Genome editing tools are allowing researchers to creatively influence signaling pathways to be able to shed light on them and are important for addressing certain issues and limitations associated with in vitro and in vivo aspects of cardiac regeneration, such as imaging and immune rejection. In this chapter, the pathways involved in cardiac regeneration will be highlighted, and the role of gene-editing tools in endogenous and exogenous approaches to regenerate injured myocardium is discussed.
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CRISPR/Cas9-based application for cancer therapy: Challenges and solutions for non-viral delivery.
CRISPR/Cas9 genome editing is a promising therapeutic technique, which makes precise and rapid gene editing technology possible on account of its high sensitivity and efficiency. CRISPR/Cas9 system has been proved to able to effectively disrupt and modify genes, which shows great potential for cancer treatment. Current researches proves that virus vectors are capable of effectively delivering the CRISPR/Cas9 system, but immunogenicity and carcinogenicity caused by virus transmission still trigger serious consequences. Therefore, the greatest challenge of CRISPR/Cas9 for cancer therapy lies on how to deliver it to the target tumor site safely and effectively. Non-viral delivery systems with specific targeting, high loading capacity, and low immune toxicity are more suitable than viral vectors, which limited by uncontrollable side effects. Their medical advances and applications have been widely concerned. Herein, we present the molecule mechanism and different construction strategies of CRISPR/Cas9 system for editing genes at the beginning of this research. Subsequently, several common CRISPR/Cas9 non-viral deliveries for cancer treatment are introduced. Lastly, based on the main factors limiting the delivery efficiency of non-viral vectors proposed in the existing researches and literature, we summarize and discuss the main methods to solve these limitations in the existing tumor treatment system, aiming to introduce further optimization and innovation of the CRISPR/Cas9 non-viral delivery system suitable for cancer treatment.
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Single-step generation of homozygous knockout/knock-in individuals in an extremotolerant parthenogenetic tardigrade using DIPA-CRISPR.
Tardigrades are small aquatic invertebrates known for their remarkable tolerance to diverse extreme stresses. To elucidate the in vivo mechanisms underlying this extraordinary resilience, methods for genetically manipulating tardigrades have long been desired. Despite our prior success in somatic cell gene editing by microinjecting Cas9 ribonucleoproteins (RNPs) into the body cavity of tardigrades, the generation of gene-edited individuals remained elusive. In this study, employing an extremotolerant parthenogenetic tardigrade species, Ramazzottius varieornatus, we established conditions that led to the generation of gene-edited tardigrade individuals. Drawing inspiration from the direct parental CRISPR (DIPA-CRISPR) technique employed in several insects, we simply injected a concentrated Cas9 RNP solution into the body cavity of parental females shortly before their initial oviposition. This approach yielded gene-edited G0 progeny. Notably, only a single allele was predominantly detected at the target locus for each G0 individual, indicative of homozygous mutations. By co-injecting single-stranded oligodeoxynucleotides (ssODNs) with Cas9 RNPs, we achieved the generation of homozygously knocked-in G0 progeny, and these edited alleles were inherited by G1/G2 progeny. This is the first example of heritable gene editing in the entire phylum of Tardigrada. This establishment of a straightforward method for generating homozygous knockout/knock-in individuals not only facilitates in vivo analyses of the molecular mechanisms underpinning extreme tolerance, but also opens up avenues for exploring various topics, including Evo-Devo, in tardigrades.
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At the Heart of Genome Editing and Cardiovascular Diseases.
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