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Genetically Modified Babies and a First Application of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas9).
The world's first babies with CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats)-edited genes were born on November 25, 2018. Dr. Jiankui He of Southern University of Science and Technology in Shenzhen performed this gene editing. Dr. He's objectives and an assessment of how well they were achieved are discussed in the context of existing research in this area.
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High GC Content Cas9-Mediated Genome-Editing and Biosynthetic Gene Cluster Activation in Saccharopolyspora erythraea.
The overexpression of bacterial secondary metabolite biosynthetic enzymes is the basis for industrial overproducing strains. Genome editing tools can be used to further improve gene expression and yield. Saccharopolyspora erythraea produces erythromycin, which has extensive clinical applications. In this study, the CRISPR-Cas9 system was used to edit genes in the S. erythraea genome. A temperature-sensitive plasmid containing the PermE promoter, to drive Cas9 expression, and the Pj23119 and PkasO promoters, to drive sgRNAs, was designed. Erythromycin esterase, encoded by S. erythraea SACE_1765, inactivates erythromycin by hydrolyzing the macrolactone ring. Sequencing and qRT-PCR confirmed that reporter genes were successfully inserted into the SACE_1765 gene. Deletion of SACE_1765 in a high-producing strain resulted in a 12.7% increase in erythromycin levels. Subsequent PermE- egfp knock-in at the SACE_0712 locus resulted in an 80.3% increase in erythromycin production compared with that of wild type. Further investigation showed that PermE promoter knock-in activated the erythromycin biosynthetic gene clusters at the SACE_0712 locus. Additionally, deletion of indA (SACE_1229) using dual sgRNA targeting without markers increased the editing efficiency to 65%. In summary, we have successfully applied Cas9-based genome editing to a bacterial strain, S. erythraea, with a high GC content. This system has potential application for both genome-editing and biosynthetic gene cluster activation in Actinobacteria.
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The Self-Inactivating KamiCas9 System for the Editing of CNS Disease Genes.
Neurodegenerative disorders are a major public health problem because of the high frequency of these diseases. Genome editing with the CRISPR/Cas9 system is making it possible to modify the sequence of genes linked to these disorders. We designed the KamiCas9 self-inactivating editing system to achieve transient expression of the Cas9 protein and high editing efficiency. In the first application, the gene responsible for Huntington's disease (HD) was targeted in adult mouse neuronal and glial cells. Mutant huntingtin (HTT) was efficiently inactivated in mouse models of HD, leading to an improvement in key markers of the disease. Sequencing of potential off-targets with the constitutive Cas9 system in differentiated human iPSC revealed a very low incidence with only one site above background level. This off-target frequency was significantly reduced with the KamiCas9 system. These results demonstrate the potential of the self-inactivating CRISPR/Cas9 editing for applications in the context of neurodegenerative diseases.
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Efficient genomic correction methods in human iPS cells using CRISPR-Cas9 system.
Precise gene correction using the CRISPR-Cas9 system in human iPS cells holds great promise for various applications, such as the study of gene functions, disease modeling, and gene therapy. In this review article, we summarize methods for effective editing of genomic sequences of iPS cells based on our experiences correcting dystrophin gene mutations with the CRISPR-Cas9 system. Designing specific sgRNAs as well as having efficient transfection methods and proper detection assays to assess genomic cleavage activities are critical for successful genome editing in iPS cells. In addition, because iPS cells are fragile by nature when dissociated into single cells, a step-by-step confirmation during the cell recovery process is recommended to obtain an adequate number of genome-edited iPS cell clones. We hope that the techniques described here will be useful for researchers from diverse backgrounds who would like to perform genome editing in iPS cells.
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Development of TALE-adenine base editors in plants.
Base editors enable precise nucleotide changes at targeted genomic loci without requiring double-stranded DNA breaks or repair templates. TALE-adenine base editors (TALE-ABEs) are genome editing tools, composed of a DNA-binding domain from transcription activator-like effectors (TALEs), an engineered adenosine deaminase (TadA8e), and a cytosine deaminase domain (DddA), that allow A•T-to-G•C editing in human mitochondrial DNA. However, the editing ability of TALE-ABEs in plants apart from chloroplast DNA has not been described, so far, and the functional role how DddA enhances TadA8e is still unclear. We tested a series of TALE-ABEs with different deaminase fusion architectures in Nicotiana benthamiana and rice. The results indicate that the double-stranded DNA-specific cytosine deaminase DddA can boost the activities of single-stranded DNA-specific deaminases (TadA8e or APOBEC3A) on double-stranded DNA. We analysed A•T-to-G•C editing efficiencies in a β-glucuronidase reporter system and showed precise adenine editing in genomic regions with high product purity in rice protoplasts. Furthermore, we have successfully regenerated rice plants with A•T-to-G•C mutations in the chloroplast genome using TALE-ABE. Consequently, TALE-adenine base editors provide alternatives for crop improvement and gene therapy by editing nuclear or organellar genomes.
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Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors.
The development of new CRISPR-Cas genome editing tools continues to drive major advances in the life sciences. Four classes of CRISPR-Cas-derived genome editing agents-nucleases, base editors, transposases/recombinases and prime editors-are currently available for modifying genomes in experimental systems. Some of these agents have also moved rapidly into the clinic. Each tool comes with its own capabilities and limitations, and major efforts have broadened their editing capabilities, expanded their targeting scope and improved editing specificity. We analyze key considerations when choosing genome editing agents and identify opportunities for future improvements and applications in basic research and therapeutics.
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Application of CRISPR-Cas systems in neuroscience.
CRISPR-Cas systems have, over the years, emerged as indispensable tools for Genetic interrogation in contexts of clinical interventions, elucidation of genetic pathways and metabolic engineering and have pervaded almost every aspect of modern biology. Within this repertoire, the nervous system comes with its own set of perplexities and mysteries. Scientists have, over the years, tried to draw up a clearer genetic picture of the neuron and how it functions in a network, mainly in an endeavor to mitigate diseases of the human nervous system like Alzheimer's, Parkinson's, Huntington's, Autism Spectrum Disorder (ASD), etc. With most being progressive in nature, these diseases have plagued mankind for centuries. In spite of our immense progress in modern biology, we are yet to get a grasp over these diseases and unraveling their mechanisms is of utmost importance. Before CRISPR-Cas systems came along, the elucidation of the complex interactome of the mammalian nervous system was attempted with erstwhile existing electrophysiological, histological and pharmacological techniques coupled with Next Generation Sequencing and cell-specific targeting technologies. Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), imparted excellent sequence specific DNA targeting capabilities but came with their huge baggage of extensive protein engineering requirements, which practically rendered them unsuitable for high throughput exercises. With the discovery of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and CRISPR Associated proteins(CAS) systems by Ishino (1987)1, the era of extensive custom made endonuclease targeting was ushered in. For the first time in 2012, Jinek et al. (2012)2 repurposed the CRISPR-Cas mediated bacterial immune system for customizable mammalian gene editing. The CRISPR-Cas technology made it possible to easily customize Cas9 endonucleases to cleave near specifically targeted sequences, thereby facilitating knock-ins or knock-outs, silencing or activating or editing any gene, at any locus of the genome, both at the base-pair level or at the epigenetic level. With this enhanced degree of freedom, decrypting the nervous system and therapeutic interventions for neuropathies became significantly less cumbersome an exercise. Here we take a brisk walk through the several endeavors of research that show how the humble bacteria's CRISPR-Cas system gave us the "nerves" to "talk" to our nerves with ease.
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Prime editing-mediated correction of the leptin receptor in muscle cells of db/db mice.
Genetic diseases can be caused by monogenic diseases, which result from a single gene mutation in the DNA sequence. Many innovative approaches have been developed to cure monogenic genetic diseases, namely by genome editing. A specific type of genomic editing, prime editing, has the potential advantage to edit the human genome without requiring double-strand breaks or donor DNA templates for editing. Additionally, prime editing does not require a precisely positioned protospacer adjacent motif (PAM) sequence, which offers flexible target and more precise genomic editing. Here we detail a novel construction of a prime editing extended guide RNA (pegRNA) to target mutated leptin receptors in B6.BKS(D)-Leprdb/J mice (db/db mice). The pegRNA was then injected into the flexor digitorum brevis (FDB) muscle of db/db mice to demonstrate in vivo efficacy, which resulted in pegRNA mediated base transversion at endogenous base transversion. Genomic DNA sequencing confirmed that prime editing could correct the mutation of leptin receptor gene in db/db mice. Furthermore, prime editing treated skeletal muscle exhibited enhanced leptin receptor signals. Thus, the current study showed in vivo efficacy of prime editing to correct mutant protein and rescue the physiology associated with functional protein.
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Increased lateral root formation by CRISPR/Cas9-mediated editing of arginase genes in cotton.
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An SpG-Cas9-based cytosine base editor expands the scope of genome editing in carrot plants.
SpG Cas9 significantly expands the genome editing scope in carrot with NGN PAM recognition.
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