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Type I-D CRISPR System-Mediated Genome Editing in Plants.
Genome editing has revolutionized plant research and plant breeding by enabling precise genome manipulation. In particular, the application of type II CRISPR-Cas9 systems to genome editing has proved an important milestone, accelerating genetic engineering and the analysis of gene function. On the other hand, the potential of other types of CRISPR-Cas systems, especially many of the most abundant type I CRISPR-Cas systems, remains unexplored. We recently developed a novel genome editing tool, TiD, based on the type I-D CRISPR-Cas system. In this chapter, we describe a protocol for genome editing of plant cells using TiD. This protocol allows the application of TiD to induce short insertion and deletions (indels) or long-range deletions at target sites with high specificity in tomato cells.
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From Genome Sequencing to CRISPR-Based Genome Editing for Climate-Resilient Forest Trees.
Due to the economic and ecological importance of forest trees, modern breeding and genetic manipulation of forest trees have become increasingly prevalent. The CRISPR-based technology provides a versatile, powerful, and widely accepted tool for analyzing gene function and precise genetic modification in virtually any species but remains largely unexplored in forest species. Rapidly accumulating genetic and genomic resources for forest trees enabled the identification of numerous genes and biological processes that are associated with important traits such as wood quality, drought, or pest resistance, facilitating the selection of suitable gene editing targets. Here, we introduce and discuss the latest progress, opportunities, and challenges of genome sequencing and editing for improving forest sustainability.
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A Nanoimprinted Photothermal Chip for On-Demand Spatiotemporal Activation of CRISPR/Cas9 Gene Editing.
Precise control of gene editing in target cells is essential for CRISPR/Cas9 applications. Here, we present a nanoimprinted photothermal chip (NPC) engineered for on-demand delivery and activation of CRISPR/Cas9 complexes with high spatial and temporal precision. Fabricated by nanoimprint lithography and subsequent surface modification, NPC features a customized PEGylated plasmonic gold nanopillar array, which provides both optimal cellular adhesion and efficient photothermal conversion. Upon NIR irradiation, NPC generates spatially confined thermal microdomains that transiently permeabilizes cell membranes, thereby facilitating cytosolic delivery of CRISPR/Cas9 complexes and synchronously modulating genome-editing kinetics. In vitro studies demonstrate robust gene knockout in both mouse and sheep cell lines while preserving high cell viability and editing fidelity. Remarkably, NPC-mediated PD-1 gene disruption in cytotoxic T cells markedly enhance their antitumor activity. Overall, this work establishes NPC as a transformative platform for precise and controllable CRISPR/Cas9 gene editing with broad therapeutic potential.
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SYNCAS based CRISPR-Cas9 gene editing in predatory mites, whiteflies and stinkbugs.
Despite the establishment of CRISPR-Cas9 gene editing protocols in a wide range of organisms, genetic engineering is still challenging for many organisms due to constraints including lethality of embryo injection, difficulties in egg/embryo collection or viviparous lifestyles. Recently, an efficient CRISPR-Cas9 method, termed SYNCAS, was developed to genetically modify spider mites and thrips species. The method is based on maternal injection of formulated CRISPR-Cas9 using saponin and BAPC. Here, we investigate whether the method can be used to perform gene editing in other arthropods such as the beneficial predatory mites Amblyseius swirskii and Phytoseiulus persimilis, and the pests Bemisia tabaci and Nezara viridula. For the predatory mites, Antp and SLC25A38 were used as target genes, while the ortholog of the Drosophila melanogaster ABCG transporter white was targeted in B. tabaci and N. viridula. All species were successfully edited with the highest efficiencies (up to 39%) being obtained for B. tabaci. For A. swirskii and P. persimilis no clear phenotypes could be observed, even though SLC25A38 was successfully knocked-out. The lack of a color phenotype in SLC25A38 mutants was confirmed in the spider mite Tetranychus urticae. Disruption of the target gene Antp is likely lethal in predatory mites, as no true null mutants could be recovered. For B. tabaci, KO of white resulted in orange eyes which diverges from the phenotype seen in white mutants of D. melanogaster. In the last species, N. viridula, a single phenotypic mutant could be detected having a patchy white body coloration with wild type eye coloration. Genotyping revealed a single amino acid deletion at the target site, suggesting the creation of a hypomorphic allele. To conclude, the protocols provided in this work can contribute to the genetic study of predatory mites used in biological control, as well as hemipteran pests.
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The State-of-the-Art of Gene Editing and its Application to Viral Infections and Diseases Including COVID-19.
Gene therapy delivers a promising hope to cure many diseases and defects. The discovery of gene-editing technology fueled the world with valuable tools that have been employed in various domains of science, medicine, and biotechnology. Multiple means of gene editing have been established, including CRISPR/Cas, ZFNs, and TALENs. These strategies are believed to help understand the biological mechanisms of disease progression. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been designated the causative virus for coronavirus disease 2019 (COVID-19) that emerged at the end of 2019. This viral infection is a highly pathogenic and transmissible disease that caused a public health pandemic. As gene editing tools have shown great success in multiple scientific and medical areas, they could eventually contribute to discovering novel therapeutic and diagnostic strategies to battle the COVID-19 pandemic disease. This review aims to briefly highlight the history and some of the recent advancements of gene editing technologies. After that, we will describe various biological features of the CRISPR-Cas9 system and its diverse implications in treating different infectious diseases, both viral and non-viral. Finally, we will present current and future advancements in combating COVID-19 with a potential contribution of the CRISPR system as an antiviral modality in this battle.
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Society for Maternal-Fetal Medicine Special Statement: Beyond the scalpel: in utero fetal gene therapy and curative medicine.
With the recent advances in gene editing with systems such as CRISPR-Cas9, precise genome editing in utero is on the horizon. Sickle cell disease is an excellent candidate for in utero fetal gene therapy, because the disease is monogenic, causes irreversible harm, and has life-limiting morbidity. Gene therapy has recently been proven to be effective in an adolescent patient. Several hurdles still impede the progress for fetal gene therapy in humans, including an incomplete understanding of the fetal immune system, unclear maternal immune responses to in utero gene therapy, risks of off-target effects from gene editing, gestational age constraints, and ethical questions surrounding fetal genetic intervention. However, none of these barriers appears insurmountable, and the journey to in utero gene therapy for sickle cell disease and other conditions should be well underway.
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Survival and Evolution of CRISPR-Cas System in Prokaryotes and Its Applications.
Prokaryotes have developed numerous innate immune mechanisms in order to fend off bacteriophage or plasmid attack. One of these immune systems is clustered regularly interspaced short palindromic repeats (CRISPR). CRISPR-associated proteins play a key role in survival of prokaryotes against invaders, as these systems cleave DNA of foreign genetic elements. Beyond providing immunity, these systems have significant impact in altering the bacterial physiology in term of its virulence and pathogenicity, as well as evolution. Also, due to their diverse nature of functionality, cas9 endoribonuclease can be easily reprogrammed with the help of guide RNAs, showing unprecedented potential and significance for gene editing in treating genetic diseases. Here, we also discuss the use of NgAgo-gDNA system in genome editing of human cells.
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Targeted Gene Editing in Human Pluripotent Stem Cells Using Site-Specific Nucleases.
Introduction of induced pluripotent stem cell (iPSC) technology and site-directed nucleases brought a major breakthrough in the development of regenerative therapies and biomedical research. With the advancement of ZFNs, TALENs, and the CRISPR/Cas9 technology, straightforward and precise manipulation of the genome of human pluripotent stem cells (PSC) became possible, allowing relatively easy and fast generation of gene knockouts, integration of transgenes, or even introduction of single nucleotide changes for correction or introduction of disease-specific mutations. We review current applications of site-specific nucleases in human PSCs and focus on trends and challenges for efficient gene editing and improvement of targeting strategies. Graphical Abstract.
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Rogues and Regulation of Germline Editing.
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Reprogramming site-specific retrotransposon activity to new DNA sites.
Retroelements have a critical role in shaping eukaryotic genomes. For instance, site-specific non-long terminal repeat retrotransposons have spread widely through preferential integration into repetitive genomic sequences, such as microsatellite regions and ribosomal DNA genes1-6. Despite the widespread occurrence of these systems, their targeting constraints remain unclear. Here we use a computational pipeline to discover multiple new site-specific retrotransposon families, profile members both biochemically and in mammalian cells, find previously undescribed insertion preferences and chart potential evolutionary paths for retrotransposon retargeting. We identify R2Tg, an R2 retrotransposon from the zebra finch, Taeniopygia guttata, as an orthologue that can be retargeted by payload engineering for target cleavage, reverse transcription and scarless insertion of heterologous payloads at new genomic sites. We enhance this activity by fusing R2Tg to CRISPR-Cas9 nickases for efficient insertion at new genomic sites. Through further screening of R2 orthologues, we select an orthologue, R2Tocc, with natural reprogrammability and minimal insertion at its natural 28S site, to engineer SpCas9H840A-R2Tocc, a system we name site-specific target-primed insertion through targeted CRISPR homing of retroelements (STITCHR). STITCHR enables the scarless, efficient installation of edits, ranging from a single base to 12.7 kilobases, gene replacement and use of in vitro transcribed or synthetic RNA templates. Inspired by the prevalence of nLTR retrotransposons across eukaryotic genomes, we anticipate that STITCHR will serve as a platform for scarless programmable integration in dividing and non-dividing cells, with both research and therapeutic applications.
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