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Conversion Tract Analysis of Homology-Directed Genome Editing Using Oligonucleotide Donors.
Homology-directed genome editing is the intentional alteration of an endogenous genetic locus using information from an exogenous homology donor. A conversion tract is defined as the amount of genetic information that is converted from the homology donor to a given strand of the targeted chromosomal locus. Because of this, conversion tract analysis retrospectively not only elucidates the mechanism of homology-directed genome editing but also provides valuable insights on the conversion efficiency of every nucleotide in the homology donor. Here we describe a blue fluorescent protein-to-green fluorescent protein conversion system that can be conveniently used to measure the efficiency and analyze the lengths of conversion tracts of homology-directed genome editing using oligonucleotide donors in mammalian cells.
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[Directing construction of CRISPR/Cas9 vector of SmPAL1 in Salvia miltiorrhiza by target efficiency detection in vitro].
To construct CRISPR/Cas9 vectors for the editing of SmPAL1 in the phenylpropane metabolic pathway of Salvia miltiorrhiza, CIRSPR/Cas9 target sites of SmPAL1 were designed by online software. Its target efficiencies were detected in vitro by enzyme digestion and sequences with highly efficiency were constructed into CRISPR/Cas9 vectors. Three possible CRISPR target sequences (SmPAL1-g1, SmPAL1-g2, SmPAL1-g3) were designed and the enzyme digestion efficiencies were 53.3%, 76.6% and 10.0%. SmPAL1-g1 and SmPAL1-g2 were constructed into vector VK005-03 named as VK005-03-g1 and VK005-03-g2. The results of sequencing showed that the two CRISPR/Cas target sequences were all constructed into VK005-03. Here we first laid the foundation for the study of SmPAL1 and provided an effective strategy for the screening of sgRNA.
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Editing the mushroom.
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Tissue-specific genome editing in Ciona embryos by CRISPR/Cas9.
The CRISPR/Cas9 system has ushered in a new era of targeted genetic manipulations. Here, we report the use of CRISPR/Cas9 to induce double-stranded breaks in the genome of the sea squirt Ciona intestinalis. We use electroporation to deliver CRISPR/Cas9 components for tissue-specific disruption of the Ebf (Collier/Olf/EBF) gene in hundreds of synchronized Ciona embryos. Phenotyping of transfected embryos in the 'F0' generation revealed that endogenous Ebf function is required for specification of Islet-expressing motor ganglion neurons and atrial siphon muscles. We demonstrate that CRISPR/Cas9 is sufficiently effective and specific to generate large numbers of embryos carrying mutations in a targeted gene of interest, which should allow for rapid screening of gene function in Ciona.
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Efficient genome editing in Caenorhabditis elegans by CRISPR-targeted homologous recombination.
Cas9 is an RNA-guided double-stranded DNA nuclease that participates in clustered regularly interspaced short palindromic repeats (CRISPR)-mediated adaptive immunity in prokaryotes. CRISPR-Cas9 has recently been used to generate insertion and deletion mutations in Caenorhabditis elegans, but not to create tailored changes (knock-ins). We show that the CRISPR-CRISPR-associated (Cas) system can be adapted for efficient and precise editing of the C. elegans genome. The targeted double-strand breaks generated by CRISPR are substrates for transgene-instructed gene conversion. This allows customized changes in the C. elegans genome by homologous recombination: sequences contained in the repair template (the transgene) are copied by gene conversion into the genome. The possibility to edit the C. elegans genome at selected locations will facilitate the systematic study of gene function in this widely used model organism.
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A streamlined CRISPR workflow to introduce mutations and generate isogenic iPSCs for modeling amyotrophic lateral sclerosis.
Amyotrophic lateral sclerosis (ALS) represents a complex neurodegenerative disorder with significant genetic heterogeneity. To date, both the genetic etiology and the underlying molecular mechanisms driving this disease remain poorly understood, although in recent years several studies have highlighted a number of genetic mutations causative for ALS. With these mutations pointing to potential pathways that may be affected within individuals with ALS, having the ability to generate human neurons and other disease relevant cells containing these mutations becomes even more critical if new therapies are to emerge. Recent developments with the advent of induced pluripotent stem cells (iPSCs) and clustered regularly interspaced short palindromic repeats (CRISPR) gene editing fields gave us the tools to introduce or correct a specific mutation at any site within the genome of an iPSC, and thus model the specific contribution of risk mutations. In this study we describe a rapid and efficient way to either introduce a mutation into a control line, or to correct an allele-specific mutation, generating an isogenic control line from patient-derived iPSCs with a given mutation. The mutations introduced were the G94A (also known as G93A) mutation into SOD1 or H517Q into FUS, and the mutation corrected was a patient iPSC line with I114T mutation in SOD1. A combination of small molecules and growth factors were used to guide a stepwise differentiation of the edited cells into motor neurons in order to demonstrate that disease-relevant cells could be generated for downstream applications. Through a combination of iPSCs and CRISPR editing, the cells generated here will provide fundamental insights into the molecular mechanisms underlying neuron degeneration in ALS.
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Hydrogen Peroxide-triggered Chemical Strategy for Controlling CRISPR systems.
The function of the CRISPR system can be conditionally controlled through rationally guided RNA engineering such that the target sequences can be precisely selected and manipulated. In particular, gRNA, as an important component of the CRISPR system, provides a unique tool for multifunctional control of the system based on the structure of the RNA itself. Therefore, we introduced here a protective group on the 2'-OH position of RNA to inhibit RNA-guided nucleic acid cleavage. Next, the modified gRNA can restore its original function under the chemical stimulation of hydrogen peroxide to realize the control of the CRISPR system. The experiment result demonstrated that the operating mechanism of this strategy may be based on chemical modifications that reduce the number of complementary base pairs between RNAs and targets, and the RNA-protein interaction. This further enriches the toolbox of conditional control of CRISPR function and has broad potential for gene editing in living cells and disease treatment using endogenous hydrogen peroxide.
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Targeted mutagenesis in cotton (Gossypium hirsutum L.) using the CRISPR/Cas9 system.
The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system has been widely used for genome editing in various plants because of its simplicity, high efficiency and design flexibility. However, to our knowledge, there is no report on the application of CRISPR/Cas9-mediated targeted mutagenesis in cotton. Here, we report the genome editing and targeted mutagenesis in upland cotton (Gossypium hirsutum L., hereafter cotton) using the CRISPR/Cas9 system. We designed two guide RNAs to target distinct sites of the cotton Cloroplastos alterados 1 (GhCLA1) and vacuolar H+-pyrophosphatase (GhVP) genes. Mutations in these two genes were detected in cotton protoplasts. Most of the mutations were nucleotide substitutions, with one nucleotide insertion and one substitution found in GhCLA1 and one deletion found in GhVP in cotton protoplasts. Subsequently, the two vectors were transformed into cotton shoot apexes through Agrobacterium-mediated transformation, resulting in efficient target gene editing. Most of the mutations were nucleotide deletions, and the mutation efficiencies were 47.6-81.8% in transgenic cotton plants. Evaluation using restriction-enzyme-PCR assay and sequence analysis detected no off-target mutations. Our results indicated that the CRISPR/Cas9 system was an efficient and specific tool for targeted mutagenesis of the cotton genome.
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Current and future delivery systems for engineered nucleases: ZFN, TALEN and RGEN.
Gene therapy by engineered nucleases is a genetic intervention being investigated for curing the hereditary disorders by targeting selected genes with specific nucleotides for establishment, suppression, abolishment of a function or correction of mutation. Here, we review the fast developing technology of targeted genome engineering using site specific programmable nucleases zinc finger nucleases (ZFNs), transcription activator like nucleases (TALENs) and cluster regulatory interspaced short palindromic repeat/CRISPR associated proteins (CRISPR/Cas) based RNA-guided DNA endonucleases (RGENs) and their different characteristics including pros and cons of genome modifications by these nucleases. We have further discussed different types of delivery methods to induce gene editing, novel development in genetic engineering other than nucleases and future prospects.
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CRISPR in Animals and Animal Models.
CRISPR-Cas9 has revolutionized the generation of transgenic animals. This system has demonstrated an unprecedented efficiency, multiplexability, and ease of use, thereby reducing the time and cost required for genome editing and enabling the production of animals with more extensive genetic modifications. It has also been shown to be applicable to a wide variety of animals, from early-branching metazoans to primates. Genome-wide screens in model organisms have been performed, accurate models of human diseases have been constructed, and potential therapies have been tested and validated in animal models. Several achievements in genetic modification of animals have been translated into products for the agricultural and pharmaceutical industries. Based on the remarkable progress to date, one may anticipate that in the future, CRISPR-Cas9 technology will enable additional far-reaching advances, including understanding the bases of diseases with complex genetic origins, engineering animals to produce organs for human transplantation, and genetically transforming entire populations of organisms to prevent the spread of disease.
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