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The Impact of Chromatin Dynamics on Cas9-Mediated Genome Editing in Human Cells.
In order to efficiently edit eukaryotic genomes, it is critical to test the impact of chromatin dynamics on CRISPR/Cas9 function and develop strategies to adapt the system to eukaryotic contexts. So far, research has extensively characterized the relationship between the CRISPR endonuclease Cas9 and the composition of the RNA-DNA duplex that mediates the system's precision. Evidence suggests that chromatin modifications and DNA packaging can block eukaryotic genome editing by custom-built DNA endonucleases like Cas9; however, the underlying mechanism of Cas9 inhibition is unclear. Here, we demonstrate that closed, gene-silencing-associated chromatin is a mechanism for the interference of Cas9-mediated DNA editing. Our assays use a transgenic cell line with a drug-inducible switch to control chromatin states (open and closed) at a single genomic locus. We show that closed chromatin inhibits binding and editing at specific target sites and that artificial reversal of the silenced state restores editing efficiency. These results provide new insights to improve Cas9-mediated editing in human and other mammalian cells.
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Using CRISPR/ttLbCas12a for in planta Gene Targeting in A. thaliana.
CRISPR/Cas systems enable gene editing through the induction of site-specific DNA double-strand breaks (DSB). However, the nature of the induced modification highly depends on the mechanism used for DNA DSB repair. Non-homologous end joining (NHEJ)-mediated targeted mutagenesis induced by CRISPR/Cas is an already standardly applied tool, which can lead to various different kinds of mutations at a specific genomic site. Nevertheless, precise genome modification using homologous donor sequences is still challenging in plants. Applications depending on the less frequent homologous recombination (HR) require further improvements to create an attractive and efficient tool for general application in plants. Focusing on this issue, we developed the in planta gene targeting (ipGT) system, which is based on the simultaneous excision of a stably integrated, homologous donor sequence and the induction of a DSB within the target site. In recent years, several improvements were achieved enhancing gene targeting (GT) frequencies. After the successful application of Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9) for ipGT, we were able to further improve the system using Lachnospiraceae bacterium Cas12a (LbCas12a), which also enables cleavage in T-rich regions. Most recently, we tested an improved, temperature-tolerant version of LbCas12a (ttLbCas12a) for ipGT and were able to further increase GT efficiencies. Here, we describe the experimental procedure of the recently published ipGT system using ttLbCas12a in Arabidopsis thaliana in detail. © 2020 The Authors. Basic Protocol 1: Construction of CRISPR/ttLbCas12a expression vector to analyze ipGT efficiencies Basic Protocol 2: Achieving heritable GT plants.
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Genetic and Epigenetic Manipulation Using CRISPR Techniques in Ewing Sarcoma.
CRISPR-based technologies have transformed experimental biology by enabling precise, efficient, and programmable manipulation of the genome and epigenome. This chapter provides a comprehensive and practical guide to genetic and epigenetic editing using CRISPR technology, with a particular focus on applications in Ewing sarcoma cell models. We describe strategies for CRISPR-mediated gene knockout (CRISPR KO) via nonhomologous end joining (NHEJ), precise gene knock-in (CRISPR KI) through homology-directed repair (HDR), and reversible transcriptional modulation using CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa). We emphasize the use of ribonucleoprotein (RNP) delivery of Cas9 and single guide RNAs, which offers improved editing efficiency, reduced off-target activity, and enhanced safety compared to plasmid-based approaches. Detailed protocols are provided for guide RNA design, RNP assembly, electroporation-based delivery, single-cell cloning, and molecular validation of edited clones. In addition, we outline best practices for HDR template design, discuss strategies to improve KI efficiency, and address common challenges such as polyploidy, variable editing rates, and DNA-mediated cytotoxicity. For CRISPRi and CRISPRa applications, we describe plasmid-based systems employing catalytically inactive Cas9 fused to transcriptional repressors or activators, enabling potentially tunable and reversible control of endogenous gene expression. Throughout the chapter, practical advice, troubleshooting tips, and critical considerations are included to facilitate successful implementation across diverse experimental settings. Collectively, this chapter serves as a detailed methodological resource for researchers aiming to employ CRISPR-based engineering for functional studies.
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Electroporation-based CRISPR/Cas9 Gene Editing in Haliotis Discus Hannai.
Abalone, a marine mollusk with significant economic and ecological value, plays a crucial role in sustainable aquaculture. The development and application of CRISPR-Cas9 gene-editing technology have opened up a new path for improving breeding efficiency. CRISPR/Cas9-mediated gene editing has been achieved in abalones via microinjection. In this study, a gene encoding myostatin MSTN in H. discus hannai; was selected as target for conducting the CRISPR-Cas9 gene editing experiment in combination with an electroporation delivery system. Our results showed that all three sgRNAs effectively targeted and cleaved the target segment, with sgRNA1 and sgRNA2 exhibiting high in vitro activity. After electroporation, the effects of transfection on embryonic development of fertilized eggs were observed and statistically analyzed. 12.7 ± 5.4% of the fertilized eggs were damaged and deformed after electroporation. Twenty-four hours after electroporation, surviving larvae were collected for DNA extraction and sequencing. Two potential mutations within the target region of MSTN were identified by sequencing. These results provide a reference for the improvement and development of CRISPR-mediated gene editing methods in marine mollusks such as abalones.
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Generation of gene edited birds in one generation using sperm transfection assisted gene editing (STAGE).
Generating transgenic and gene edited mammals involves in vitro manipulation of oocytes or single cell embryos. Due to the comparative inaccessibility of avian oocytes and single cell embryos, novel protocols have been developed to produce transgenic and gene edited birds. While these protocols are relatively efficient, they involve two generation intervals before reaching complete somatic and germline expressing transgenic or gene edited birds. Most of this work has been done with chickens, and many protocols require in vitro culturing of primordial germ cells (PGCs). However, for many other bird species no methodology for long term culture of PGCs exists. Developing methodologies to produce germline transgenic or gene edited birds in the first generation would save significant amounts of time and resource. Furthermore, developing protocols that can be readily adapted to a wide variety of avian species would open up new research opportunities. Here we report a method using sperm as a delivery mechanism for gene editing vectors which we call sperm transfection assisted gene editing (STAGE). We have successfully used this method to generate GFP knockout embryos and chickens, as well as generate embryos with mutations in the doublesex and mab-3 related transcription factor 1 (DMRT1) gene using the CRISPR/Cas9 system. The efficiency of the method varies from as low as 0% to as high as 26% with multiple factors such as CRISPR guide efficiency and mRNA stability likely impacting the outcome. This straightforward methodology could simplify gene editing in many bird species including those for which no methodology currently exists.
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Genome Editing with CRISPR-Cas9 in Lactobacillus plantarum Revealed That Editing Outcomes Can Vary Across Strains and Between Methods.
Lactic-acid bacteria such as Lactobacillus plantarum are commonly used for fermenting foods and as probiotics, where increasingly sophisticated genome-editing tools are employed to elucidate and enhance these microbes' beneficial properties. The most advanced tools to date utilize an oligonucleotide or double-stranded DNA donor for recombineering and Cas9 for targeted DNA cleavage. As the associated methods are often developed in isolation for one strain, it remains unclear how different Cas9-based editing methods compare across strains. Here, this work directly compares two methods in different strains of L. plantarum: one utilizing a plasmid-encoded recombineering template and another utilizing an oligonucleotide donor and an inducible DNA recombinase. This comparison reveals one instance in which only the recombineering-template method generates desired edits and another instance in which only the oligo method generates desired edits. It is further found that both methods exhibit highly variable success editing the same site across multiple L. plantarum strains. Finally, failure modes are identified for the recombineering-template method, including a consistent genomic deletion and reversion of a point mutation in the recombineering template. This study therefore highlights surprising differences for Cas9-mediated genome editing between methods and related strains, arguing for the need for multiple, distinct methods when performing CRISPR-based editing in bacteria.
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Continual improvement of CRISPR-induced multiplex mutagenesis in Arabidopsis.
CRISPR/Cas9 is currently the most powerful tool to generate mutations in plant genomes and more efficient tools are needed as the scale of experiments increases. In the model plant Arabidopsis, the choice of the promoter driving Cas9 expression is critical to generate germline mutations. Several optimal promoters have been reported. However, it is unclear which promoter is ideal as they have not been thoroughly tested side by side. Furthermore, most plant vectors still use one of the two Cas9 nuclear localization sequence (NLS) configurations initially reported. We genotyped more than 6000 Arabidopsis T2 plants to test seven promoters and six types of NLSs across 14 targets to systematically improve the generation of single and multiplex inheritable mutations. We found that the RPS5A promoter and bipartite NLS were individually the most efficient components. When combined, 99% of T2 plants contained at least one knockout (KO) mutation and 84% contained 4- to 7-plex KOs, the highest multiplexing KO rate in Arabidopsis to date. These optimizations will be useful to generate higher-order KOs in the germline of Arabidopsis and will likely be applicable to other CRISPR systems as well.
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CRISPR-Cas9-Mediated Gene Silencing in Cultured Human Epithelia.
CRISPR/Cas9 technology enables rapid and efficient genome editing in a variety of experimental systems. Genome editing using CRISPR/Cas9 has become an increasingly popular genetic engineering tool due to (1) an extensive array of commercial ready-to-use CRIPSR/Cas9 systems, (2) improved efficiency of cell delivery, and (3) the possibility to do multigene editing. Here, we describe a method to introduce single gene disruption in lung bronchial epithelial cells. This approach can be used to study host factors important for pathogen interaction or to identify and study genetic markers determining susceptibility to fungal disease.
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CRISPR-Cas9 Causes Chromosomal Instability and Rearrangements in Cancer Cell Lines, Detectable by Cytogenetic Methods.
CRISPR-Cas9 has quickly become the method of choice for genome editing, with multiple publications describing technical advances and novel applications. It has been widely adopted as a tool for basic research and has significant translational and clinical potential. However, its usage has outpaced the establishment of essential and rigorous controls for unwanted off-target effects, manifested as small mutations, large deletions of target loci, or large-scale chromosomal rearrangements. A common application of CRISPR-Cas9 is as a tool for creating isogenic cell-line models to study the effects of precise mutations, or variants, on disease traits. Here, we describe the effect of standard CRISPR-Cas9 mutagenesis protocols on well characterized cancer cell lines. We demonstrate that commonly used methods for detecting correctly mutated clones fail to uncover large-scale rearrangements. We show that simple cytogenetic methods can be used to identify clones carrying chromosomal abnormalities and large mutations at target loci. These methods are quick and cost-efficient, and we suggest that such controls should be performed prior to publication of studies based on novel CRISPR-Cas9 mutated cancer cell lines.
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CRISPR-mediated megabase-scale transgene de-duplication to generate a functional single-copy full-length humanized DMD mouse model.
BACKGROUND: The development of sequence-specific precision treatments like CRISPR gene editing therapies for Duchenne muscular dystrophy (DMD) requires sequence humanized animal models to enable the direct clinical translation of tested strategies. The current available integrated transgenic mouse model containing the full-length human DMD gene, Tg(DMD)72Thoen/J (hDMDTg), has been found to have two copies of the transgene per locus in a tail-to-tail orientation, which does not accurately simulate the true (single) copy number of the DMD gene. This duplication also complicates analysis when testing CRISPR therapy editing outcomes, as large genetic alterations and rearrangements can occur between the cut sites on the two transgenes. RESULTS: To address this, we performed long read nanopore sequencing on hDMDTg mice to better understand the structure of the duplicated transgenes. Following that, we performed a megabase-scale deletion of one of the transgenes by CRISPR zygotic microinjection to generate a single-copy, full-length, humanized DMD transgenic mouse model (hDMDTgSc). Functional, molecular, and histological characterisation shows that the single remaining human transgene retains its function and rescues the dystrophic phenotype caused by endogenous murine Dmd knockout. CONCLUSIONS: Our unique hDMDTgSc mouse model simulates the true copy number of the DMD gene, and can potentially be used for the further generation of DMD disease models that would be better suited for the pre-clinical assessment and development of sequence specific CRISPR therapies.
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