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Flow cytometry-based quantification of genome editing efficiency in human cell lines using the L1CAM gene.
CRISPR/Cas9 is a powerful genome editing system that has remarkably facilitated gene knockout and targeted knock-in. To accelerate the practical use of CRISPR/Cas9, however, it remains crucial to improve the efficiency, precision, and specificity of genome editing, particularly targeted knock-in, achieved with this system. To improve genome editing efficiency, researchers should first have a molecular assay that allows sensitive monitoring of genome editing events with simple procedures. In the current study, we demonstrate that genome editing events occurring in L1CAM, an X-chromosome gene encoding a cell surface protein, can be readily monitored using flow cytometry (FCM) in multiple human cell lines including neuroblastoma cell lines. The abrogation of L1CAM was efficiently achieved using Cas9 nucleases which disrupt exons encoding the L1CAM extracellular domain, and was easily detected by FCM using anti-L1CAM antibodies. Notably, L1CAM-abrogated cells could be quantified by FCM in four days after transfection with a Cas9 nuclease, which is much faster than an established assay based on the PIGA gene. In addition, the L1CAM-based assay allowed us to measure the efficiency of targeted knock-in (correction of L1CAM mutations) accomplished through different strategies, including a Cas9 nuclease-mediated method, tandem paired nicking, and prime editing. Our L1CAM-based assay using FCM enables rapid and sensitive quantification of genome editing efficiencies and will thereby help researchers improve genome editing technologies.
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Generation of homozygous and heterozygous REEP1 knockout induced pluripotent stem cell lines by CRISPR/Cas9 gene editing.
REEP1 is a transmembrane protein in the endoplasmic reticulum (ER) membrane that is involved in shaping and remodeling of the ER. Mutations in REEP1 cause SPG31, an autosomal dominant form of hereditary spastic paraplegia (HSP). Here we show the generation of a homozygous and a heterozygous REEP1 knockout induced pluripotent stem cell line suitable for in vitro disease modelling using the CRISPR/Cas9 editing system.
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Reboot ethics governance in China.
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VUS no more: Decoding inborn errors of immunity for clinical action.
Saturation genome editing meets functional phenotyping to turn sequencing ambiguity into actionable diagnoses.
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CRISPR/Cas9-mediated gene editing in an exogenous transgene and an endogenous sex determination gene in the Caribbean fruit fly, Anastrepha suspensa.
CRISPR/Cas9-mediated gene-editing, using injected Cas9 protein, was achieved in the Caribbean fruit fly, Anastrepha suspensa, by initially targeting an exogenous transgene, polyubiquitin-regulated EGFP (PUb-EGFP), for heritable non-homologous end-joining (NHEJ) knock-outs using an individual sgRNA. Multiple deletion mutations, ranging from two to five nts proximal to the target site, were identified phenotypically by the loss of green fluorescence in transgenic flies that were also marked with PUb-DsRed. This represented a relatively high efficiency rate of 29% for germ-line mutations. Similar conditions were then used to target an endogenous sex-determination gene, As-transformer-2 (Astra-2), using two sgRNAs that targeted independent exon sequences 671 bp apart. Somatic mutations were identified phenotypically in G0 adult flies at a frequency of 81% based upon intersexual genital morphology, expected to occur only in XX females since Astra-2 knock-outs by dsRNA do not have a phenotypic effect in XY males. Consistent with this expectation, twelve types of short indels, ranging from -15 nts to +5 nts, were identified proximal to the 5' sgRNA-1 target site in intersexual adults. However, the 3' sgRNA-2 target was only associated with a single 774 bp deletion extending from the sgRNA-1 target site to 100 bp downstream of the sgRNA-2 target. This is encouraging for the eventual use of dual target sites for homology-directed repair (HDR) insertions, but suggests that the sgRNA-2 target site tested may not be optimal for Astra-2 HDR modification.
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Allele-Specific Epigenome Editing.
The discovery and adaptation of the CRISPR/Cas system for epigenome editing has allowed for a straightforward design of targeting modules which can direct epigenetic editors to virtually any genomic site. This advancement in DNA-targeting technology brings allele-specific epigenome editing into reach, a "super-specific" variation of epigenome editing whose goal is an alteration of chromatin marks at only one selected allele of the target genomic locus. This technology would be useful for the treatment of diseases caused by a mutant allele with a dominant effect, because allele-specific epigenome editing allows the specific silencing of the mutated allele leaving the healthy counterpart expressed. Moreover, it may allow the direct correction of aberrant imprints in imprinting disorders where editing of DNA methylation is needed in one allele only. Here, we describe some principal setups of allele-specific epigenome editing systems and present exemplary data illustrating the feasibility of the concept.
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Genetic engineering: CREATE-ing genome-wide designed mutations.
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An RNA-Directed Gene Editing Strategy for Attenuating the Infectious Potential of Feline Immunodeficiency Virus-Infected Cells: A Proof of Concept.
Modern antiretroviral therapy for immunodeficiency viruses, although remarkably effective in controlling viral transcription, and overt virus-associated morbidity, has failed to absolutely eradicate retroviruses from their infected hosts as a result of proviral integration in long-lived reservoir cells. Immunodeficiency virus-infected patients are therefore consigned to lifelong antiviral therapy as a means to control viremia, viral transmission, and infection-associated morbidity. Unfortunately, lifelong antiviral therapies can be difficult for patients to continuously maintain and may be associated with therapy-specific morbidities. Patient advocates have argued for new methods to achieve retroviral eradication. As a proof-of-concept study, a lentivirus-delivered RNA-directed gene editing strategy was utilized in a series of in vitro experiments in an attempt to attenuate the feline immunodeficiency virus (FIV) proviral load, viral transcription, and production of infectious virions. We found that a feline T lymphocyte cell line (MCH5-4) treated with an FIV-specific clustered regularly interspersed short palindromic repeats (CRISPR)-associated protein 9 (Cas9) gene editing tool resulted in a reduction of cell-free viral RNA relative to control cells. Decreased infectious potential was demonstrated in a two-step FIV infection study-naïve MCH5-4 cells infected with cell-free FIV harvested from FIV-infected and CRISPR lentivirus-treated cells had less integrated proviral DNA than control cells. This study represents the initial steps towards the development of an effective method of proviral eradication in an immunodeficiency virus-infected host.
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Advances in CRISPR-Cas technology and its applications: revolutionising precision medicine.
CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated proteins) has undergone marked advancements since its discovery as an adaptive immune system in bacteria and archaea, emerged as a potent gene-editing tool after the successful engineering of its synthetic guide RNA (sgRNA) toward the targeting of specific DNA sequences with high accuracy. Besides its DNA editing ability, further-developed Cas variants can also edit the epigenome, rendering the CRISPR-Cas system a versatile tool for genome and epigenome manipulation and a pioneering force in precision medicine. This review explores the latest advancements in CRISPR-Cas technology and its therapeutic and biomedical applications, highlighting its transformative impact on precision medicine. Moreover, the current status of CRISPR therapeutics in clinical trials is discussed. Finally, we address the persisting challenges and prospects of CRISPR-Cas technology.
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High-fidelity endonuclease variant HypaCas9 facilitates accurate allele-specific gene modification in mouse zygotes.
CRISPR/Cas9 has been widely used for the efficient generation of genetically modified animals; however, this system could have unexpected off-target effects. In the present study, we confirmed the validity of a high-fidelity Cas9 variant, HypaCas9, for accurate genome editing in mouse zygotes. HypaCas9 efficiently modified the target locus while minimizing off-target effects even in a single-nucleotide mismatched sequence. Furthermore, by applying HypaCas9 to the discrimination of SNP in hybrid strain-derived zygotes, we accomplished allele-specific gene modifications and successfully generated mice with a monoallelic mutation in an essential gene. These results suggest that the improved accuracy of HypaCas9 facilitates the generation of genetically modified animals.
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