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Biochemistry Dec 2023CRISPR systems mediate adaptive immunity in bacteria and archaea through diverse effector mechanisms and have been repurposed for versatile applications in therapeutics... (Review)
Review
CRISPR systems mediate adaptive immunity in bacteria and archaea through diverse effector mechanisms and have been repurposed for versatile applications in therapeutics and diagnostics thanks to their facile reprogramming with RNA guides. RNA-guided CRISPR-Cas targeting and interference are mediated by effectors that are either components of multisubunit complexes in class 1 systems or multidomain single-effector proteins in class 2. The compact class 2 CRISPR systems have been broadly adopted for multiple applications, especially genome editing, leading to a transformation of the molecular biology and biotechnology toolkit. The diversity of class 2 effector enzymes, initially limited to the Cas9 nuclease, was substantially expanded via computational genome and metagenome mining to include numerous variants of Cas12 and Cas13, providing substrates for the development of versatile, orthogonal molecular tools. Characterization of these diverse CRISPR effectors uncovered many new features, including distinct protospacer adjacent motifs (PAMs) that expand the targeting space, improved editing specificity, RNA rather than DNA targeting, smaller crRNAs, staggered and blunt end cuts, miniature enzymes, promiscuous RNA and DNA cleavage, etc. These unique properties enabled multiple applications, such as harnessing the promiscuous RNase activity of the type VI effector, Cas13, for supersensitive nucleic acid detection. class 1 CRISPR systems have been adopted for genome editing, as well, despite the challenge of expressing and delivering the multiprotein class 1 effectors. The rich diversity of CRISPR enzymes led to rapid maturation of the genome editing toolbox, with capabilities such as gene knockout, base editing, prime editing, gene insertion, DNA imaging, epigenetic modulation, transcriptional modulation, and RNA editing. Combined with rational design and engineering of the effector proteins and associated RNAs, the natural diversity of CRISPR and related bacterial RNA-guided systems provides a vast resource for expanding the repertoire of tools for molecular biology and biotechnology.
Topics: CRISPR-Cas Systems; Gene Editing; Bacteria; RNA, Bacterial; DNA
PubMed: 37192099
DOI: 10.1021/acs.biochem.3c00159 -
Molecular Therapy : the Journal of the... Nov 2021
Topics: Animals; CRISPR-Cas Systems; Clinical Trials as Topic; Gene Editing; Genetic Therapy; Humans; Research; Translational Research, Biomedical
PubMed: 34699779
DOI: 10.1016/j.ymthe.2021.10.016 -
Plant Communications Mar 2021The recent discovery of the mode of action of the CRISPR/Cas9 system has provided biologists with a useful tool for generating site-specific mutations in genes of...
The recent discovery of the mode of action of the CRISPR/Cas9 system has provided biologists with a useful tool for generating site-specific mutations in genes of interest. In plants, site-targeted mutations are usually obtained by the stable transformation of a Cas9 expression construct into the plant genome. The efficiency of introducing mutations in genes of interest can vary considerably depending on the specific features of the constructs, including the source and nature of the promoters and terminators used for the expression of the Cas9 gene and the guide RNA, and the sequence of the Cas9 nuclease itself. To optimize the efficiency of the Cas9 nuclease in generating mutations in target genes in , we investigated several features of its nucleotide and/or amino acid sequence, including the codon usage, the number of nuclear localization signals (NLSs), and the presence or absence of introns. We found that the Cas9 gene codon usage had some effect on its activity and that two NLSs worked better than one. However, the highest efficiency of the constructs was achieved by the addition of 13 introns into the Cas9 coding sequence, which dramatically improved the editing efficiency of the constructs. None of the primary transformants obtained with a Cas9 gene lacking introns displayed a knockout mutant phenotype, whereas between 70% and 100% of the primary transformants generated with the intronized Cas9 gene displayed mutant phenotypes. The intronized Cas9 gene was also found to be effective in other plants such as and .
Topics: Arabidopsis; Arabidopsis Proteins; CRISPR-Associated Protein 9; CRISPR-Cas Systems; Gene Editing; Genome, Plant; Introns
PubMed: 33898975
DOI: 10.1016/j.xplc.2020.100135 -
Biotechnology Letters Jun 2016To identify the best lipid nanoparticles for delivery of purified Cas9 protein and gRNA complexes (Cas9 RNPs) into mammalian cells and to establish the optimal...
OBJECTIVES
To identify the best lipid nanoparticles for delivery of purified Cas9 protein and gRNA complexes (Cas9 RNPs) into mammalian cells and to establish the optimal conditions for transfection.
RESULTS
Using a systematic approach, we screened 60 transfection reagents using six commonly-used mammalian cell lines and identified a novel transfection reagent (named Lipofectamine CRISPRMAX). Based on statistical analysis, the genome modification efficiencies in Lipofectamine CRISPRMAX-transfected cell lines were 40 or 15 % higher than those in Lipofectamine 3000 or RNAiMAX-transfected cell lines, respectively. Upon optimization of transfection conditions, we observed 85, 75 or 55 % genome editing efficiencies in HEK293FT cells, mouse ES cells, or human iPSCs, respectively. Furthermore, we were able to co-deliver donor DNA with Cas9 RNPs into a disrupted EmGFP stable cell line, resulting in the generation of up to 17 % EmGFP-positive cells.
CONCLUSION
Lipofectamine CRISPRMAX was characterized as the best lipid nanoparticles for the delivery of Cas9 RNPs into a variety of mammalian cell lines, including mouse ES cells and iPSCs.
Topics: Animals; CRISPR-Cas Systems; Cell Line; Electroporation; Gene Editing; Gene Targeting; Green Fluorescent Proteins; Humans; Induced Pluripotent Stem Cells; Lipids; Mice; Transfection
PubMed: 26892225
DOI: 10.1007/s10529-016-2064-9 -
Nature Reviews. Genetics Jan 2022Gene drives are selfish genetic elements that are transmitted to progeny at super-Mendelian (>50%) frequencies. Recently developed CRISPR-Cas9-based gene-drive systems... (Review)
Review
Gene drives are selfish genetic elements that are transmitted to progeny at super-Mendelian (>50%) frequencies. Recently developed CRISPR-Cas9-based gene-drive systems are highly efficient in laboratory settings, offering the potential to reduce the prevalence of vector-borne diseases, crop pests and non-native invasive species. However, concerns have been raised regarding the potential unintended impacts of gene-drive systems. This Review summarizes the phenomenal progress in this field, focusing on optimal design features for full-drive elements (drives with linked Cas9 and guide RNA components) that either suppress target mosquito populations or modify them to prevent pathogen transmission, allelic drives for updating genetic elements, mitigating strategies including trans-complementing split-drives and genetic neutralizing elements, and the adaptation of drive technology to other organisms. These scientific advances, combined with ethical and social considerations, will facilitate the transparent and responsible advancement of these technologies towards field implementation.
Topics: Alleles; Animals; CRISPR-Cas Systems; Gene Drive Technology; Gene Editing; Genetics, Population; Humans; Models, Genetic; Mutation; RNA, Guide, CRISPR-Cas Systems
PubMed: 34363067
DOI: 10.1038/s41576-021-00386-0 -
Molecular Therapy : the Journal of the... Jan 2022Most gene editing technologies introduce breaks or nicks into DNA, leading to the generation of mutagenic insertions and deletions by non-homologous end-joining repair....
Most gene editing technologies introduce breaks or nicks into DNA, leading to the generation of mutagenic insertions and deletions by non-homologous end-joining repair. Here, we report a new, cleavage-free gene editing approach based on replication interrupted template-driven DNA modification (RITDM). The RITDM system makes use of sequence-specific DLR fusion molecules that are specifically designed to enable localized, temporary blockage of DNA replication fork progression, thereby exposing single-stranded DNA that can be bound by DNA sequence modification templates for precise editing. We evaluate the use of zinc-finger arrays for sequence recognition. We demonstrate that RITDM can be used for gene editing at endogenous genomic loci in human cells and highlight its safety profile of low indel frequencies and undetectable off-target side effects in RITDM-edited clones and pools of cells.
Topics: CRISPR-Cas Systems; DNA Breaks, Double-Stranded; DNA End-Joining Repair; Gene Editing; Genome, Human; Humans
PubMed: 34864205
DOI: 10.1016/j.ymthe.2021.12.001 -
Biomolecules Sep 2021As a vertebrate model, zebrafish () plays a vital role in the field of life sciences. Recently, gene-editing technology has become increasingly innovative, significantly... (Review)
Review
As a vertebrate model, zebrafish () plays a vital role in the field of life sciences. Recently, gene-editing technology has become increasingly innovative, significantly promoting scientific research on zebrafish. However, the implementation of these methods in a reasonable and accurate manner to achieve efficient gene-editing remains challenging. In this review, we systematically summarize the development and latest progress in zebrafish gene-editing technology. Specifically, we outline trends in double-strand break-free genome modification and the prospective applications of fixed-point orientation transformation of any base at any location through a multi-method approach.
Topics: Animals; Animals, Genetically Modified; DNA Breaks, Double-Stranded; Gene Editing; Gene Targeting; Templates, Genetic; Zebrafish
PubMed: 34572513
DOI: 10.3390/biom11091300 -
Nature Biotechnology May 2020Cytosine base editors (CBEs) enable targeted C•G-to-T•A conversions in genomic DNA. Recent studies report that BE3, the original CBE, induces a low frequency of...
Cytosine base editors (CBEs) enable targeted C•G-to-T•A conversions in genomic DNA. Recent studies report that BE3, the original CBE, induces a low frequency of genome-wide Cas9-independent off-target C•G-to-T•A mutation in mouse embryos and in rice. Here we develop multiple rapid, cost-effective methods to screen the propensity of different CBEs to induce Cas9-independent deamination in Escherichia coli and in human cells. We use these assays to identify CBEs with reduced Cas9-independent deamination and validate via whole-genome sequencing that YE1, a narrowed-window CBE variant, displays background levels of Cas9-independent off-target editing. We engineered YE1 variants that retain the substrate-targeting scope of high-activity CBEs while maintaining minimal Cas9-independent off-target editing. The suite of CBEs characterized and engineered in this study collectively offer ~10-100-fold lower average Cas9-independent off-target DNA editing while maintaining robust on-target editing at most positions targetable by canonical CBEs, and thus are especially promising for applications in which off-target editing must be minimized.
Topics: CRISPR-Associated Protein 9; Cytosine; Escherichia coli; Gene Editing; HEK293 Cells; Humans; Mutation; Whole Genome Sequencing
PubMed: 32042165
DOI: 10.1038/s41587-020-0414-6 -
Human Gene Therapy Mar 2021Precise gene manipulation by gene editing approaches facilitates the potential to cure several debilitating genetic disorders. Gene modification stimulated by engineered... (Review)
Review
Precise gene manipulation by gene editing approaches facilitates the potential to cure several debilitating genetic disorders. Gene modification stimulated by engineered nucleases induces a double-stranded break (DSB) in the target genomic locus, thereby activating DNA repair mechanisms. DSBs triggered by nucleases are repaired either by the nonhomologous end-joining or the homology-directed repair pathway, enabling efficient gene editing. While there are several ongoing genome editing clinical trials, current research underscores the therapeutic potential of CRISPR/Cas-based (clustered regularly interspaced short palindrome repeats-associated Cas nuclease) gene editing. In this review, we provide an overview of the CRISPR/Cas-mediated genome therapy applications and explore their prospective clinical translatability to treat human monogenic disorders. In addition, we discuss the various challenges associated with genome editing technologies and strategies used to circumvent them. Despite the robust and precise nuclease-mediated gene editing, a promoterless, nuclease-independent gene targeting strategy has been utilized to evade the drawbacks of the nuclease-dependent system, such as off-target effects, immunogenicity, and cytotoxicity. Thus, the rapidly evolving paradigm of gene editing technologies will continue to foster the progress of gene therapy applications.
Topics: CRISPR-Cas Systems; Endonucleases; Gene Editing; Gene Targeting; Humans; Prospective Studies
PubMed: 33750221
DOI: 10.1089/hum.2021.013 -
Heart Failure Clinics Apr 2018With an increasing understanding of genetic defects leading to cardiomyopathy, focus is shifting to correcting these underlying genetic defects. One approach involves... (Review)
Review
With an increasing understanding of genetic defects leading to cardiomyopathy, focus is shifting to correcting these underlying genetic defects. One approach involves treating mutant RNA through antisense oligonucleotides; the first drug has received regulatory approval to treat specific mutations associated with Duchenne muscular dystrophy. Gene editing is being evaluated in the preclinical setting. For inherited cardiomyopathies, genetic correction strategies require tight specificity for the mutant allele. Gene-editing methods are being tested to create deletions that may be useful to restore protein expression by through the bypass of mutations that restore protein production. Site-specific gene editing, which is required to correct many point mutations, is a less efficient process than inducing deletions.
Topics: Animals; Cardiomyopathies; Gene Editing; Genetic Therapy; Humans
PubMed: 29525646
DOI: 10.1016/j.hfc.2017.12.006