Somatic genome editing with CRISPR/Cas9 generates and corrects a metabolic disease. Examining sources of error in PCR by single-molecule sequencing. Inference of CRISPR edits from Sanger trace data. Easy quantitative assessment of genome editing by sequence trace decomposition. Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly. Mutation detection using Surveyor nuclease. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR–Cas nucleases. Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases. CRISPR/Cas9 systems targeting beta-globin and CCR5 genes have substantial off-target activity. High-frequency off-target mutagenesis induced by CRISPR–Cas nucleases in human cells. DNA targeting specificity of RNA-guided Cas9 nucleases. Engineered materials for in vivo delivery of genome-editing machinery. AAV-CRISPR gene editing is negated by pre-existing immunity to Cas9. Prevalence of pre-existing antibodies to CRISPR-associated nuclease Cas9 in the USA population. Identification of preexisting adaptive immunity to Cas9 proteins in humans. High prevalence of Streptococcus pyogenes Cas9-reactive T cells within the adult human population.
Therapeutic genome editing: prospects and challenges. TALENs: a widely applicable technology for targeted genome editing. Genome engineering with zinc-finger nucleases. CRISPR–Cas systems for editing, regulating and targeting genomes. Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors. Base editing: precision chemistry on the genome and transcriptome of living cells. Search-and-replace genome editing without double-strand breaks or donor DNA. High-efficiency multiplex genome editing of streptomyces species using an engineered CRISPR/Cas system. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity. A TALE nuclease architecture for efficient genome editing. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Chimeric nucleases stimulate gene targeting in human cells. Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Future work to improve off-target analysis includes expanding the true off-target editing dataset to evaluate new experimental techniques and to train machine learning algorithms performing analysis using the particular genome of the cells in question rather than the reference genome and applying novel NGS techniques to improve the sensitivity of amplicon-based off-target editing quantification. Therefore, we recommend that at least one in silico tool and one experimental tool should be used together to identify potential off-target sites, and amplicon-based next-generation sequencing (NGS) should be used as the gold standard assay for assessing the true off-target effects at these candidate sites. However, no single tool is able to accurately predict low-frequency off-target editing, presenting a bottleneck in therapeutic genome editing, because even a small number of cells with off-target editing can be detrimental. Recent advances in both in silico and experimental tools for off-target analysis have generated remarkably concordant results for sites with high off-target editing activity. Here we review tools developed for identifying potential off-target editing sites and compare the ability of these tools to properly analyze off-target effects. Off-target editing by these nucleases remains a considerable concern, especially in therapeutic applications. Genome editing using programmable nucleases is revolutionizing life science and medicine.
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