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      CRISPR-Pass: Gene Rescue of Nonsense Mutations Using Adenine Base Editors

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          Abstract

          A nonsense mutation is a substitutive mutation in a DNA sequence that causes a premature termination during translation and produces stalled proteins, resulting in dysfunction of a gene. Although it usually induces severe genetic disorders, there are no definite methods for inducing read through of premature termination codons (PTCs). Here, we present a targeted tool for bypassing PTCs, named CRISPR-pass, that uses CRISPR-mediated adenine base editors. CRISPR-pass, which should be applicable to 95.5% of clinically significant nonsense mutations in the ClinVar database, rescues protein synthesis in patient-derived fibroblasts, suggesting potential clinical utility.

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          Abstract

          Lee et al. showed that CRISPR-pass, based on adenine base editors, would be a targeted tool for bypassing premature termination codons. This system could be applicable to ∼95% of clinically significant nonsense mutations, related to genetic diseases, in the ClinVar database.

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          Most cited references 24

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          Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements

          CRISPR-Cas9 is poised to become the gene editing tool of choice in clinical contexts. Thus far, exploration of Cas9-induced genetic alterations has been limited to the immediate vicinity of the target site and distal off-target sequences, leading to the conclusion that CRISPR-Cas9 was reasonably specific. Here we report significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitors and a human differentiated cell line. Using long-read sequencing and long-range PCR genotyping, we show that DNA breaks introduced by single-guide RNA/Cas9 frequently resolved into deletions extending over many kilobases. Furthermore, lesions distal to the cut site and crossover events were identified. The observed genomic damage in mitotically active cells caused by CRISPR-Cas9 editing may have pathogenic consequences.
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            Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome.

            Isogenic settings are routine in model organisms, yet remain elusive for genetic experiments on human cells. We describe the use of designed zinc finger nucleases (ZFNs) for efficient transgenesis without drug selection into the PPP1R12C gene, a "safe harbor" locus known as AAVS1. ZFNs enable targeted transgenesis at a frequency of up to 15% following transient transfection of both transformed and primary human cells, including fibroblasts and hES cells. When added to this locus, transgenes such as expression cassettes for shRNAs, small-molecule-responsive cDNA expression cassettes, and reporter constructs, exhibit consistent expression and sustained function over 50 cell generations. By avoiding random integration and drug selection, this method allows bona fide isogenic settings for high-throughput functional genomics, proteomics, and regulatory DNA analysis in essentially any transformed human cell type and in primary cells.
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              Functional Anatomy of the Human Microprocessor.

              MicroRNA (miRNA) maturation is initiated by Microprocessor composed of RNase III DROSHA and its cofactor DGCR8, whose fidelity is critical for generation of functional miRNAs. To understand how Microprocessor recognizes pri-miRNAs, we here reconstitute human Microprocessor with purified recombinant proteins. We find that Microprocessor is an ∼364 kDa heterotrimeric complex of one DROSHA and two DGCR8 molecules. Together with a 23-amino acid peptide from DGCR8, DROSHA constitutes a minimal functional core. DROSHA serves as a "ruler" by measuring 11 bp from the basal ssRNA-dsRNA junction. DGCR8 interacts with the stem and apical elements through its dsRNA-binding domains and RNA-binding heme domain, respectively, allowing efficient and accurate processing. DROSHA and DGCR8, respectively, recognize the basal UG and apical UGU motifs, which ensure proper orientation of the complex. These findings clarify controversies over the action mechanism of DROSHA and allow us to build a general model for pri-miRNA processing.
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                Author and article information

                Contributors
                Journal
                Mol Ther
                Mol. Ther
                Molecular Therapy
                American Society of Gene & Cell Therapy
                1525-0016
                1525-0024
                07 August 2019
                24 May 2019
                : 27
                : 8
                : 1364-1371
                Affiliations
                [1 ]Department of Chemistry, Seoul National University, Seoul 08826, South Korea
                [2 ]Center for Genome Engineering, Institute for Basic Science, Seoul 08826, South Korea
                [3 ]Fight against Angiogenesis-Related Blindness (FARB) Laboratory, Clinical Research Institute, Seoul National University Hospital, Seoul 03080, South Korea
                [4 ]Department of Chemistry, Hanyang University, Seoul 04763, South Korea
                [5 ]Research Institute for Convergence of Basic Sciences, Hanyang University, Seoul 04763, South Korea
                [6 ]Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul 03080, South Korea
                [7 ]Department of Ophthalmology, Seoul National University College of Medicine, Seoul 03080, South Korea
                Author notes
                []Corresponding author: Jeong Hun Kim, Fight against Angiogenesis-Related Blindness (FARB) Laboratory, Clinical Research Institute, Seoul National University Hospital, Seoul 03080, South Korea. steph25@ 123456snu.ac.kr
                [∗∗ ]Corresponding author: Department of Chemistry, Hanyang University, Seoul 04763, South Korea. sangsubae@ 123456hanyang.ac.kr
                [8]

                These authors contributed equally to this work.

                Article
                S1525-0016(19)30227-8
                10.1016/j.ymthe.2019.05.013
                6698196
                31164261
                © 2019 The Author(s)

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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