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Response to “Unexpected mutations after CRISPR–Cas9 editing in vivo ”

Published on Apr 1, 2018in Nature Methods28.467
· DOI :10.1038/nmeth.4553
Lauryl M. J. Nutter13
Estimated H-index: 13
,
Jason D. Heaney12
Estimated H-index: 12
(BCM: Baylor College of Medicine)
+ 6 AuthorsMark W. Moore31
Estimated H-index: 31
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Abstract
  • References (24)
  • Citations (13)
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References24
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Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas) systems employ the dual RNA–guided DNA endonuclease Cas9 to defend against invading phages and conjugative plasmids by introducing site-specific double-stranded breaks in target DNA. Target recognition strictly requires the presence of a short protospacer adjacent motif (PAM) flanking the target site, and subsequent R-loop formation and strand scission are driven by complementary base pairin...
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Genome instability, defined as higher than normal rates of mutation, is a double-edged sword. As a source of genetic diversity and natural selection, mutations are beneficial for evolution. On the other hand, genomic instability can have catastrophic consequences for age-related diseases such as cancer. Mutations arise either from inactivation of DNA repair pathways or in a repair-competent background due to genotoxic stress from celluar processes such as transcription and replication that overw...
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Background Nuclease-based technologies have been developed that enable targeting of specific DNA sequences directly in the zygote. These approaches provide an opportunity to modify the genomes of inbred mice, and allow the removal of strain-specific mutations that confound phenotypic assessment. One such mutation is the Cdh23ahl allele, present in several commonly used inbred mouse strains, which predisposes to age-related progressive hearing loss.
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Knockin (KI) mouse carrying a point mutation has been an invaluable tool for disease modeling and analysis. Genome editing technologies using the CRISPR/Cas system has emerged as an alternative way to create KI mice. However, if the mice carry nucleotide insertions and/or deletions (InDels) in other genes, which could have unintentionally occurred during the establishment of the KI mouse line and potentially have larger impact than a point mutation, it would confound phenotyping of the KI mice. ...
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Massive parallel sequencing enables identification of numerous genetic variants in mutant organisms, but determining pathogenicity of any one mutation can be daunting. The most commonly studied preclinical model of retinitis pigmentosa called the "rodless" ( rd1 ) mouse is homozygous for two mutations: a nonsense point mutation (Y347X) and an intronic insertion of a leukemia virus ( Xmv-28 ). Distinguishing which mutation causes retinal degeneration is still under debate nearly a century after t...
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Background Phenotypic variability among inbred littermates reared in controlled environments remains poorly understood. Metastable epialleles refer to loci that intrinsically behave in this way and a few examples have been described. They display differential methylation in association with differential expression. For example, inbred mice carrying the agouti viable yellow (Avy) allele show a range of coat colours associated with different DNA methylation states at the locus. The availability of...
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Germline mutations are the ultimate source of congenital diseases, individual phenotypic variations, and evolutionary phenotypic changes. The per generation de novo mutation rate affects genetic variability and the speed of evolution (Kimura 1983; Drake et al. 1998). Advancements in high-throughput sequencing have made it possible to determine the mutation rates in various organisms. For example, the mean germline base-substitution mutation rate is calculated at 1.2 × 10−8 per nucleotide per gen...
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Abstract Genome editing tools have already revolutionised biomedical research and are also expected to have an important impact in the clinic. However, their extensive use in research has revealed much unpredictability, both off- and on-target, in the outcome of their application. We discuss the challenges associated with this unpredictability, both for research and in the clinic. For the former, an extensive validation of the model is essential. For the latter, potential unpredicted activity do...
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Deaminase base editing has emerged as a tool to install or correct point mutations in the genomes of living cells in a wide range of organisms. However, the genome-wide off-target effects introduced by base editors in the mammalian genome have been examined in only one study. Here, we have investigated the fidelity of cytosine base editor 4 (BE4) and adenine base editors (ABE) in mouse embryos using unbiased whole-genome sequencing of a family-based trio cohort. The same sgRNA was used for BE4 a...
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CRISPR-Cas9 is a widely-used genome editing tool, but its off-target effect and on-target complex mutations remain a concern, especially in view of future clinical applications. Non-human primates (NHPs) share close genetic and physiological similarities with humans, making them an ideal preclinical model for developing Cas9-based therapies. However, to our knowledge no comprehensive in vivo off-target and on-target assessment has been conducted in NHPs. Here, we perform whole genome trio sequen...
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