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Mol Cells
2017 Nov 30;4011:823-827. doi: 10.14348/molcells.2017.0262.
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Targeted Base Editing via RNA-Guided Cytidine Deaminases in Xenopus laevis Embryos.
Park DS
,
Yoon M
,
Kweon J
,
Jang AH
,
Kim Y
,
Choi SC
.
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Genome editing using programmable nucleases such as CRISPR/Cas9 or Cpf1 has emerged as powerful tools for gene knock-out or knock-in in various organisms. While most genetic diseases are caused by point mutations, these genome-editing approaches are inefficient in inducing single-nucleotide substitutions. Recently, Cas9-linked cytidine deaminases, named base editors (BEs), have been shown to convert cytidine to uridine efficiently, leading to targeted single-base pair substitutions in human cells and organisms. Here, we first report on the generation of Xenopus laevis mutants with targeted single-base pair substitutions using this RNA-guided programmable deaminase. Injection of base editor 3 (BE3) ribonucleoprotein targeting the tyrosinase (tyr) gene in early embryos can induce site-specific base conversions with the rates of up to 20.5%, resulting in oculocutaneous albinism phenotypes without off-target mutations. We further test this base-editing system by targeting the tp53 gene with the result that the expected single-base pair substitutions are observed at the target site. Collectively, these data establish that the programmable deaminases are efficient tools for creating targeted point mutations for human disease modeling in Xenopus.
Fig. 1. Generation of tyrosinase-deficient X. laevis by a RNA-guided programmable deaminase, BE3(A) The target sequences at the tyra and tyrb loci. The PAM and target sequences for gRNAs are shown in red and black bar, respectively. The nucleotides substituted by programmable deaminase are shown in red. (B) In vitro assay for Cas9 protein-mediated cleavage of the target sequences at tyra and tyrb. (C) In vitro deamination assay for BE3 RNP with gRNA #1 targeting both tyra and tyrb. Arrowheads indicate cleavage products for each target site. (D, E) Targeted deep sequencing analysis of frequencies of indel mutation (D) and C-to-T substitution (E) that were induced by Cas9 or BE3, respectively, in X. laevis. UN, uninjected control embryos. (F) Percentages of phenotypes of the Cas9 or BE3 RNP-injected embryos. With the phenotypically abnormal (cyclopic, strongly kinked axes) embryos being discarded, the phenotypes were categorized into 3 groups (severe, moderate, weak) according to the degree of loss of pigmentation in melanocytes and RPE. (G) Compared to wild-type (WT) control embryos, the BE3 RNP-injected embryos exhibited significant reduction of pigmentation in the eyes and melanocytes along the antero-posterior body axis. (H) Alignment of the most frequent mutant sequences from the BE3-injected pooled X. laevis embryos. Target sequences are underlined. The nucleotides substituted by programmable deaminase are shown in red. Dashes (−) denote gaps. The nature of the mutations is indicated in the right column. WT, wild-type; sub, substitutions; Δ, deletion. The number of deleted base pairs is shown after the symbol, Δ. (I) On-target and off-target sequences of tyr gene are shown on the left column. The PAM site and mismatched bases are shown in blue and red, respectively. The UN, uninjected control embryo.
Fig. 2. Generation of premature stop codon in tp53 via a programmable deaminase, BE3(A) The target sequences at the tp53 locus. The PAM and target sequences for gRNAs #1 and #2 are shown in red and black bar, respectively. The nucleotides substituted by programmable deaminase are shown in red. (B) In vitro cleavage and (C) in vitro deamination assays for the target sequences in PCR amplicons from X. laevis genomic DNA. Arrowheads indicate cleavage products for each target site. (D, E) Targeted deep sequencing analysis of frequencies of indel mutations (D) and C-to-T substitution (E) in Cas9 or BE3 RNP-injected X. laevis embryos. UN, uninjected control embryo. (F) Alignment of mutant sequences from the BE3-injected pooled X. laevis embryos. The target sequences are underlined. The nucleotides substituted by programmable deaminase are shown in red. The nature of the mutations is shown in the right column. WT, wild-type; Δ, deletion; sub, substitutions. The number of deleted base pairs is indicated after the symbol, Δ.
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