Targeted Base Editing via RNA-Guided Cytidine Deaminases in Xenopus laevis Embryos

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 substitu...

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Published inMolecules and cells Vol. 40; no. 11; pp. 823 - 827
Main Authors Park, Dong-Seok, Yoon, Mijung, Kweon, Jiyeon, Jang, An-Hee, Kim, Yongsub, Choi, Sun-Cheol
Format Journal Article
LanguageEnglish
Published United States Korean Society for Molecular and Cellular Biology 30.11.2017
한국분자세포생물학회
Subjects
Albinism, Oculocutaneous - genetics
Amino Acid Substitution
Animals
Cytidine Deaminase - metabolism
Gene Editing - methods
Monophenol Monooxygenase - genetics
Mutation Rate
Phenotype
Point Mutation
Rapid Report
RNA, Guide, CRISPR-Cas Systems - genetics
Xenopus laevis - embryology
Xenopus laevis - genetics
Xenopus Proteins - genetics
생물학
CRISPR/Cas9
base editing
Xenopus laevis
genome engineering
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Summary: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.
Bibliography:These authors contributed equally to this work.
ISSN:1016-8478
0219-1032
DOI:10.14348/molcells.2017.0262