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RNA editing is a post-transcriptional modification where a precursor mRNA (pre-mRNA) nucleotide sequence is changed by base insertion, deletion, or modification. The extent of RNA editing varies from a few hundred bases, in mitochondrial DNA of trypanosomes, to a just single base, in nuclear genes of mammals. Even a single base change in the pre-mRNA can convert a codon for one amino acid into the codon for another amino acid or a stop codon. This type of re-coding can significantly affect the...
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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
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Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors
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Improved cytosine base editors generated from TadA variants.

Dieter K Lam1, Patricia R Feliciano1, Amena Arif1

  • 1Beam Therapeutics, Cambridge, MA, USA.

Nature Biotechnology
|January 9, 2023
PubMed
Summary
This summary is machine-generated.

Engineered cytosine base editors (CBEs) achieve precise C-to-T DNA edits with high on-target accuracy and no detectable off-target mutations. New cytosine and adenine base editors (CABEs) enable all transition mutations for versatile genome editing applications.

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Area of Science:

  • Genetics
  • Molecular Biology
  • Biotechnology

Background:

  • Cytosine base editors (CBEs) are tools for programmable C·G-to-T·A mutations, typically using CRISPR-Cas and cytidine deaminase.
  • Naturally occurring deaminases in CBEs can lead to unintended genome-wide edits, while improved versions may compromise on-target efficiency.

Purpose of the Study:

  • To develop novel CBEs with enhanced on-target activity and reduced off-target effects.
  • To engineer cytosine and adenine base editors (CABEs) for broader transition mutation capabilities.

Main Methods:

  • Generation and characterization of CBEs utilizing engineered TadA variants (CBE-Ts).
  • Assessment of on-target editing efficiency across diverse genomic loci.
  • Evaluation of genome-wide off-target mutations and editor activity in primary cells.
  • Development of CABEs capable of both A-to-I and C-to-U editing.

Main Results:

  • Engineered CBE-Ts demonstrated high on-target C·G-to-T·A editing efficiency in sequence-diverse genomic sites.
  • No detectable increase in genome-wide mutations was observed with the engineered CBE-Ts.
  • CABEs were developed, enabling both A-to-I and C-to-U editing.
  • The engineered TadA variants offer improved properties compared to previous CBEs.

Conclusions:

  • Engineered TadA variants provide highly accurate and efficient CBEs with minimal off-target activity.
  • CBE-Ts and CABE-Ts, alongside ABEs, expand the toolkit for programmable installation of all transition mutations.
  • These advancements offer improved precision and versatility in base editing technologies.