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RNA Editing02:23

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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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Synthesis of new DNA molecules is carried out by the enzyme DNA polymerase, which adds nucleotides on the daughter strand complementary to the template DNA strand. DNA polymerase has a higher affinity to add the correct base and ensures fidelity during DNA replication. Furthermore,  it exhibits proofreading activity during replication, using an exonuclease domain that cuts off incorrect nucleotides from the nascent DNA strand.
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Rationally Designed APOBEC3B Cytosine Base Editors with Improved Specificity.

Shuai Jin1, Hongyuan Fei1, Zixu Zhu1

  • 1State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome Editing, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China; College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China.

Molecular Cell
|July 30, 2020
PubMed
Summary

Cytosine base editors (CBEs) can cause unintended DNA changes. A new R-loop assay in rice identified highly specific CBE variants, A3Bctd-VHM-BE3 and A3Bctd-KKR-BE3, with reduced off-target effects.

Keywords:
A3Bctd deaminasecytosine base editorsnSaCas9off-target editingrationally designingspecificitywhole-genome sequencing

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Area of Science:

  • Molecular Biology
  • Plant Science
  • Genome Editing

Background:

  • Cytosine base editors (CBEs) enable precise C-to-T substitutions without double-strand breaks.
  • Off-target mutations, particularly sgRNA-independent deamination by CBEs like BE3, pose a significant challenge.
  • Identifying and mitigating these off-target effects is crucial for safe and effective genome editing applications.

Purpose of the Study:

  • To develop a high-throughput assay for assessing sgRNA-independent off-target effects of CBEs in rice.
  • To screen and identify CBE variants with enhanced specificity and reduced off-target activity.
  • To validate the efficacy of newly developed CBE variants in eliminating unwanted DNA modifications.

Main Methods:

  • Utilized SaCas9 nickase to generate orthogonal R-loops, mimicking transcriptionally active loci susceptible to deamination.
  • Established a high-throughput assay in rice protoplasts to evaluate CBE off-target activity.
  • Employed whole-genome sequencing (WGS) to confirm assay reliability and analyze off-target edits in regenerated rice plants.

Main Results:

  • The R-loop assay demonstrated reliability, confirmed by WGS analysis of 10 base editors in rice.
  • Screening identified two efficient CBE variants, A3Bctd-VHM-BE3 and A3Bctd-KKR-BE3, with significantly improved specificity.
  • WGS analysis confirmed that these novel CBEs eliminated sgRNA-independent off-target DNA edits in rice plants.
  • The new variants also exhibited enhanced precision at target sites, reducing multiple C-to-T edits.

Conclusions:

  • The developed R-loop assay provides a robust platform for assessing CBE specificity in plants.
  • A3Bctd-VHM-BE3 and A3Bctd-KKR-BE3 represent highly specific and precise cytosine base editors for rice genome editing.
  • These findings contribute to the development of safer and more accurate genome editing tools for crop improvement.