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Related Concept Videos

Mismatch Repair01:20

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Organisms are capable of detecting and fixing nucleotide mismatches that occur during DNA replication. This sophisticated process requires identifying the new strand and replacing the erroneous bases with correct nucleotides. Mismatch repair is coordinated by many proteins in both prokaryotes and eukaryotes.
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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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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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Base complementarity between the three base pairs of mRNA codon and the tRNA anticodon is not a failsafe mechanism. Inaccuracies can range from a single mismatch to no correct base pairing at all. The free energy difference between the correct and nearly correct base pairs can be as small as 3 kcal/ mol. With complementarity being the only proofreading step, the estimated error frequency would be one wrong amino acid in every 100 amino acids incorporated. However, error frequencies observed in...
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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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DNA replication is a well-evolved process that copies millions of base pairs with high fidelity during each cell division. Occasionally a wrong base or a long stretch of wrong bases may get added to the daughter strands. If the errors are left unchecked, cells might accumulate several mutations that might endanger their  survival. Therefore, the copying errors are checked and repaired at three levels.
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Updated: Sep 28, 2025

Efficient PAM-Less Base Editing for Zebrafish Modeling of Human Genetic Disease with zSpRY-ABE8e
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Highly efficient prime editing by introducing same-sense mutations in pegRNA or stabilizing its structure.

Xiaosa Li1,2,3, Lina Zhou4,5,6,7, Bao-Qing Gao8

  • 1Department of Ophthalmology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, 200080, Shanghai, China. xiaosa.li@shgh.cn.

Nature Communications
|March 30, 2022
PubMed
Summary
This summary is machine-generated.

Researchers enhanced prime editing (PE) efficiency using novel RNA designs. spegRNA and apegRNA significantly boost base and indel editing, enabling precise genetic modifications at previously inaccessible sites.

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Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
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Area of Science:

  • Molecular Biology
  • Gene Editing Technologies

Background:

  • Prime editing (PE) combines Cas9 nickase and reverse transcriptase for precise DNA modifications.
  • Current PE systems exhibit limitations in editing efficiency, restricting their application.

Purpose of the Study:

  • To develop novel guide RNA structures to enhance prime editing efficiency.
  • To improve both base substitution and indel editing capabilities of prime editors.

Main Methods:

  • Designed spegRNA (single-sense prime editing guide RNA) by modifying the reverse-transcription template of pegRNA.
  • Developed apegRNA (anti-prime editing guide RNA) by altering pegRNA secondary structure.
  • Integrated spegRNA and apegRNA into PE3 and PE5 prime editing systems.

Main Results:

  • spegRNA increased base-editing efficiency up to 4,976-fold (average 353-fold).
  • apegRNA enhanced indel-editing efficiency up to 10.6-fold (average 2.77-fold).
  • Combined strategies in sPE3, aPE3, sPE5, and aPE5 systems showed significant efficiency improvements.

Conclusions:

  • Novel spegRNA and apegRNA strategies substantially enhance prime editing efficiency.
  • These advancements enable highly efficient prime editing at previously challenging genomic sites.
  • The developed methods expand the utility of prime editing for precise genetic engineering.