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Related Concept Videos

Conservative Site-specific Recombination and Phase Variation02:53

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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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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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Translesion (TLS) polymerases rescue stalled DNA polymerases at sites of damaged bases by replacing the replicative polymerase and installing a nucleotide across the damaged site. Doing so, TLS allows additional time for the cell to repair the damage before resuming regular DNA replication.
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DNA-only transposons are called autonomous transposons since they code for the enzyme transposase that is required for the transposition mechanism. Insertion of transposons can alter gene functions in multiple ways. They can mutate the gene, alter gene expression by introducing a novel promoter or insulator sequence, introduce new splice sites, and change the mRNA transcripts produced, or remodel chromatin structure.
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The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...
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Related Experiment Video

Updated: Nov 15, 2025

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A versatile platform for locus-scale genome rewriting and verification.

Ran Brosh1, Jon M Laurent1, Raquel Ordoñez1

  • 1Institute for Systems Genetics, NYU Langone Health, New York, NY 10016.

Proceedings of the National Academy of Sciences of the United States of America
|March 2, 2021
PubMed
Summary
This summary is machine-generated.

Big-IN is a new platform for large DNA integration in mammalian cells. This versatile tool enables scalable functional genomic analysis by efficiently engineering stem cells with large DNA constructs.

Keywords:
genome engineeringgenome writingregulatory genomicsstem cellssynthetic biology

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Area of Science:

  • Genomics
  • Molecular Biology
  • Biotechnology

Background:

  • Functional analysis of human traits and diseases requires efficient DNA rewriting in specific genomic loci.
  • Current DNA integration methods lack scalability and portability across different genomic regions and cell types.

Purpose of the Study:

  • To develop a versatile platform, named Big-IN, for targeted integration of large DNA sequences into mammalian cells.
  • To overcome the limitations of existing DNA integration approaches in terms of scalability and portability.

Main Methods:

  • Utilized CRISPR/Cas9 technology to target and insert a landing pad into the genome.
  • Employed recombinase-mediated delivery for introducing large DNA payloads (up to 143 kb) into the landing pad.
  • Implemented efficient positive/negative selection strategies for isolating correctly engineered mammalian stem cells.
  • Developed a verification pipeline combining PCR genotyping and targeted capture sequencing.

Main Results:

  • Demonstrated successful integration of large DNA constructs up to 143 kb using the Big-IN platform.
  • Showcased a one-step scarless DNA delivery method.
  • Established an economical and comprehensive verification process for engineered stem cells.

Conclusions:

  • The Big-IN platform offers a versatile and scalable solution for targeted large DNA integration in mammalian cells.
  • This approach facilitates functional genomic studies by enabling efficient engineering of stem cells with large DNA constructs.
  • Big-IN supports combinatorial interrogation of genomic elements and systematic locus-scale genome function analysis.