JoVE - Journal of Visualized Experiments
JoVE Visualize
Contact Us

Long sequence insertion via CRISPR/Cas gene-editing with transposase, recombinase, and integrase

  • 0Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA.
Current Opinion in Biomedical Engineering +

|

March 29, 2024

|

March 29, 2024

View abstract on PubMed

Summary

This summary is machine-generated.

CRISPR gene editing now enables inserting large DNA sequences into genomes. This review covers new methods and enzymes driving advancements in genome engineering for biomedical applications.

Area Of Science

  • Genome Science
  • Molecular Biology
  • Biotechnology

Background

  • CRISPR/Cas technology revolutionized genome science.
  • Early CRISPR applications focused on gene knock-out and base editing.
  • Recent advancements target large DNA fragment insertion (>1 kb).

Purpose Of The Study

  • To review the development of CRISPR/Cas-based sequence insertion methodologies.
  • To highlight novel editing enzymes used for large sequence insertion.
  • To discuss the potential of these technologies in biomedical applications.

Main Methods

  • Survey of CRISPR/Cas-based sequence insertion techniques.
  • Examination of novel enzyme families including transposases, recombinases, and integrases.
  • Analysis of current challenges and future directions in long-sequence editing.

Main Results

  • Development of CRISPR/Cas systems for inserting large DNA fragments.
  • Integration of new enzyme families to enhance editing efficiency and specificity.
  • Identification of key challenges hindering widespread application.

Conclusions

  • CRISPR/Cas-based large sequence insertion is a rapidly evolving field.
  • Novel enzymes are crucial for advancing genome engineering capabilities.
  • This technology promises to drive significant biomedical breakthroughs.

Keywords:

Related Concept Videos

CRISPR 01:59

50.8K

Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...

Conservative Site-specific Recombination and Phase Variation 02:53

6.0K

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.
The recognition sites for Cre recombinase called LoxP...

Homologous Recombination 02:31

50.5K

The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...

Overview of Transposition and Recombination 02:13

15.5K

Transposons make up a significant part of genomes of various organisms. Therefore, it is believed that transposition played a major evolutionary role in speciation by changing genome sizes and modifying gene expression patterns. For example, in bacteria, transposition can lead to conferring antibiotic resistance. Movement of transposable elements within the genetic pool of pathogenic bacteria can aid in transfer of antibiotic-resistant genetic elements. In eukaryotes, transposons can carry out...

DNA-only Transposons 02:57

14.5K

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.
The donor site from where the transposon is excised is either degraded or...