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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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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...
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Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
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CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats is a adaptive immune system found in bacteria and archaea that protects against viral infections. This system enables prokaryotic cells to identify, remember, and neutralize foreign genetic elements, primarily bacteriophages, by storing fragments of the invader’s DNA as a genetic memory.The CRISPR immune response begins during an initial infection. Cas (CRISPR-associated) proteins play a central role in this...
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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...
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Rapidly evolving homing CRISPR barcodes.

Reza Kalhor1, Prashant Mali2, George M Church1,3

  • 1Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.

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Summary
This summary is machine-generated.

Scientists engineered evolving DNA barcodes in living cells using a homing CRISPR-Cas9 system. This genetic barcode diversifies its sequence, enabling controlled lineage tracing and molecular recording for various biological applications.

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Area of Science:

  • Synthetic Biology
  • Genetics
  • Molecular Biology

Background:

  • Genetic barcodes are crucial for tracking cellular history.
  • Existing methods for lineage tracing have limitations in resolution and scalability.
  • CRISPR-Cas9 technology offers precise genome editing capabilities.

Purpose of the Study:

  • To develop an engineered, evolving DNA barcode system within living cells.
  • To demonstrate the utility of this system for recording cellular lineage.
  • To establish a foundation for in situ sequencing applications.

Main Methods:

  • Utilized a homing guide RNA (hgRNA) scaffold to direct a Cas9-hgRNA complex to the hgRNA's DNA locus.
  • Engineered the CRISPR-Cas9 system to function as a self-diversifying genetic barcode.
  • Controlled the rate of barcode sequence diversification in cultured cells.
  • Evaluated barcode performance in cell populations for lineage history recording.
  • Demonstrated in situ amplification of barcode RNA for subsequent sequencing.

Main Results:

  • Successfully engineered an evolving DNA barcode system in living cells.
  • Showcased the self-diversifying nature of the homing CRISPR-Cas9 system as a genetic barcode.
  • Demonstrated controllable diversification rates in cultured cells.
  • Validated the barcodes' ability to record lineage history in cell populations.
  • Confirmed the feasibility of in situ amplification of barcode RNA.

Conclusions:

  • The homing CRISPR-Cas9 system provides a novel approach for engineering evolving DNA barcodes.
  • This system enables precise and scalable lineage tracing and molecular recording.
  • The technology holds significant potential for applications in cancer biology, connectomics, and deep lineage tracing.