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Related Concept Videos

CRISPR01:59

CRISPR

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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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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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CRISPR and crRNAs02:53

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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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Updated: Jun 28, 2025

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9
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Cas9 RNP Physiochemical Analysis for Enhanced CRISPR-AuNP Assembly and Function.

Daniel D Lane1, Karthikeya S V Gottimukkala1,2, Rachel A Cunningham1,2

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Researchers developed improved CRISPR-gold nanoparticles for in vivo gene editing in hematopoietic stem cells. This approach enhances CRISPR-Cas9 delivery and activity, potentially overcoming limitations of current ex vivo therapies for blood disorders.

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Area of Science:

  • Gene editing and nanomedicine for hematological disorders.

Background:

  • Current CRISPR therapies for beta thalassemia and sickle cell anemia require ex vivo manipulation of hematopoietic stem and progenitor cells (HSPC), involving toxic electroporation and chemotherapy conditioning.
  • In vivo delivery of CRISPR gene editing tools via nanocarriers offers a promising alternative to mitigate complexity and toxicity, but faces challenges in overcoming HSPC restriction factors.
  • Previous CRISPR-gold nanoparticle (CRISPR-AuNP) systems showed inconsistent Cas9 activity due to RNA instability during loading.

Approach:

  • Developed a second-generation CRISPR-AuNP by preforming Cas9 or Cas12a ribonucleoprotein complexes (RNP) before loading onto gold nanoparticles.
  • Optimized RNP loading chemistry and conditions to enhance nanoparticle loading efficiency and stability, incorporating PEGylation for a hydrophilic surface.
  • Evaluated the in vitro activity and HSPC uptake of the modified CRISPR-AuNPs.

Key Points:

  • Optimized loading strategies resulted in significantly higher Cas9 RNP/AuNP (39.6 ± 7.0) and Cas12a RNP/AuNP (10-fold increase) compared to previous methods, without compromising RNP activity.
  • The second-generation CRISPR-AuNP exhibits favorable characteristics for in vivo administration, including a stable, hydrophilic, and neutral surface.
  • In vitro studies demonstrated high uptake (72.5 ± 7.37%) of the nanoparticles by primary human HSPCs, although endosomal accumulation and limited gene editing were observed.

Conclusions:

  • Preformation of RNPs and optimized loading chemistry are crucial for efficient in vivo delivery of CRISPR-Cas9 and Cas12a gene editing systems via gold nanoparticles.
  • The developed CRISPR-AuNP nanoformulation shows potential for in vivo gene editing in HSPCs, but further improvements are needed to enhance endosomal escape and gene editing efficiency.