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Related Concept Videos

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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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A gene is a stretch of DNA that serves as the blueprint for functional RNAs and proteins. Since DNA is comprised  of nucleotides and proteins are comprised of amino acids, a mediator is required to convert the information encoded in DNA into proteins. This mediator is the messenger RNA (mRNA). mRNA copies the blueprint from DNA by a process called transcription. In eukaryotes, transcription occurs in the nucleus by complementary base-pairing with the DNA template. The mRNA is then...
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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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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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Multicellular organisms contain a variety of structurally and functionally distinct cell types, but the DNA in all the cells originated from the same parent cells. The differences in the cells can be attributed to the differential gene expression. Liver cells, whose functions include detoxification of blood, production of bile to metabolize fats, and synthesis of proteins essential for metabolism, must express a specific set of genes to perform their functions. Gene expression also varies with...
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Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9
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A Simple Cloning-free Method to Efficiently Induce Gene Expression Using CRISPR/Cas9.

Lyujie Fang1, Sandy S C Hung2, Jennifer Yek3

  • 1Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne, VIC, Australia; Ophthalmology, Department of Surgery, University of Melbourne, East Melbourne, VIC, Australia; Jinan University, Guangzhou, China.

Molecular Therapy. Nucleic Acids
|December 31, 2018
PubMed
Summary
This summary is machine-generated.

This study introduces a cloning-free CRISPR/Cas9 activation method for rapid gene expression studies. This technique efficiently induces endogenous genes, accelerating gain-of-function research in molecular and cell biology.

Keywords:
CRISPRCRISPR activationSaCas9SpCas9cloning freegene activationmultiplex gene activationtranscriptional activator

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Area of Science:

  • Molecular Biology
  • Gene Regulation
  • CRISPR Technology

Background:

  • Gain-of-function studies traditionally require laborious cDNA cloning for gene overexpression.
  • CRISPR/Cas technology offers innovative solutions for gene manipulation and expression studies.

Purpose of the Study:

  • To develop and validate a simple, cloning-free method for inducing endogenous gene expression using CRISPR/Cas9 activators.
  • To establish a rapid workflow for initiating gain-of-function studies.

Main Methods:

  • Utilized synthesized sgRNA expression cassettes to direct a nuclease-null Cas9 complex fused with transcriptional activators (VP64, p65, Rta).
  • Tested two CRISPR activation systems, dSpCas9VPR and dSaCas9VPR, for site-specific induction of endogenous genes in human and rat cells.
  • Assessed the induction efficiency of multiple neural development genes.

Main Results:

  • Demonstrated efficient induction of six neural development genes (CRX, RORB, RAX, OTX2, ASCL1, NEUROD1) in human cells using both CRISPR activators.
  • Observed variable and less efficient gene induction in rat cells for three genes (Ascl1, Neurod1, Nrl).
  • Confirmed the method's ability to initiate gain-of-function studies on the same day.

Conclusions:

  • The developed CRISPR/Cas9 activation strategy provides a simple and efficient means to activate endogenous gene expression.
  • This cloning-free approach serves as a rapid workflow for gain-of-function studies across various biological disciplines.
  • The system shows high efficiency in human cells, with potential for optimization in other cell types like rat cells.